The Impact of Controlled Ovarian Stimulation on Serum Oxidative Stress Markers in Infertile Women with Endometriosis Undergoing ICSI.

Endometriosis-related infertility is associated with oxidative stress (OS). The present study aims to compare serum OS markers of infertile women with endometriosis and controls during the follicular phase of the natural cycle (D1), after pituitary downregulation using a GnRH agonist (D2), after controlled ovarian stimulation (COS) on the day of human chorionic gonadotropin administration (D3), and on the day of oocyte retrieval (D4). One hundred and eight serum samples (58 controls and 35 early and 18 advanced endometriosis cases) were collected at these four timepoints. OS markers were compared among the groups and timepoints using a linear regression model with mixed effects and a post-test using orthogonal contrasts. The significance was set at 5%. We observed altered OS markers in the endometriosis patients during the D1, D2, D3, and D4 timepoints compared to the controls. The evidence of systemic OS in infertile patients with endometriosis during COS suggests the mobilization of potent antioxidants in an attempt to protect the oocyte from oxidative damage, especially on the day of oocyte retrieval.

Systemic oxidative stress as a possible mechanism underlying the pathogenesis of mild endometriosis-related infertility.

RESEARCH QUESTION: Does systemic oxidative stress occur during the early follicular phase of the menstrual cycle in infertile women with minimal (stage I) or mild (stage II) endometriosis? Are serum oxidative stress markers during the early follicular phase of the menstrual cycle good predictors of successful gestation in these women who undergo ovarian stimulation for intracytoplasmic sperm injection (ICSI)? MATERIALS AND METHODS: A pilot study (prospective case-control study) was conducted in a University Hospital. Serum samples were obtained during the early follicular phase of the natural cycle preceding ovarian stimulation for ICSI of infertile women (with and without stage I and II endometriosis, the latter having male factor infertility). Total hydroperoxides (FOX1), malondialdehyde, advanced oxidation protein products, reduced glutathione, superoxide dismutase, total antioxidant capacity (TAC), 8-hydroxy-2'-deoxyguanosine (8OHdG) and vitamin E were analysed in serum from 35 women with stage I or II endometriosis and 60 control women. The accuracy of oxidative stress markers for predicting clinical pregnancy and live births was determined by receiver operator characteristic curves. RESULTS: Women with stage I and II endometriosis showed lower serum 8OHdG concentrations (16.02 ng/ml) compared with the control group (22.08 ng/ml). The best predictor for clinical pregnancy and live births was TAC, whereas FOX1 was the best predictor of clinical pregnancy in the control group. CONCLUSIONS: Infertile women with stage I and II endometriosis present systemic oxidative stress during the early follicular phase of the menstrual cycle. Some oxidative stress markers were good predictors of clinical pregnancy and live births after ICSI. Serum TAC was predictive of clinical pregnancy and live births after ICSI in women with stage I or II endometriosis.

Prematuration of bovine oocytes with butyrolactone I reversibly arrests meiosis without increasing meiotic abnormalities after in vitro maturation.

OBJECTIVES: Asynchrony between nuclear and cytoplasmic maturation, and possibly damage to the oocyte meiotic spindle, limits the application of in vitro maturation (IVM) in assisted reproduction. Several studies have suggested that Prematuration with meiosis blockers may improve oocyte quality after IVM, favoring early embryogenesis. Thus, we investigated the effect of Prematuration with the nuclear maturation inhibitor butyrolactone I (BLI) on the meiotic spindle and chromosomal configuration of bovine oocytes. STUDY DESIGN: Immature oocytes obtained from cows slaughtered in a slaughterhouse (n=840) were divided into the following groups: (1) control (n=325), submitted only to IVM in TCM199 for 24h; (2) BLI 18h (n=208) submitted to meiotic blockage with 100 microM BLI for 24h (Prematuration) and then induction of IVM in TCM199 for 18h; and (3) BLI 24h (n=307), pre-matured with 100 microM BLI for 24h followed by 24h of IVM in TCM199. The oocytes were then fixed, stained by immunofluorescence for morphological visualization of both microtubules and chromatin, and evaluated. RESULTS: Meiotic arrest occurred in 90.2% of the oocytes cultured with BLI. Maturation rates were similar for all groups (80.3%, 73.6% and 82.7% for the control, BLI 18h and BLI 24h groups, respectively). We observed 81.3% normal oocytes in metaphase II in the control group, and 80.0% and 81.2% in the BLI 18h and BLI 24h groups, respectively. The incidence of meiotic anomalies did not differ between groups (18.7%, 20.0% and 18.8% for the control, BLI 18h and BLI 24h, respectively). CONCLUSION: Prematuration with butyrolactone I reversibly arrests meiosis without damaging the meiotic spindle or the chromosome distribution of bovine oocytes after in vitro maturation.

Bloqueio da meiose sem aumento da incidência de anomalias meióticas / Blockage of meiosis without increase of meiotic abnormalities incidence

Objetivo: investigar o efeito do pré-maturação com o inibidor de maturação nuclear, butirolactona-I (BLI), sobre o fuso meiótico e distribuição cromossômica de oócitos bovinos, o que poderia melhorar a qualidade oocitária e embriogênese. Material e métodos: oócitos imaturos, obtidos de vacas abatidas em matadouro (n igual 610) foram submetidos nos grupos: Controle (n igual 208) submetidos à maturação in vitro (MI) em TCM 199 por 24 horas; BLI -18 horas (n igual 208) submetidos à pré-maturação com 100 miuM de BLI por 24 horas e posterior indução da MI em TCM 199, por 18 horas; BLI 24 horas (n igual 195), pré-maturados com 100 miuM de BLI, por 24 horas, seguida por 24 horas de MIV em TCM 199. Em seguida, os oócitos foram fixados, corados por imunofluorescência e avaliados. Resultados: o bloqueio meiótico ocorreu em 88,8% dos oócitos pré-maturados. As taxas de maturaçao foram similares (79,3; 73,5 e 82%, respectivamente, para Controle, BLI 18 horas e BLI 24 horas). Observou-se 82,5% oócitos normais em metáfase II no Controle e 80 e 82% nos grupos BLI 18 horas e BLI 24 horas, respectivamente. A incidência de anomalias meióticas não diferiu (17,5; 20 e 18%, respectivamente, para Controle, BLI 18 horas e BLI 24 horas) - p menor que 0,05, teste do x2. Conclusões: a BLI bloqueia a meiose sem promover danos ao fuso meiótico e distribuição cromossômica oocitária após a MIV

Anomalias meióticas de oócitos de pacientes com endometriose submetidas à estimulação ovariana / Meiotic abnormalities of oocytes from patients with endometriosis submitted to ovarian stimulation

OBJETIVO: avaliar o fuso meiótico e a distribuição cromossômica de oócitos maturados in vitro, obtidos de ciclos estimulados de mulheres inférteis com endometriose e fatores masculino e/ou tubário de infertilidade (Grupo Controle), comparando as taxas de maturação in vitro (MIV) entre os dois grupos avaliados. MÉTODOS: quatorze pacientes com endometriose e oito com fator tubário ou masculino, submetidas à estimulação ovariana para injeção intracitoplasmática de espermatozóide, foram selecionadas, prospectiva e consecutivamente, e constituíram os Grupos de Estudo e Controle, respectivamente. Oócitos imaturos (46 e 22, respectivamente, dos Grupos Endometriose e Controle) foram submetidos à MIV. Oócitos que apresentaram a extrusão do primeiro corpúsculo polar foram fixados e corados para avaliação dos microtúbulos e cromatina por técnica de imunofluorescência. A análise estatística foi realizada utilizando o teste exato de Fisher, com significância estatística quando p<0,05. RESULTADOS: não se observou diferença significativa nas taxas de MIV entre os dois grupos avaliados (45,6 e 54,5 por cento, respectivamente, nos Grupos Endometriose e Controle). A organização cromossômica e do fuso meiótico foi observada em 18 e 11 oócitos dos Grupos Endometriose e Controle, respectivamente. No Grupo Endometriose, oito oócitos (44,4 por cento) se apresentavam como metáfase II (MII) normais, três (16,7 por cento) MII anormais, cinco (27,8 por cento) estavam em estágio de telófase I e dois (11,1 por cento) sofreram ativação partenogenética. No Grupo Controle, cinco oócitos (45,4 por cento) se apresentavam como MII normais, três (27,3 por cento) MII anormais, um (9,1 por cento) estava em estágio de telófase I e dois (18,2 por cento) sofreram ativação partenogenética. Não se observou diferença significativa na porcentagem de anomalias meióticas entre os oócitos em MII dos dois grupos avaliados. CONCLUSÕES: os dados do presente estudo não demonstraram diferença...

PURPOSE: to evaluate the meiotic spindle and the chromosome distribution of in vitro mature oocytes from stimulated cycles of infertile women with endometriosis, and with male and/or tubal infertility factors (Control Group), comparing the rates of in vitro maturation (IVM) between the two groups evaluated. METHODS: fourteen patients with endometriosis and eight with male and/or tubal infertility factors, submitted to ovarian stimulation for intracytoplasmatic sperm injection have been prospectively and consecutively selected, and formed a Study and Control Group, respectively. Immature oocytes (46 and 22, respectively, from the Endometriosis and Control Groups) were submitted to IVM. Oocytes presenting extrusion of the first polar corpuscle were fixed and stained for microtubules and chromatin evaluation through immunofluorescence technique. Statistical analysis has been done by the Fisher's exact test, with statistical significance at p<0.05. RESULTS: there was no significant difference in the IVM rates between the two groups evaluated (45.6 and 54.5 percent for the Endometriosis and Control Groups, respectively). The chromosome and meiotic spindle organization was observed in 18 and 11 oocytes from the Endometriosis and Control Groups, respectively. In the Endometriosis Group, eight oocytes (44.4 percent) presented themselves as normal metaphase II (MII), three (16.7 percent) as abnormal MII, five (27.8 percent) were in telophase stage I and two (11.1 percent) underwent parthenogenetic activation. In the Control Group, five oocytes (45.4 percent) presented themselves as normal MII, three (27.3 percent) as abnormal MII, one (9.1 percent) was in telophase stage I and two (18.2 percent) underwent parthenogenetic activation. There was no significant difference in meiotic anomaly rate between the oocytes in MII from both groups. CONCLUSIONS: the present study data did not show significant differences in the IVM or in the meiotic anomalies...

Avaliação de anomalias meióticas de oócitos em pacientes com síndrome dos ovários policísticos submetidas à estimulação ovariana / Evaluation of meiotic abnormalities of oocytes from polycystic ovary syndrome patients submitted to ovarian stimulation

OBJETIVO: avaliar o fuso meiótico e a distribuição cromossômica de oócitos maturados in vitro, obtidos de ciclos estimulados de mulheres inférteis com síndrome dos ovários policísticos (SOP) e fatores masculino e/ou tubário de infertilidade (Grupo Controle) e comparar as taxas de maturação in vitro (MIV) entre os dois grupos avaliados. MÉTODOS: cinco pacientes inférteis com SOP e oito pacientes controles, submetidas à estimulação ovariana para injeção intracitoplasmática de espermatozóide, foram selecionadas prospectiva e consecutivamente, e constituíram os grupos de estudo e Controle, respectivamente. Oócitos imaturos captados após estimulação ovariana para a realização de injeção intracitoplasmática de espermatozóide (21 e 29, respectivamente, nos Grupos SOP e Controle) foram submetidos à MIV. Apenas os oócitos que apresentaram a extrusão do primeiro corpúsculo polar após a MIV foram fixados e submetidos à imunocoloração e análise por microscopia de fluorescência para avaliação morfológica do fuso e da distribuição cromossômica. A análise estatística foi realizada utilizando o teste exato de Fisher, com significância estatística quando p<0,05. RESULTADOS: as taxas de MIV foram similares entre os dois grupos (47,6 e 44,8 por cento, respectivamente, nos Grupos SOP e Controle). Seis dos dez oócitos (60 por cento) analisados do grupo de estudo e quatro dos 12 oócitos (33,3 por cento) analisados do Grupo Controle apresentaram anomalias meióticas, caracterizadas por anomalias do fuso e/ou distribuição cromossômica oocitária, sem diferença significativa entre os grupos. CONCLUSÕES: os dados do presente estudo não demonstraram diferença significativa nas taxas de MIV e nas proporções de anomalias meióticas entre os oócitos maturados in vitro, provenientes de ciclos estimulados de pacientes com SOP, quando comparados aos controles.

PURPOSE: to evaluate the meiotic spindle and the chromosome distribution of in vitro matured oocytes obtained from stimulated cycles of infertile women with polycystic ovary syndrome (PCOS) and with male factor and/or tubal infertility (Control Group) and compare in vitro maturation (IVM) rates between the groups analyzed. METHODS: five infertile patients with PCOS and eight controls, submitted to stimulated cycles for intracytoplasmic sperm injection, were selected prospectively and consecutively, and respectively assigned to the study group and the Control Group. Immature oocytes (21 and 29, respectively, from PCOS and Control Group) were submitted to IVM. After IVM, oocytes with first polar body extruded were fixed and submitted to immunostaining and fluorescence microscopy for morphological evaluation of the spindle and of chromosome distribution. Statistical analysis was performed by the Fisher test with significance, when p<0.05. RESULTS: IVM rates were similar between groups (47.6 e 44.8 percent, respectively, for PCOS and Control Group). Six of the ten oocytes (60 percent) from the study group and four of the 12 oocytes (33.3 percent) from the Control Group presented meiotic anomalies of the spindle and/or anomalous chromosome distribution, without statistical difference between groups. CONCLUSIONS: data from the present study did not demonstrate significant difference neither in IVM rates nor in the proportions of meiotic anomalies between in vitro matured oocytes obtained from stimulated cycles from PCOS patients and control ones.

Proteínas ósseas morfogenéticas (BMPs): regulação da foliculogêne e infertilidade / Bone morphogenetic proteins (BMPs): regulation of folliculogenesis and infertility

A expressão e função das proteínas ósseas morfogênicas (BMPs) no ovário humano têm despertado grande interesse a partir das recentes evidências da função desses fatores de crescimento na foliculogênese e infertilidade. No entanto, ainda se sabe muito pouco sobre sítios de expressão dessas importantes moléculas regulatórias e como sua expressão gênica é modulada nas células ovarianas durante o ciclo menstrual. Mutações nos genes gdf9 (fator de crescimento e diferenciação-9) e bmp 15 (proteína óssea morfogenética-15) têm sido associadas a patologias com conhecida correlação com infertilidade como falência ovariana prematura e a síndrome dos ovarios policísticos. A função ovariana é regulada pelas interações entre gonadotrofinas, hormônio folículo estimulante e hormônio luteinizante, e os fatores ovarianos locais, como as inibinas, activinas e as BMPs, membros da superfamília do fator de crescimento e transformação-beta (TGF-beta). As BMPs estão emergindo como uma família de proteínas críticas para a fertilidade em diversas espécies de mamíferos, com inúmeros genes envolvidos no desenvolvimento folicular normal e infertilidade.

[Evaluation of meiotic abnormalities of oocytes from polycystic ovary syndrome patients submitted to ovarian stimulation]. / Avaliação de anomalias meióticas de oócitos em pacientes com síndrome dos ovários policísticos submetidas à estimulação ovariana.

PURPOSE: to evaluate the meiotic spindle and the chromosome distribution of in vitro matured oocytes obtained from stimulated cycles of infertile women with polycystic ovary syndrome (PCOS) and with male factor and/or tubal infertility (Control Group) and compare in vitro maturation (IVM) rates between the groups analyzed. METHODS: five infertile patients with PCOS and eight controls, submitted to stimulated cycles for intracytoplasmic sperm injection, were selected prospectively and consecutively, and respectively assigned to the study group and the Control Group. Immature oocytes (21 and 29, respectively, from PCOS and Control Group) were submitted to IVM. After IVM, oocytes with first polar body extruded were fixed and submitted to immunostaining and fluorescence microscopy for morphological evaluation of the spindle and of chromosome distribution. Statistical analysis was performed by the Fisher test with significance, when p< or =0.05. RESULTS: IVM rates were similar between groups (47.6 e 44.8%, respectively, for PCOS and Control Group). Six of the ten oocytes (60%) from the study group and four of the 12 oocytes (33.3%) from the Control Group presented meiotic anomalies of the spindle and/or anomalous chromosome distribution, without statistical difference between groups. CONCLUSIONS: data from the present study did not demonstrate significant difference neither in IVM rates nor in the proportions of meiotic anomalies between in vitro matured oocytes obtained from stimulated cycles from PCOS patients and control ones.

[Meiotic abnormalities of oocytes from patients with endometriosis submitted to ovarian stimulation]. / Anomalias meióticas de oócitos de pacientes com endometriose submetidas à estimulação ovariana.

PURPOSE: to evaluate the meiotic spindle and the chromosome distribution of in vitro mature oocytes from stimulated cycles of infertile women with endometriosis, and with male and/or tubal infertility factors (Control Group), comparing the rates of in vitro maturation (IVM) between the two groups evaluated. METHODS: fourteen patients with endometriosis and eight with male and/or tubal infertility factors, submitted to ovarian stimulation for intracytoplasmatic sperm injection have been prospectively and consecutively selected, and formed a Study and Control Group, respectively. Immature oocytes (46 and 22, respectively, from the Endometriosis and Control Groups) were submitted to IVM. Oocytes presenting extrusion of the first polar corpuscle were fixed and stained for microtubules and chromatin evaluation through immunofluorescence technique. Statistical analysis has been done by the Fisher's exact test, with statistical significance at p<0.05. RESULTS: there was no significant difference in the IVM rates between the two groups evaluated (45.6 and 54.5% for the Endometriosis and Control Groups, respectively). The chromosome and meiotic spindle organization was observed in 18 and 11 oocytes from the Endometriosis and Control Groups, respectively. In the Endometriosis Group, eight oocytes (44.4%) presented themselves as normal metaphase II (MII), three (16.7%) as abnormal MII, five (27.8%) were in telophase stage I and two (11.1%) underwent parthenogenetic activation. In the Control Group, five oocytes (45.4%) presented themselves as normal MII, three (27.3%) as abnormal MII, one (9.1%) was in telophase stage I and two (18.2%) underwent parthenogenetic activation. There was no significant difference in meiotic anomaly rate between the oocytes in MII from both groups. CONCLUSIONS: the present study data did not show significant differences in the IVM or in the meiotic anomalies rate between the IVM oocytes from stimulated cycles of patients with endometriosis, as compared with controls. Nevertheless, they have suggested a delay in the outcome of oocyte meiosis I from patients with endometriosis, shown by the higher proportion of oocytes in telophase I observed in this group.