L.A. Dib

Marcadores séricos de estresse oxidativo em mulheres inférteis com endometriose

OBJETIVO: comparar marcadores sericos de estresse oxidativo entre pacientes inferteis com e sem endometriose e avaliar a associacao destes marcadores com o estadiamento da doenca. METODOS: estudo prospectivo envolvendo a inclusao consecutiva de 112 pacientes inferteis, nao-obesas, com idade inferior a 39 anos, divididas em dois grupos: Endometriose (n=48, sendo 26 com endometriose minima e leve - Estadio I/II e 22 com endometriose moderada e grave - Estadio III/IV) e Controle (n=64, com fator tubario e/ou masculino de infertilidade). Durante a fase folicular precoce do ciclo menstrual, foram coletadas amostras sanguineas para analise dos niveis sericos de malondialdeido, glutationa e niveis totais de hidroperoxidos, por espectrofotometria e vitamina E, por cromatografia liquida de alto desempenho. Os resultados obtidos foram comparados da seguinte forma: os grupos endometriose versus controle; endometriose estadio I/II e controle, endometriose estadio III/IV e controle e entre os dois subgrupos de endometriose. Em todas as analises, foi considerado o nivel de significância de 5% (p<0,05). RESULTADOS: os niveis de vitamina E e glutationa foram mais baixos no soro de mulheres inferteis com endometriose moderada/grave (21,7±6,0 µMol/L e 159,6±77,2 nMol/g proteina, respectivamente) quando comparadas a mulheres com endometriose minima e leve (28,3±14,4 µMol/L e 199,6±56,1 nMol/g proteina, respectivamente). Os niveis totais de hidroperoxidos foram significativamente mais elevados no grupo endometriose (8,9±1,8 µMol/g proteina) em relacao ao Grupo Controle (8,0±2 µMol/g proteina) e nas portadoras de doenca III/IV (9,7±2,3 µMol/g proteina) em relacao a I/II (8,2±1,0 µMol/g proteina). Nao se observou diferenca significativa nos niveis sericos de malondialdeido entre os diversos grupos. CONCLUSOES: foi evidenciada uma associacao positiva entre infertilidade relacionada a endometriose, avanco do estadiamento da doenca e aumento dos niveis sericos de hidroperoxidos, sugerindo aumento da producao de especies reativas em portadoras de endometriose. Esses dados, associados a reducao dos niveis sericos de vitamina E e glutationa, sugerem a ocorrencia de estresse oxidativo sistemico em portadoras de infertilidade associada a endometriose.

Noninvasive imaging of the meiotic spindle of in vivo matured oocytes from infertile women with endometriosis.

The objectives of this prospective study were to evaluate the nuclear maturation stage and the presence and location of meiotic spindles of in vivo matured oocytes from infertile women with and without endometriosis (male or tubal causes of infertility) undergoing stimulated cycles for intracytoplasmic sperm injection (ICSI). We also compared the ICSI outcomes among groups. We analyzed the meiotic spindles of oocytes from 36 patients with endometriosis I/II, 24 with endometriosis III/IV, and 60 without endometriosis (male or tubal causes of infertility). The oocytes were imaged using polarization microscopy. There were no differences in the number of oocytes in telophase I (mean [standard deviation]: 0.1 [0.5], 0.2 [0.4], and 0.2 [0.5], respectively, in the endometriosis I/II, endometriosis III/IV, and control groups), in metaphase II with visible spindles (4.2 [2.5], 3.1 [2.0], and 3.6 [2.2], respectively, in the endometriosis I/II, endometriosis III/IV, and control groups), and in spindle location among groups. We can conclude from this study that noninvasive analysis of spindles from in vivo matured oocytes of infertile patients with endometriosis did not demonstrate significant differences in terms of the nuclear maturation stage, the percentage of oocytes in metaphase II with visible spindles, and the spindle localization when compared to the control group. However, it is important to state that there are no studies evaluating the accuracy of polarization microscopy for the detection of meiotic anomalies in human oocytes, which would need to be better evaluated in future studies using an appropriate methodology.

Serum markers of oxidative stress and assisted reproduction procedures results in infertile patients with polycystic ovary syndrome and controls

PURPOSE To compare the serum levels of five markers of oxidative stress and assisted reproduction (AR) outcomes among infertile patients, with tubal and/or male factor and with polycystic ovary syndrome (PCOS). METHODS 70 patients were included, 58 with tubal and/or male factor infertility and 12 with PCOS, who underwent controlled ovarian stimulation to perform intracytoplasmic sperm injection (ICSI). A blood sample was collected between the third and fifth day of the menstrual cycle in the month prior to ovarian stimulation. We analyzed the levels of malondialdehyde, hydroperoxides, protein oxidation products, glutathione and vitamin E, by reading the absorbance with a spectrophotometer and by high performance liquid chromatography (HPLC). Data were analyzed statistically by the Students t-test and Fishers exact test. RESULTS Significant increases in the body mass index, ovarian volume and number of antral follicles were observed in PCOS patients, as well as the use of a lower total dose of follicle stimulating hormone for these patients. There were no differences in the response to ovarian stimulation, in the results of AR or serum levels of malondialdehyde, hydroperoxides, advanced oxidation protein products, glutathione and vitamin E between groups. CONCLUSIONS The present data did not demonstrate a difference in the levels of serum markers of oxidative stress or in AR results when comparing non-obese infertile patients with PCOS and controls. These data suggest that the results of AR may not be compromised in this specific subgroup of patients with PCOS. However, interpretations of the action of oxidative stress on the results of AR are still not clear and the reproductive implications of oxidative stress need to be better evaluated.

Oócitos aparentemente maduros injetados em telófase I apresentam piores resultados de reprodução assistida

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 Fishers 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.

Comparative Analysis of the Spindle of Fresh In Vivo-Matured Human Oocytes Through Polarized Light and Confocal Microscopy A Pilot Study

Background: Considering the scarcity of literature data about the predictive capacity of polarization microscopy (PM) in identifying meiotic normality in human in vivo-matured oocytes, the main objective of the present study was to determine whether qualitative analysis of the spindle through PM can predict meiotic normality in fresh in vivo-matured human oocytes. Methods: Infertile patients undergoing controlled ovarian stimulation to intracytoplasmic sperm injection (ICSI) were selected. Fresh in vivo-matured oocytes were evaluated by PM and, immediately afterward, fixed for confocal microscopy (CM) analysis for evaluation of the spindle and chromosome distribution. We evaluated the percentage of oocytes with meiotic normality and abnormality determined by CM among oocytes with visible and nonvisible spindle and between oocytes with the spindle located at 0° to 60° angle to the first polar body (PB; normal position) and at 60° to 90° angle to the first PB (risky position) according to PM. Results: From 23 patients, 73 oocytes were analyzed. There were no significant differences in the percentage of oocytes with meiotic abnormalities among oocytes with visible and nonvisible spindles and among oocytes with the spindle in the normal and risky positions. Conclusions: Under the conditions tested, qualitative evaluation of the spindle through PM is not consistent with CM analysis and has limited predictive value of meiotic normality in fresh in vivo-matured human oocytes, which needs to be confirmed in larger studies. Our findings make questionable the usefulness of this methodology as a tool for noninvasive oocyte selection for ICSI.

Hormônio anti-Mülleriano sérico para predição da resposta ovariana em ciclos de reprodução assistida

OBJETIVOS: Comparar as concentracoes sericas do hormonio anti- Mulleriano (AMH) no setimo dia de estimulacao ovariana em pacientes boas e mas respondedoras. METODOS: Foram incluidas 19 mulheres com idade ≥35 anos, ciclos menstruais regulares e que foram submetidas a estimulacao ovariana para reproducao assistida. Foram excluidas mulheres portadoras de endometriose ou sindrome dos ovarios policisticos ou aquelas submetidas previamente a cirurgia ovariana. Foram coletadas amostras de sangue periferico no dia basal e no setimo dia de estimulacao para dosagens de AMH, hormonio foliculo estimulante (FSH) e estradiol. Os niveis de AMH foram avaliados pelo metodo de enzimoimunoensaio (ELISA) e os niveis de FSH e estradiol foram avaliados por imunoquimioluminescencia. Ao final do ciclo, as pacientes foram classificadas como normo (obtencao de quatro ou mais oocitos durante a captacao) ou mas respondedoras (obtencao de menos de quatro oocitos ou cancelamento do ciclo por ma resposta) e analisadas comparativamente em relacao aos niveis hormonais, duracao da estimulacao ovariana, numero de foliculos recrutados, embrioes produzidos e transferidos atraves do teste t. A associacao entre os niveis de AMH e os parâmetros acima tambem foi avaliada pelo teste de correlacao de Spearman. RESULTADOS: Nao houve diferenca significativa entre os grupos para os niveis de AMH, FSH e estradiol no dia basal e no setimo dia de estimulacao ovariana, sendo observada correlacao significativa entre os niveis de AMH do setimo dia e a quantidade total de FSH exogeno utilizada (p=0,02). CONCLUSOES: Os niveis de AMH obtidos no setimo dia do ciclo de estimulacao ovariana nao parecem predizer o padrao de resposta ovariana, nao sendo recomendadas as suas dosagens para esta finalidade.

Análise invasiva e não invasiva do fuso meiótico de oócitos humanos obtidos de ciclos estimulados: dados preliminares

PURPOSE: To evaluate the concordance between polarization microscopy and confocal microscopy techniques in the evaluation of the meiotic spindle of human oocytes matured in vivo. METHODS: Prospective study that evaluated oocytes with the first polar extruded body obtained from infertile women who had undergone ovarian stimulation for intracytoplasmic sperm injection. The oocytes with the first polar extruded body were evaluated by polarization microscopy and were then immediately fixed and stained for microtubule and chromatin evaluation by high-performance confocal microscopy. We determined the correlation of polarization microscopy with confocal microscopy in the detection of meiotic oocyte anomalies, and we also evaluated the percentage of oocytes with a visible and non-visible cell spindle by polarization microscopy and with meiotic normality and abnormalities by confocal microscopy. Confidence intervals, Kappas index and concordance between the methodologies were calculated, considering immunofluorescence microscopy analysis as the golden-standard for evaluating normal spindle and oocyte chromosome distribution. RESULTS: We observed that 72.7% of metaphase II oocytes with a nonvisible meiotic spindle by polarization microscopy showed no meiotic abnormalities by confocal analysis and 55.6% of metaphase II oocytes with a visible meiotic spindle by polarization microscopy were found to be abnormal oocytes by the confocal analysis. Only 44.4% of oocytes with a visible meiotic spindle by polarization microscopy were found to be normal by confocal analysis. Concordance between the methods was 51.1% (Kappa: 0.11; 95%CI -0.0958 - 0.319). CONCLUSIONS: The low correlation between polarization microscopy and confocal microscopy in the assessment of oocyte meiotic spindle suggests that visualization of the meiotic spindle of human oocytes at metaphase II by polarization microscopy is not a good indicator of oocyte meiotic normality.

Influence of pituitary suppression and controlled ovarian hyperstimulation (COH) on oxidative stress markers of infertile patients with endometriosis and controls

INFLUENCE OF PITUITARY SUPPRESSION AND CONTROLLED OVARIAN HYPERSTIMULATION (COH) ON OXIDATIVE STRESS MARKERS OF INFERTILE PATIENTS WITH ENDOMETRIOSIS AND CONTROLS. F. C. Donabela, A. Z. Andrade, J. K. Rodrigues, L. A. Dib, A. A. Jord~ao, Jr, P. A. Navarro. Dept. of Ob/Gyn, Faculty of Medicine of Ribeir~ao Preto, University of S~ao Paulo, Ribeir~ao Preto, SP, Brazil; National Institute of Hormones and Woman’s Health, CNPq, Ribeir~ao Preto, SP, Brazil; Nutrition and Metabolism Laboratory, Faculty of Medicine of Ribeir~ao Preto, University of Sao Paulo, Ribeir~ao Preto, SP, Brazil.