J.K. Rodrigues

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.

Direct actions of androgens on the survival, growth and secretion of steroids and anti-Müllerian hormone by individual macaque follicles during three-dimensional culture

STUDY QUESTION What are the direct effects of androgens on primate follicular development and function at specific stages of folliculogenesis? SUMMARY ANSWER Androgen addition altered primate follicle survival, growth, steroid and anti-Müllerian hormone (AMH) production, and oocyte quality in vitro, in a dose- and stage-dependent manner. WHAT IS KNOWN ALREADY Androgens have local actions in the ovary, particularly in the developing follicles. It is hypothesized that androgen promotes early follicular growth, but becomes detrimental to the antral follicles in primates. STUDY DESIGN, SIZE, DURATION In vitro follicle maturation was performed using rhesus macaques. Secondary (125-225 µm) follicles were mechanically isolated from 14 pairs of ovaries, encapsulated into alginate (0.25% w/v), and cultured for 40 days. PARTICIPANTS/MATERIALS, SETTING, METHODS Individual follicles were cultured in a 5% O2 environment, in alpha minimum essential medium supplemented with recombinant human FSH. Follicles were randomly assigned to experiments of steroid ablation by trilostane (TRL), testosterone (T) replacement and dihydrotestosterone (DHT) replacement. Follicle survival and growth were assessed. Follicles with diameters ≥500 μm at Week 5 were categorized as fast-grow follicles. Pregnenolone (P5), progesterone (P4), estradiol (E2) and AMH concentrations in media were measured. Meiotic maturation and fertilization of oocytes from recombinant human chorionic gonadotrophin-treated follicles were assessed at the end of culture. MAIN RESULTS AND THE ROLE OF CHANCE Compared with controls, TRL exposure reduced (P < 0.05) follicle survival, antrum formation rate and follicle diameters at Week 5. While P5 concentrations increased (P < 0.05) following TRL treatment, P4 levels decreased (P < 0.05) in fast-grow follicles at Week 5. Few healthy oocytes were retrieved from antral follicles developed in the presence of TRL. T replacement with TRL increased (P < 0.05) follicle survival and antrum formation at Week 5, compared with TRL alone, to levels comparable to controls. However, high-dose T with TRL decreased (P < 0.05) diameters of fast-grow follicles. Although P4 concentrations produced by fast-grow follicles were not altered by T in the presence of TRL, there was a dose-dependent increase (P < 0.05) in E2 levels at Week 5. High-dose T with TRL decreased (P < 0.05) AMH production by fast-grow follicles at Week 3. More healthy oocytes were retrieved from antral follicles developed in TRL+T compared with TRL alone. DHT had the similar effects to those of high-dose T, except that DHT replacement decreased (P < 0.05) E2 concentrations produced by fast-grow follicles at Week 5 regardless of TRL treatment. LIMITATION, REASONS FOR CAUTION This study reports T and DHT actions on in vitro-developed individual primate (macaque) follicles, which are limited to the interval from the secondary to small antral stage. WIDER IMPLICATION OF THE FINDINGS The above findings provide novel information on the role(s) of androgens in primate follicular development and oocyte maturation. We hypothesize that androgens promote pre-antral follicle development, but inhibit antral follicle growth and function in primates. While androgens can act positively, excess levels of androgens may have negative impacts on primate folliculogenesis. STUDY FUNDING/COMPETING INTERESTS NIH U54 RR024347/RL1HD058294/PL1EB008542 (Oncofertility Consortium), NIH U54 HD071836 (SCCPIR), NIH ORWH/NICHD 2K12HD043488 (BIRCWH), NIH FIC TW/HD-00668, ONPRC 8P51OD011092. There are no conflicts of interest.

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.

Oxidative stress and polycystic ovary syndrome: an evaluation during ovarian stimulation for intracytoplasmic sperm injection

Oxidative stress (OS) may affect natural fertility and the results of assisted reproduction techniques (ARTs). Subfertility associated with polycystic ovary syndrome (PCOS) may be related to OS. This process may intensify during controlled ovarian stimulation (COS) for ARTs because of increased ovarian metabolic activity and hypoestrogenism with the use of gonadotropin-releasing hormone agonists (GnRHas). The objective of this study was to investigate the presence of systemic OS in non-stimulated cycles and to determine OS markers (malondialdehyde [MDA], advanced oxidation protein products [AOPP], hydroperoxides [FOX], glutathione [GSH], and vitamin E) during COS in non-obese infertile women with and without PCOS who were subjected to ARTs. A prospective cohort study was conducted on non-obese women (16 with PCOS, and 60 ovulatory patients with infertility due to male and/or tubal factors). The OS markers were determined during the following time-points: the follicular phase of the natural cycle (D1), after pituitary downregulation with GnRHa and before the use of gonadotropins (D2), on the day of administration of human chorionic gonadotropin (D3), and at oocyte retrieval (D4). Intergroup analysis showed that serum MDA concentrations were higher in the PCOS group at D3 (P=0.048) and D4 (P=0.002). On an intragroup analysis, the control group had higher MDA concentrations at D2 than at D1 (P=0.01) or D4 (P=0.004). The AOPP concentrations were higher at D2 (P<0.0001), D3 (P<0.001) and D4 (P<0.0001) compared to D1. The FOX concentrations were lower at D2 (P<0.0001), D3 (P<0.0001), and D4 (P<0.001) than at D1. Serum GSH concentrations were significantly higher at D4 than at D1 (P=0.02). An intragroup analysis of the PCOS subjects showed that the five OS markers did not differ significantly among the four time-points when they were analyzed (D1, D2, D3 and D4). In conclusion, non-obese infertile women with PCOS showed evidence of systemic OS after COS with gonadotropins for ICSI. On the other hand, non-obese ovulatory infertile women, and women with infertility due to male and/or tubal factors showed a possible systemic oxidative balance until the final of COS.

Strategies to preserve the reproductive future of women after cancer

ABSTRACT Malignant and cardiovascular diseases are the main causes of death in Brazil. Estimates for 2013 predict the occur-rence of 189,150 new cases of cancer in Brazilian women. With advanced detection tools, patients are diagnosed and treated for cancer at a younger age and are more likely to survive. The cytotoxic action of chemotherapeutic agents and radiotherapy very frequently implies serious damage to the gonads, and consequences due to the hypoestro-genism, such as osteoporosis, infertility and premature ovarian failure, are expected. Oncofertility, then, appears as a new area of reproductive medicine, which is dedicated to the development of strategies for the reduction of ther-apeutic sequels in cancer survivals, ultimately aiming the maintenance of their quality of life and the possibility of bi-ological maternity. This article aims to present an overview of possible options for female fertility preservation after cancer and future perspectives in oncofertility. Keywords:

Fertility preservation in women with cancer: update and perspectives

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, and humans. 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.

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.

Follicle Viability after Vitrification of Bovine Ovarian Tissue

Purpose The present study aimed to evaluate the impact of vitrification on the viability of follicles using a three-dimensional (3D) in vitro culture. Methods Bovine ovarian tissue samples (n = 5) obtained from slaughterhouses were utilized. The cortex was cut into small fragments of 2 × 3 × 0.5 mm using a tissue slicer. From these fragments, secondary follicles were first isolated by mechanical and enzymatic methods, then encapsulated in alginate gel and individually cultured for 20 days. Additional fragments of the same ovarian tissue were vitrified in a solution containing 25% glycerol and 25% ethylene glycol. After warming, the follicles underwent the same follicular isolation process that was performed for the fresh follicles. Results A total of 61 follicles were isolated, 51 from fresh ovarian tissue, and 10 from vitrified tissue. After the culture, the vitrified and fresh follicles showed 20% and 43.1% survival rates respectively (p = 0.290), with no significant differences. At the end of the culture, there were no significant differences in follicular diameter between the vitrified (422.93 ± 85.05 µm) and fresh (412.99 ± 102.55 µm) groups (p = 0.725). Fresh follicles showed higher mean rate of antrum formation when compared with vitrified follicles (47.1% and 20.0% respectively), but without significant difference (p = 0.167). Conclusions The follicles were able to develop, grow and form antrum in the 3D system after vitrification, despite the lower results obtained with the fresh tissue.

Managing Fertility Preservation in Childhood Cancer Patients in Brazilian Scenario

The preservation of fertility in young and adolescent population is a matter of great relevance at the present time. For many young children, there are many psychological stress factors that affect them in the context of their lives at the time of illness, and the risk of losing their fertility is a major concern. The ability to preserve fertility can contribute positively to the emotional aspects of the disease and its treatment. In adulthood, reproductive capacity is for most individuals, one of the main determinants of their quality of life [5, 15].

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.