Retinoic acid (all-trans) presents antioxidant properties within human ovary and reduces progesterone production by human granulosa cells.

Both vitamin A and E support female reproduction and embryonic development. These vitamins have been associated with decreased fertility or failure to end the pregnancy in animals. An observational study was conducted on follicular fluid (FF) samples to determine the concentrations of fat-soluble vitamins of women undergoing in vitro fertilization and its correlation with assisted reproductive technology characteristics and pregnancy outcomes. Moreover, the effects of all-trans-retinoic acid (atRA) and alpha-tocopherol on granulosa cell viability, apoptosis, autophagy and hormonal production were evaluated. No association was identified between fat-soluble vitamin concentrations in FF and infertility aetiology, body mass index or woman's age. There were differences in follicular antioxidant profiles and ovarian response stimulation. In vitro evaluation of atRA and alpha-tocopherol reveals that, at physiological concentrations, both compounds may affect the viability of granulosa cells. In addition, these compounds are able to protect granulosa cells from oxidative stress, as well as to affect estradiol and progesterone production. Our data suggest that atRA and alpha-tocopherol levels should be well controlled as they may have implications in the function and viability of granulosa cells and highlights retinol as a marker of the oxidative defenses within ovary environment.

Increased expression of NLRP3 inflammasome components in granulosa cells and follicular fluid interleukin(IL)-1beta and IL-18 levels in fresh IVF/ICSI cycles in women with endometriosis.

The inflammasomes are a family of recently described multi-protein cytoplasmic sensors that orchestrate the inflammatory response and participate in a variety of inflammatory conditions. We hypothesized that the activation of pyrin domain­containing protein 3 (NLRP3) inflammasome by granulosa cells (hGCs) may be activated in women with endometriosis and influence oocyte maturation and IVF outcomes. We performed a cross-sectional study to investigate the NLRP3 inflammasome status in follicular fluid (FF) and in hGCs from 44 women undergoing controlled ovarian stimulation for IVF/ICSI. Study subjects were divided into two groups according to the infertility etiology: group with tubal or male factor (control, n = 22) vs. group with endometriosis (n = 22). The FF IL-1beta and IL-18 levels in the endometriosis group were significantly higher than those in the non-endometriosis group, i.e., 5010 pg/mL and 2738 pg/mL, respectively (p < 0.05). No correlation was found between clinical pregnancy and live birth rate and analyzed inflammasome component levels (p > 0.05). In addition, the hGCs from endometriosis women demonstrated high expression of NLRP3 inflammasome at both protein and mRNA levels. Higher expression of inflammasome components within the ovary compartment may result from the exaggerated inflammatory state associated with endometriosis and thus impact the fertility of these women.

Low Doses of Resveratrol Protect Human Granulosa Cells from Induced-Oxidative Stress.

Resveratrol is a phytoalexin present in plant-derived foods, including grape's skin, cocoa, and peanuts. Evidence suggests that it has beneficial effects on human health because of its antioxidant properties. However, there is limited knowledge about the part played by resveratrol in ovarian function. In this paper, the influence of resveratrol on granulosa cells (GC) was evaluated. In addition to being the main estradiol producers, GC are in direct contact with the oocyte, playing a fundamental role in its growth and development. The cell line COV434 and human granulosa cells (hGC), obtained from women undergoing assisted reproductive technology (ART), were used. GC were treated with resveratrol (0.001-20 µM) at different times (24-72 h). Low concentrations of this compound suggest a protective role, as they tend to reduce ROS/RNS formation after inducement of stress. On the contrary, high concentrations of resveratrol affect GC viability and steroidogenic function. As it may act as a direct modulator of GC oxidative balance, this work may help to clarify the impact of resveratrol on GC and the usefulness of this antioxidant as adjunct to infertility treatments.

Concentrations of the endocannabinoid N-arachidonoylethanolamine in the follicular fluid of women with endometriosis: the role of M1 polarised macrophages.

Although N-arachidonoylethanolamine (AEA; also known as anandamide) is present in human follicular fluid (FF), its regulation remains unknown. Therefore, the aims of the present study were to: (1) investigate the relationships between FF AEA concentrations in women undergoing assisted reproductive technology and their age, body mass index, ART characteristics and fertility treatment outcomes; and (2) assess how different inflammatory patterns may trigger AEA production by human granulosa cells (hGCs). FF AEA concentrations were higher in women undergoing IVF than in those undergoing intracytoplasmic sperm injection group. FF AEA median concentrations were lower in women undergoing ART because of male factor infertility than in women with endometriosis (1.6 vs 2.5nM respectively), but not women with tubal, hormonal or unexplained infertility (1.6, 2.4 and 1.9nM respectively). To evaluate the effects of macrophages on AEA production by hGCs, hGCs were cocultured with monocyte-derived macrophages. The conditioned medium from M1 polarised macrophages increased AEA production by hGCs. This was accompanied by an increase in AEA-metabolising enzymes, particularly N-acyl phosphatidylethanolamine-specific phospholipase D. The results of the present study show that high FF AEA concentrations in patients with endometriosis may be associated with the recruitment of inflammatory chemokines within the ovary, which together may contribute to the decreased reproductive potential of women with endometriosis. Collectively, these findings add a new player to the hormone and cytokine networks that regulate fertility in women.

Preservação da fertilidade masculina no Centro Hospitalar de Vila Nova de Gaia/Espinho EPE - 17 anos de experiência / Preserving male fertility at the Centro Hospitalar de Vila Nova de Gaia/Espinho EPE -17 years of experience

Objetivo. Reportar a nossa experiência nos últimos 17 anos na preservação da fertilidade masculina por doença oncológica, genética, imunológica e endocrinológica, assim como por alterações graves da espermatogénese. Material e métodos. avaliação de parâmetros demográficos, características do esperma e resultados obtidos com amostras descongeladas em ciclos de procriação medicamente assistida (PMA). Resultados. Duzentos e setenta e um homens com uma idade média de 31,5±6,3 anos (16‐52 anos) foram referenciados para PF. A doença oncológica foi a causa mais comum (34,3%), seguida de azoospermia secretora ou não obstrutiva, oligoastenoteratozoospermia (TAO) grave, doença genética, hipogonadismo/atrofia testicular, doença auto‐imune, doença endócrino‐metabólica e pré‐cirurgia potencialmente comprometedora da fertilidade. Dentro da doença oncológica, o tumor do testículo foi a neoplasia mais comum (64,5%), seguido do cancro hematológico e de etiologias variadas. Na doença genética, a síndrome de Klinefelter foi a condição mais frequente (50%). No total 194 homens ficaram com amostras criopreservadas (1.099 amostras no total; 5,7±3,4 por paciente). Os homens com doença oncológica tiveram, em média, um número significativo superior de amostras congeladas (p<0,001). Os casos de tumor do testículo apresentaram uma concentração média de espermatozoides/ml significativamente inferior face aos outros tumores (p<0,005). Durante estes 17 anos, 58 homens (29,9%) procuraram o centro para realização de técnicas de PMA com amostras descongeladas. Foram realizados 87 ciclos, com 19 gestações (21,8%). Nasceram 15 recém‐nascidos (RN) saudáveis. Conclusões. Ainda que o número de homens que tenham procurado o uso das amostras armazenadas, não se pode desvalorizar o impacto psicológico positivo da PF, tendo em conta que pode ser a única possibilidade de paternidade biológica (AU)

Objective. A report is presented of 17 years of experience in male fertility preservation due to oncological, genetic, immunological and endocrinological disease, as well as severe spermatogenesis alterations. Material and methods. An evaluation is made of demographic parameters, sperm characteristics and results of assisted reproduction techniques cycles with thawed samples. Results. A total of 271 men with a mean age 31.5±6.3 years (16 to 52 years) were referred to sperm banking. The most common cause was oncological disease (34.3%), followed by secretory azoospermia, oligoasthenoteratozoospermia, genetic disease, testicular atrophy/hypogonadism, auto‐immune disease, endocrine‐metabolic disease, and prior to potentially fertility damage surgery. Among oncological disease, testicular tumour was the most common cancer (64.5%), followed by haematological cancer, and other aetiologies. Klinefelter syndrome was the most common (50%) among the genetic diseases. A total of 194 men had samples cryopreserved (1099 total samples; 5.7±3.4 per patient). Men with oncological disease had a significantly higher number of cryopreserved samples (p<.001). Testicular tumour cases had a mean lower sperm concentration relative to other tumours (p<.005). During these 17 years, 58 men (29.9%) asked to have assisted reproduction techniques performed with their thawed samples. The 87 cycles performed resulted in 19 pregnancies (21.8%) and 15 healthy newborns. Conclusions. Although the number of men asking to use their cryopreserved samples is low, we cannot undervalue the positive psychological impact of fertility preservation, taking in consideration that it can be the only possibility to biological fatherhood (AU)

Trombofilia e terapêutica com heparina de baixo peso molecular na falha de implantação / Thrombophilia and therapy with low-molecular-weight heparin in implantation failure

Introdução: Estudos recentes demonstraram a associação de trombofilias e com falha de implantação. O objectivo deste trabalho foi avaliar o impacto na taxa de gravidez da pesquisa de trombofilias e terapêutica com heparina de baixo peso molecular (HBPM) nas mulheres em que se verifique falha de implantação. Material E Métodos: Estudo retrospectivo observacional controlado de todos os casos de falha de implantação durante 3 anos. Foram constituídos 2 grupos: grupo A, em que foi efectuada pesquisa de trombofilias, tendo sido instituída terapêutica com HBPM nos casos alterados, a partir da data da punção folicular; grupo B, em que não foi efectuada pesquisa de trombofilias nem terapêutica com HBPM. Foi realizada a comparação da taxa de implantação, taxa de gravidez e taxa de nados vivos entre ambos os grupos. Resultados: Foram incluídos 33 casos no grupo A e 24 casos no grupo B. O resultado da pesquisa de trombofilias, efectuada apenas no grupo A, foi alterada em todos os casos, com presença de mutação para o gene da MTHFR em 87,88 % dos casos e mutações do gene do PAI-1 em 75,76 % dos casos. Verificou- se um aumento estatisticamente significativo da taxa de implantação e da taxa de gravidez no grupo A (29,09 % vs 10,26 %, p <0,05, e 45, 45 % vs 12,50 %, p <0,05). A taxa de abortamento foi sobreponível em ambos os grupos. A terapêutica com HBPM associou-se a um aumento significativo da taxa de nados vivos por transferência (36,36 % vs 12,50 % respectivamente no grupo A e B, p <0,05). Não se verificaram complicações associadas à terapêutica anticoagulante. Conclusão: As trombofilias parecem representar um importante papel no sucesso/insucesso da implantação embrionária. A terapêutica com HBPM nas mulheres com trombofilia associou-se a um aumento significativo da taxa de implantação, da taxa de gravidez e da taxa de nados vivos (AU)

Introduction: Recent studies have reported an association between thrombophilia and implantation failure. The aim of this study was to evaluate the impact on the pregnancy rate of thrombophilia research and therapy with low-molecularweight heparin (LMWH) in women with implantation failure. Material and Methods: Retrospective observational controlled study of all cases of implantation failure during 3 years. Cases were divided into two groups: Group A, where thrombophilic defects were searched, and therapy with LMWH was started in altered cases, from the date of ovary punction; Group B, in which thrombophilia research wasn’t performed nor anticoagulant therapy was started. Primary outcomes were the implantation, pregnancy and live birth rates. Results: There were 33 patients included in group A and 24 patients in group B. The search for thrombophilia, performed only in group A, was altered in all cases, with the presence of mutations in the MTHFR gene in 87.88 % of cases and PAI- 1 gene mutations in 75.76 % of cases. There was a statistically significant increase in the implantation and pregnancy rates in group A (29.10 % vs. 10.26 %, p <0.05, and 45.45 % vs. 12.50 %, p <0.05). The abortion rate was similar in both groups. LMWH therapy was associated with a significant increase in live birth rate per transfer (36.36 % vs. 12.50 % in group A and B respectively, p <0.05). There were no complications associated with anticoagulant therapy. Conclusion: Thrombophilia seems to play an important role in the success/failure of embryo implantation. LMWH therapy in women with thrombophilia is associated with a significantly increased in implantation, pregnancy and live birth rates (AU)

Outcomes from ovarian hyperstimulation following the sole administration of gonadotrophin-releasing hormone agonist in the context of in vitro fertilization: report of two cases and review of the literature.

OBJECTIVE: To report the outcomes from two cases of ovarian stimulation following the sole administration of gonadotrophin-releasing hormone agonist (GnRHa) in the context of in vitro fertilization (IVF). DESIGN: A case study was conducted. SETTING: National Referral Unit of Reproductive Medicine. PATIENTS: Two infertile women undergoing IVF participated in the study. INTERVENTIONS: Controlled ovarian hyperstimulation using a long protocol. GnRHa (Buserelin) was started in the luteal phase, in a dose of 600 µg/day, for 12 days. MAIN OUTCOME MEASURES: Number of retrieved oocytes, fertilization rate, number of embryos transferred, implantation rate, ongoing pregnancy, and live birth. RESULTS: Both women underwent egg retrieval and transfer of good quality embryos. One of them conceived and recently gave birth to a healthy full-term baby. CONCLUSIONS: The ovarian hyperstimulation after the sole administration of GnRHa is a rare condition. Oocyte retrieval may be a reasonable treatment under these circumstances instead of cycle cancellation. As far as it is known, this is the third case reported of a live birth following the sole administration of GnRHa in the context of IVF.

Estimulação ovariana controlada e inseminação intrauterina: uma terapia atual? / Controlled ovarian stimulation and intrauterine insemination: an actual therapy?

OBJETIVO: Analisar a taxa de gravidez na inseminação intrauterina (IIU), aferindo eventuais fatores prognósticos de sucesso. MÉTODOS: Estudo retrospectivo de ciclos de IIU ocorridos de janeiro de 2007 a julho de 2010 em uma Unidade de Medicina da Reprodução do Hospital Vila Nova de Gaia. Os ciclos foram precedidos por estimulação ovárica controlada e monitorizada por ecografia endovaginal. Avaliou-se a taxa de gravidez em função da idade da mulher, tipo e duração da infertilidade, motilidade no espermograma, número de folículos e fármaco usado na estimulação ovárica. A análise estatística foi efetuada com o Statistical Package for the Social Sciences (SPSS 17), com um nível de significância 5 por cento (p<0,05). RESULTADOS: O estudo integrou 139 casais submetidos a 220 ciclos de IIU. A taxa global de gravidez por ciclo foi de 18,6 por cento e, das 41 grávidas, 5 foram gemelares (12,1 por cento). Ocorreu maior taxa de gravidez abaixo dos 30 anos (28,5 vs 15,7 por cento; p=0,02), duração de infertilidade <3 anos (23,8 vs 13,9 por cento; p=0.05), espermograma sem alterações da motilidade (23,2 vs 10,3 por cento; p=0,01) e na presença de dois folículos no momento da ovulação face ao desenvolvimento monofolicular (27,7 vs 14,2 por cento; p=0,030). As taxas gravídicas com citrato de clomifeno, gonadotrofinas e a sua associação foram de 13,0, 26,1 e 28,6 por cento, respectivamente, com diferença significativa na taxa de gravidez clínica entre citrato de clomifeno e gonadotrofinas. CONCLUSÕES: A IIU mantém-se como um natural ponto de partida para casais convenientemente selecionados. Idades mais jovens, menor duração da infertilidade e espermograma sem alterações na motilidade constituem fatores de bom prognóstico e a estimulação com gonadotrofinas contribui para melhoria da taxa de gravidez.

PURPOSE: To evaluate the pregnancy rate in intrauterine insemination (IUI), and to determine possible prognostic factors of successful pregnancy. METHODS: A retrospective study of IUI cycles performed in the Reproductive Medicine Unit of Vila Nova de Gaia Hospital, between January 2007 and July 2010. The IUI cycles were preceded by ovarian stimulation and monitored by vaginal ultrasound. Clinical pregnancy rates were analyzed according to the woman’s age, type and duration of infertility, spermatozoa parameters assessed in the spermogram, number of mature follicles and the drug used for ovarian stimulation. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS17), with the level of significance set at p<0.05. RESULTS: The study comprised 139 couples who underwent 220 IUI cycles. The absolute pregnancy rate per cycle was 18.6 percent. Of the 41 clinical pregnancies, 5 were twin pregnancies (12.1 percent). The pregnancy rate was higher at ages <30 years (28.5 vs 15.7 percent; p=0.024), duration of infertility <3 years (23.8 vs 13.9 percent; p=0.05), normal sperm motility (23.2 vs 10.3 percent; p=0.01) and with two follicles at the time of insemination (27.7 vs 14.2 percent for monofollicular growth; p=0.030). The pregnancy rates obtained with clomiphene citrate, gonadotropins and combined clomiphene citrate/gonadotropin were 13.0, 26.1 and 28.6 percent, respectively, with a statistically significant difference in clinical pregnancy rate between clomiphene citrate and gonadotropin. CONCLUSIONS: IUI remains a natural starting point for conveniently selected couples with infertility. Younger age and normal sperm motility are good prognostic factors. Gonadotrophin stimulation seems to be an important tool for improving the pregnancy rate of IUI.

[Controlled ovarian stimulation and intrauterine insemination: an actual therapy?]. / Estimulação ovariana controlada e inseminação intrauterina: uma terapia atual?

PURPOSE: To evaluate the pregnancy rate in intrauterine insemination (IUI), and to determine possible prognostic factors of successful pregnancy. METHODS: A retrospective study of IUI cycles performed in the Reproductive Medicine Unit of Vila Nova de Gaia Hospital, between January 2007 and July 2010. The IUI cycles were preceded by ovarian stimulation and monitored by vaginal ultrasound. Clinical pregnancy rates were analyzed according to the woman's age, type and duration of infertility, spermatozoa parameters assessed in the spermogram, number of mature follicles and the drug used for ovarian stimulation. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS17), with the level of significance set at p<0.05. RESULTS: The study comprised 139 couples who underwent 220 IUI cycles. The absolute pregnancy rate per cycle was 18.6%. Of the 41 clinical pregnancies, 5 were twin pregnancies (12.1%). The pregnancy rate was higher at ages <30 years (28.5 vs 15.7%; p=0.024), duration of infertility <3 years (23.8 vs 13.9%; p=0.05), normal sperm motility (23.2 vs 10.3%; p=0.01) and with two follicles at the time of insemination (27.7 vs 14.2% for monofollicular growth; p=0.030). The pregnancy rates obtained with clomiphene citrate, gonadotropins and combined clomiphene citrate/gonadotropin were 13.0, 26.1 and 28.6%, respectively, with a statistically significant difference in clinical pregnancy rate between clomiphene citrate and gonadotropin. CONCLUSIONS: IUI remains a natural starting point for conveniently selected couples with infertility. Younger age and normal sperm motility are good prognostic factors. Gonadotrophin stimulation seems to be an important tool for improving the pregnancy rate of IUI.