Impact of Extraction Methods and Transportation Conditions on Lipid Profiles of Bovine Oocytes.

Lipids play numerous pivotal physiological roles in mammalian reproduction, being indispensable for oocyte competence acquisition and post-fertilization embryonic development. Profiling lipids in minute samples, such as oocytes, presents challenges but has been accomplished through mass spectrometry technologies like Multiple Reaction Monitoring (MRM) profiling. With the dual objectives of simplifying workflow and examining the influence of preanalytical conditions, we assessed whether transportation at room temperature affects the lipid profile of bovine oocytes. To this end, samples were prepared using either monophasic (methanol only) or biphasic liquid extraction protocols (Bligh & Dyer method) and transported either on dry ice or at room temperature inside sealed-vacuum packages to prevent lipid oxidation. Subsequently, employing a comprehensive method, we screened a list of 316 MRMs from 10 different lipid subclasses in oocyte lipid extracts. Principal Component Analysis (PCA) revealed similar lipid profiles concerning temperature during transportation, whereas clear differentiation among samples was observed based on the lipid extraction method. Univariate analysis indicated that the one-phase methanol extraction resulted in higher relative abundances of phospholipids, except for phosphatidylserines. Conversely, the Bligh & Dyer extraction favored the detection of neutral intracellular lipids (triacylglycerols, free fatty acids, cholesteryl esters, and acyl-carnitines). Consequently, lipid recovery was directly correlated with the polarity of lipid class and the extraction method. Regarding transportation temperature, phosphatidylethanolamine, triacylglycerol, and free fatty acids exhibited lower abundances when samples were transported at room temperature. Based on multivariate and univariate analyses, we conclude that if samples undergo the same lipid extraction protocol and are transported in the same batch at room temperature inside vacuum-sealed bags, it is feasible to analyze lipid extracts of bovine oocytes and still obtain informative lipid profiling results.

Effect of lipid extraction and room temperature transportation of bovine oocytes determined by MRM profiling.

Lipids play many important physiological roles in mammalian reproduction, being essential for the acquisition of oocyte competence and post-fertilization embryonic development. Lipid profiling in samples of minute size, such as oocytes, is challenging but has been achieved by mass spectrometry technologies such as multiple reaction monitoring (MRM) profiling. With the goals of further simplifying sample workflow and investigating the influence of pre-analytical conditions, we have evaluated how different extraction methods and transportation of lipid extracts in vacuum and at room temperature impacted the lipid profile of bovine oocytes. Using a comprehensive method, 316 MRMs associated with lipids of 10 different classes were screened in oocyte lipid extracts prepared by 2 extraction methods (one-step methanol addition or Bligh and Dyer) and transporting them in dry ice or at room temperature inside vacuum packages. No changes in the multivariate analysis (PCA) were noticeable due to transportation temperature, while lipid profiles were more affected by the lipid extraction protocol. Sample extraction using pure methanol favored the detection of phospholipids uniformly, while Bligh and Dyer favored the detection of neutral intracellular lipids. Triacylglycerol lipids and free fatty acids yielded decreased abundances when samples were transported at room temperature. We conclude that if samples are submitted to the same lipid extraction protocol and same transportation batch at room temperature coupled with vacuum conditions it is possible to analyze lipid extracts of bovine oocytes and still obtain informative lipid profiling results.

Comparing the effects of a commercial and a prototype vitrification medium on meiotic spindle morphology and survival rate of mouse oocytes.

OBJECTIVE: To compare oocyte survival and meiotic spindle normality between vitrified-warmed oocytes in a mouse embryo assay using Tvitri-4 or Ingámed vitrification media. METHODS: C57BL/6 female mice aged 8-12 weeks were submitted to superovulation with pregnant mare's serum gonadotropin and human chorionic gonadotropin (hCG) for obtaining of in vivo matured oocytes. The oocytes were randomly distributed into one of the following three groups: CTR - control (fresh oocytes); ING - oocytes vitrified-warmed in a standard commercial kit supplied by Ingámed, and T4 - oocytes vitrified-warmed in the novel prototype Tvitri-4 medium. After warming and recovery culture, oocytes were assessed with respect to survival rate (SR) and both meiotic spindle morphology and chromosome alignment of each oocyte fixed in the sagittal position after immunostaining and analysis by confocal microscopy. RESULTS: A total of 354 mature oocytes were vitrified in ING (n=178) and T4 (n=176), out of which 299 (85%) survived after warming. Oocyte survival rates were not statistically different (p=0.08) between ING (145/178=81.5%) and T4 (154/176=87.5%). Regarding meiotic normality, there were no significant changes in the proportion of oocytes with normal meiotic spindle morphology and chromosome structure between ING (52,2%) and T4 (63.4%) after warming (RR: 0.95, 95% CI: 0.92-1.607). When the meiotic normality was assessed using the CTR group as a reference in the analysis of relative risk, no significant differences were observed between T4 (63.4%) and CTR (70.5%) (RR: 0.95, 95% CI: 0.72-1.12). On the other hand, the percentage of oocytes retaining normal meiotic spindle morphology and chromosome configuration in ING (52.2%) was lower than in the CTR group (RR: 0.95, 95% CI: 0.57-0.97). CONCLUSIONS: The novel prototype Tvitri-4 medium was efficient in preserve survival rate and meiotic spindle normality of oocytes and, with further verification, may be able to replace commercially available media in future clinical applications.

Phosphatidylcholine and L-acetyl-carnitine-based freezing medium can replace egg yolk and preserves human sperm function.

BACKGROUND: Conventional cryopreservation methods induce chemical and mechanical damage to the sperm membranes. The cryoprotectant potential of phospholipids of vegetal origin as soybean lecithin has been investigated as a substitute for egg yolk in diluents used for the cryopreservation of human spermatozoa. Therefore, the objective of this study was comparing the efficacy of a synthetic cryoprotectant supplemented with L-α-phosphatidylcholine (PC) and L-acetyl-carnitine (ANTIOX-PC) and the standard egg-based TEST-yolk buffer (TYB) in preserving sperm motility and chromatin quality in cryopreserved semen samples. METHODS: Prospective experimental study in which semen samples from 63 men with normal sperm motility and 58 men with low sperm motility were included and analyzed both before and after cryopreservation using ANTIOX-PC or TYB freezing media. Sperm quality was evaluated by routine semen analysis and DNA fragmentation index using the Terminal deoxynucleotidyl transferase dUTP nick end labeling assay. RESULTS: Differences in the post-thaw progressive motility and DNA fragmentation index were not detected between TYB and ANTIOX-PC cryoprotectants in both normal and low sperm motility groups (P>0.05). However, ANTIOX-PC medium retained higher non-progressive motility and lower percentage of immotile sperm when compared to TYB medium, resulting in a greater total motile sperm count (P<0.05), regardless baseline values of motility characteristic of the normospermic or asthenozoospermic samples. CONCLUSIONS: ANTIOX-PC medium was effective to protect human sperm during a freeze-thaw cycle compared to the TYB medium. A clinically relevant advantage in better preserving kinetic parameters as higher total motility and lower immotile post-thawed sperm from ANTIOX-PC, in normal and low motility semen samples, demonstrated the positive impact of phospholipid and antioxidant treatment on sperm cryotolerance with high potential for egg yolk lipids replacement and biosafety.

Driving Human Granulosa-Luteal Cells Recovered From In Vitro Fertilization Cycles Toward the Follicular Phase Phenotype.

Culture systems are available for human granulosa cells (GCs) that perpetuate luteinization. The present study examines the plating density effects and long-term serum-free culture on the in vitro dynamics differentiation of luteinizing human GCs. Cells were cultured in serum-free α-minimum essential medium (α-MEM) or serum-based tissue culture medium (TCM). The time course of GCs morphology and secretion of estradiol (E2), progesterone (P4), and relaxin were analyzed after 48, 96, and 144 hours of culture. Other functional markers as follicle-stimulating hormone/luteinizing hormone receptors and steroidogenic enzymes were investigated at the end of culture. The morphology of an α-MEM cell rather than a TCM cell resembles more closely that seen in vivo. Compared to TCM cultures, α-MEM cells secreted 93.7% and 87.2% more E2 and approximately 7% and 17% of the amount of P4 when cultured at densities of 2 × 10(4) or 4 × 10(4) cells/well, respectively. Relaxin secretion was significantly reduced in α-MEM cultures. α-MEM cells were estrogenic and expressed the CYP19 gene. Levels of CYP17 increased about 8-fold in α-MEM cells above the levels found in TCM cells. Our results reveal new insights into human GCs differentiation in vitro and demonstrate the critical importance of the culture system and cell-plating density on the establishment of estrogenic or progestogenic GC phenotypes.

Cultura de células da granulosa humanas com fenótipo de fase folicular: influência do sistema quimicamente definido na morfologia, ultraestrutura, secreção de esteroides e relaxina / Culture of human granulosa cells with follicular phase phenotype:influence of chemically defined system on morphology, ultrastructure,secretion of steroids and relaxin

Está bem descrito na literatura o padrão de cultivo de células da granulosa (CG) humanas que perpetua a luteinização, simulando a fase lútea do ciclo. Nesse sistema, há redução na secreção de estradiol (E2) e aumento na síntese de progesterona (P4) e relaxina (RLN). Objetivamos padronizar um sistema de cultura livre de soro, com o intuito de reverter o processo de luteinização de CG obtidas em ciclos de fertilização in vitro (FIV), pré-luteinizadas pela gonadotrofina coriônica humana (hCG), para aplicação na maturação in vitro de folículos ovarianos pré-antrais. Foi feito estudo experimental com GC obtidas de 10 mulheres em tratamento de reprodução assistida. As CG foram cultivadas em α-MEM contendo IGF-I, ITS, androstenediona, PVP-40 (meio quimicamente definido) ou TCM-199 contendo FSH/soro. Após 48, 96 e 144 horas, foram avaliados: morfologia das culturas, produção de E2, P4 (Quimioluminescência/Immulite), RLN (Elisa) e ultraestrutura (Microscopia Eletrônica). Os dados foram analisados por Anova e regressão linear com efeitos mistos (SAS versão 9.0). Células cultivadas em α-MEM apresentam alta capacidade estrogênica e padrão de produção hormonal característico da fase folicular, mantendo características morfológicas/ultraestruturais semelhantes a células in vivo. No sistema de cultura padronizado, as CG não completam in vitro o processo de luteinização deflagrado pela hCG, assumindo fenótipo de fase folicular.

It is well described in the literature the granulosa cells (GC) culture pattern that perpetuates human luteinizing simulating the luteal phase of the cycle. In this system, there is a reduction in the secretion of estradiol (E2) and increased synthesis of progesterone (P4) and relaxin (RLN). We aim to standardize a serum-free culture system, in order to reverse the luteinization process of GC obtained in IVF cycles, pre-luteinized by hCG, for use in in vitro maturation of preantral ovarian follicles. An experimental study was conducted with GC obtained from10 women undergoing treatment for assisted reproduction. The GC were cultured in α-MEM containing IGF-I, STI, androstenedione, PVP-40 (chemically defined medium) or TCM-199 containing FSH/serum. After 48, 96 and 144 h were analyzed: culture morphology, concentrations of E2, P4 (Chemioluminescence/Immulite), and RLN (Elisa), and ultrastructure (ElectronMicroscopy). Data were analyzed by Anova and linear mixed-effects regression (SAS version9.0). Cells cultured in α-MEM present estrogenic capacity and pattern of hormone production characteristic of the follicular phase, maintaining morphological/ultrastructural features similar that in vivo cell. In standard culture system, the CG not completes in vitro luteinization process triggered by hCG, assuming follicular phase phenotype.

Single-cell expression analysis of BMP15 and GDF9 in mature oocytes and BMPR2 in cumulus cells of women with polycystic ovary syndrome undergoing controlled ovarian hyperstimulation.

PURPOSE: To detect expression of bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) in oocytes, and their receptor type 2 receptor for BMPs (BMPR2) in cumulus cells in women with polycystic ovary syndrome (PCOS) undergoing in vitro fertilization (IVF), and determine if BMPR2, BMP15, and GDF9 expression correlate with hyperandrogenism in FF of PCOS patients. METHODS: Prospective case-control study. Eighteen MII-oocytes and their respective cumulus cells were obtained from 18 patients with PCOS, and 48 MII-oocytes and cumulus cells (CCs) from 35 controls, both subjected to controlled ovarian hyperstimulation (COH), and follicular fluid (FF) was collected from small (10-14 mm) and large (>18 mm) follicles. RNeasy Micro Kit (Qiagen) was used for RNA extraction and gene expression was quantified in each oocyte individually and in microdissected cumulus cells from cumulus-oocyte complexes retrieved from preovulatory follicles using qRT-PCR. Chemiluminescence and RIA assays were used for hormone assays. RESULTS: BMP15 and GDF9 expression per oocyte was higher among women with PCOS than the control group. A positive correlation was found between BMPR2 transcripts and hyperandrogenism in FF of PCOS patients. Progesterone values in FF were lower in the PCOS group. CONCLUSION: We inferred that BMP15 and GDF9 transcript levels increase in mature PCOS oocytes after COH, and might inhibit the progesterone secretion by follicular cells in PCOS follicles, preventing premature luteinization in cumulus cells. BMPR2 expression in PCOS cumulus cells might be regulated by androgens.

Cryopreservation time does not decrease follicular viability in ovarian tissue frozen for fertility preservation.

OBJECTIVE: To determine the effect of storage duration on cryopreserved ovarian tissue using fresh and frozen-thawed samples. METHODS: Seventeen fertile patients underwent an ovarian biopsy during elective laparoscopic tubal ligation. The tissue sample was divided into three parts: one part was processed fresh (FG), and two were slowly frozen, cryopreserved for 30 (G30) or 180 days (G180), thawed and analyzed. Follicular density, follicular viability, and steroidogenic capacity were assessed. RESULTS: We observed no differences between the groups in follicular density, which was assessed in hematoxylin and eosin-stained tissue sections. A heterogeneous follicular distribution was observed in the parenchyma, with a mean density of 361.3±255.4, 454.9±676.3, and 296.8±269.0 follicles/mm(3) for FG, G30 and G180, respectively (p = 0.46). Follicular viability was greater in FG (93.4%) when compared with the cryopreserved tissues (70.8% for G30 (p<0.001) and 78.4% for G180 (p<0.001)), with no difference in viability between the frozen samples (p>0.05). The steroidogenic capacity of the tissue was not significantly reduced following cryopreservation. CONCLUSION: The slow freezing procedures used for ovarian cryopreservation are capable of preserving follicular viability and maintaining the steroidogenic capacity of the tissue despite a roughly 30% decrease in follicular viability. Furthermore, short-term storage of ovarian tissue does not appear to compromise follicle integrity.

Influência da interação entre o oócito e as células da granulosa nos resultados dos procedimentos de reprodução assistida / The influence of interaction between oocyte and granulosa cells on the results of procedures in assisted reproduction

A interação oócito-células da granulosa in vivo e sua influência na qualidade oocitária e embrionária tem sido alvo de inúmeros estudos, mas muitas questões ainda necessitam ser esclarecidas. O objetivo deste trabalho foi revisar a importância dessa comunicação, estabelecendo uma relação com a questão da maturação in vitro de oócitos imaturos humanos aplicando esses conhecimentos para definir possíveis marcadores moleculares que poderiam melhorar a seleção de oócitos e, consequentemente, selecionar embriões de boa qualidade para posterior transferência e sucesso de gravidez de pacientes submetidas ao tratamento da infertilidade conjugal. As células da granulosa têm um importante papel na maturação oocitária in vitro e os benefícios da presença dessas células durante essa etapa podem ser atribuídos à formação de um microambiente favorável (bioquímico e metabólico) ao redor do oócito. Foram identificados nesta revisão vários marcadores em potencial nas células do cumulus de oócitos competentes, incluindo vários genes que poderiam ser usados como preditores da competência oocitária, o que pode contribuir para a formulação de critérios mais objetivos e confiáveis para a seleção de oócitos e embriões, e consequente aprimoramento e otimização das técnicas em reprodução humana assistida que são aplicadas nos procedimentos clínicos atuais de fertilização in vitro.

The interaction of oocyte-granulosa cells in vivo and in vitro and its influence on oocyte and embryo quality has been the subject of numerous studies, but many issues still need to be clarified. The objective of this study was to promote a review about the importance of this communication establishing a connection with the issue of in vitro maturation of immature human oocytes by applying this knowledge to define potential molecular markers that could improve the selection of oocytes and consequently select good quality embryos for later transfer and success of pregnancy in patients undergoing treatment of infertility. The granulosa cells also have an important role in oocyte maturation in vitro and the venefits from the presence of these cells during this process can be atributed to the formation of a favorable micro-environment (biochemical and metabolic) around the oocyte. In this review, we identified several potential markers in the cumulus cells of competent oocytes, including several genes that could be used as predictors of oocyte competence, which contributes for more objective and reliable criteria for the selection of oocytes and embryos, thus improving and optimizing techniques in assisted human reproduction that are applied in current clinical in vitro fertilization.

Cryopreservation time does not decrease follicular viability in ovarian tissue frozen for fertility preservation

OBJECTIVE: To determine the effect of storage duration on cryopreserved ovarian tissue using fresh and frozenthawed samples. METHODS: Seventeen fertile patients underwent an ovarian biopsy during elective laparoscopic tubal ligation. The tissue sample was divided into three parts: one part was processed fresh (FG), and two were slowly frozen, cryopreserved for 30 (G30) or 180 days (G180), thawed and analyzed. Follicular density, follicular viability, and steroidogenic capacity were assessed. RESULTS: We observed no differences between the groups in follicular density, which was assessed in hematoxylin and eosin-stained tissue sections. A heterogeneous follicular distribution was observed in the parenchyma, with a mean density of 361.3±255.4, 454.9±676.3, and 296.8±269.0 follicles/mm3 for FG, G30 and G180, respectively (p = 0.46). Follicular viability was greater in FG (93.4 percent) when compared with the cryopreserved tissues (70.8 percent for G30 (p<0.001) and 78.4 percent for G180 (p<0.001)), with no difference in viability between the frozen samples (p>0.05). The steroidogenic capacity of the tissue was not significantly reduced following cryopreservation. CONCLUSION: The slow freezing procedures used for ovarian cryopreservation are capable of preserving follicular viability and maintaining the steroidogenic capacity of the tissue despite a roughly 30 percent decrease in follicular viability. Furthermore, short-term storage of ovarian tissue does not appear to compromise follicle integrity.

Concentração dos hormônios esteroides no fluido folicular de folículos ovarianos maduros e imaturos de pacientes com síndrome dos ovários policísticos submetidas à fertilização in vitro / Concentration of steroid hormones in the follicular fluid of mature and immature ovarian follicles of patients with polycystic ovary syndrome submitted to in vitro fertilization

OBJETIVO: avaliar a concentração dos hormônios esteroides no fluido folicular (FF) de folículos pequenos (10-14 mm) e grandes (> 18 mm) de mulheres com síndrome dos ovários policísticos (SOP) submetidas à hiperestimulação ovariana controlada (HOC) e ciclos de fertilização in vitro (FIV). MÉTODOS: estudo caso-controle foi conduzido em 13 mulheres inférteis com SOP (17 ciclos) e 31 mulheres inférteis por fator masculino - Grupo Controle (31 ciclos). Os FF foram aspirados individualmente e dividos em 4 grupos: G1 (FF pequeno do Grupo Controle), G2 (FF pequeno do grupo SOP), G3 (FF grande do Grupo Controle) e G4 (FF grande do grupo SOP). A metodologia utilizada para as dosagens de estradiol, progesterona e β-hCG foi a quimioluminescência, e de testosterona e androstenediona o radioimunoensaio. Para a análise das dosagens hormonais no FF entre os grupos SOP e Controle utilizou-se o teste t não-pareado, e para a comparação entre os quatro grupos, o ANOVA. Para a taxa de gravidez, foi utilizado o teste exato de Fisher. RESULTADOS: os folículos pequenos dos dois grupos tiveram valores menores de progesterona (8.435±3.305 ng/mL) comparados aos grandes (10.280±3.475 ng/mL), com valor de p<0,01. Os níveis de progesterona de todos os folículos do grupo SOP (8.095±4.151 ng/mL) foram inferiores ao Controle (9.824±3.128 ng/mL), com valor de p=0,03. Os níveis de testosterona diferiram entre G1 (326,6±124,4 ng/dL) e G3 (205,8±98,91 ng/dL), com valor de p<0,001, e entre G3 (205,8±98,91 ng/dL) e G4 (351,10±122,1 ng/dL), com valor de p<0,001. Os folículos pequenos (508,9±266 ng/dL) apresentaram valores superiores de testosterona comparados aos grandes (245,10±123 ng/dL), com valor de p<0,0001. As taxas de gravidez não diferiram entre os grupos SOP (5/13, 38,5 por cento) e Controle (9/31, 40,9 por cento), com valor de p=072. CONCLUSÕES: mulheres com SOP apresentam altas concentrações de testosterona no FF, independentemente do estágio de desenvolvimento folicular, e níveis de progesterona diminuídos, sugerindo que fatores parácrinos podem inibir sua secreção pelas células foliculares. As taxas de gravidez mostraram que o tratamento de HOC e FIV é uma boa opção para mulheres com infertilidade secundária à SOP.

PURPOSE: to evaluate the concentration of steroid hormones in follicular fluid (FF) of small (10-14 mm) and large (> 18 mm) follicles of women with polycystic ovary syndrome (PCOS) submitted to controlled ovarian hyperstimulation (COH) and in vitro fertilization (IVF) cycles. METHODS: a case-control study was conducted on 13 infertile women with PCOS (17 cycles) and 31 infertile women due to male factor - Control Group (31 cycles). FF was aspirated individually and divided into four groups: G1 (FF of small follicles of the Control Group), G2 (FF of small follicles of the PCOS group), G3 (FF of large follicles of the Control Group) and G4 (FF of large follicles of the PCOS group). Estrogen, progesterone and β-hCG were determined by chemiluminescence, and testosterone and androstenedione by radioimmunoassay. The unpaired t-test was used to compare the hormone determinations in the FF of the PCOS and Control Groups, and the four groups were compared by ANOVA. Fisher's exact test was used to compare the pregnancy rates. RESULTS: the small follicles of the two groups had lower progesterone levels (8,435±3,305 ng/mL) than large follicles (10,280±3,475 ng/mL), p-value <0.01. The progesterone levels of all follicles of group PCOS (8,095±4,151 ng/mL) were lower than Control (9,824±3,128 ng/mL), p-value =0.03. Testosterone differed between G1 (326.6±124.4 ng/dL) and G3 (205.8±98.91 ng/dL), p-value <0.001, and between G3 (205.8±98.91 ng/dL) and G4 (351.10±122.1ng/dL), p-value <0.001. Small follicles had higher testosterone levels (508.9±266 ng/dL) than large follicles (245.10±123 ng/dL), p-value <0.0001. The pregnancy rates did not differ between the PCOS (5/13, 38.5 percent) and the Control groups (9/31, 40.9 percent), p-value =072. CONCLUSIONS: women with PCOS had high testosterone concentrations in the FF, regardless of the stage of follicle development, and reduced progesterone levels, suggesting that paracrine factors may inhibit the secretion of the latter by follicular cells. The pregnancy rates showed that treatment with COH and IVF is a good option for women with infertility secondary to PCOS.

[Concentration of steroid hormones in the follicular fluid of mature and immature ovarian follicles of patients with polycystic ovary syndrome submitted to in vitro fertilization]. / Concentração dos hormônios esteroides no fluido folicular de folículos ovarianos maduros e imaturos de pacientes com síndrome dos ovários policísticos submetidas à fertilização in vitro.

PURPOSE: to evaluate the concentration of steroid hormones in follicular fluid (FF) of small (10-14 mm) and large (> 18 mm) follicles of women with polycystic ovary syndrome (PCOS) submitted to controlled ovarian hyperstimulation (COH) and in vitro fertilization (IVF) cycles. METHODS: a case-control study was conducted on 13 infertile women with PCOS (17 cycles) and 31 infertile women due to male factor - Control Group (31 cycles). FF was aspirated individually and divided into four groups: G1 (FF of small follicles of the Control Group), G2 (FF of small follicles of the PCOS group), G3 (FF of large follicles of the Control Group) and G4 (FF of large follicles of the PCOS group). Estrogen, progesterone and ß-hCG were determined by chemiluminescence, and testosterone and androstenedione by radioimmunoassay. The unpaired t-test was used to compare the hormone determinations in the FF of the PCOS and Control Groups, and the four groups were compared by ANOVA. Fisher's exact test was used to compare the pregnancy rates. RESULTS: the small follicles of the two groups had lower progesterone levels (8,435 ± 3,305 ng/mL) than large follicles (10,280 ± 3,475 ng/mL), p-value <0.01. The progesterone levels of all follicles of group PCOS (8,095 ± 4,151 ng/mL) were lower than Control (9,824 ± 3,128 ng/mL), p-value =0.03. Testosterone differed between G1 (326.6 ± 124.4 ng/dL) and G3 (205.8 ± 98.91 ng/dL), p-value <0.001, and between G3 (205.8 ± 98.91 ng/dL) and G4 (351.10 ± 122.1 ng/dL), p-value <0.001. Small follicles had higher testosterone levels (508.9 ± 266 ng/dL) than large follicles (245.10 ± 123 ng/dL), p-value <0.0001. The pregnancy rates did not differ between the PCOS (5/13, 38.5%) and the Control groups (9/31, 40.9%), p-value =072. CONCLUSIONS: women with PCOS had high testosterone concentrations in the FF, regardless of the stage of follicle development, and reduced progesterone levels, suggesting that paracrine factors may inhibit the secretion of the latter by follicular cells. The pregnancy rates showed that treatment with COH and IVF is a good option for women with infertility secondary to PCOS.

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

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.

Análogos do GnRH: bases moleculares e aplicações em reprodução assistida / GnRH analogues: molecular basis and applications in assisted reproduction

O decapeptídeo GnRH é o iniciador central da cascata hormonal reprodutiva. É gerado em neurônios hipotalâmicos a partir de um polipeptídeo precursor por processamento enzimático e liberado de maneira pulsátil na circulação portal para estimular a biossíntese e secreção dos hormônios luteinizante (LH) e folículo-estimulante (FSH) pela hipófise. Baixas doses de GnRH aplicadas de maneira pulsátil equivalem à liberação fisiológica portal e restauram a fertilidade em diferentes condições de anovulação. Por outro lado, altas doses de GnRH causam desensibilização do gonadotrofo e inibem a liberação hipofisária com conseqüente inibição da função ovariana. Pequenas modificações na molécula original do GnRH originam substâncias conhecidas como análogos do GnRH, que podem ser agonistas e antagonistas. Os agonistas provocam uma liberação inicial de gonadotrofinas (flare-up) seguida de desensibilização do gonadotrofo (down regulation). Já os antagonistas agem diretamente sobre o gonadotrofo, através de inibição competitiva dos receptores do GnRH. Esse fenômeno de quiescência hipofisária provocada pelos análogos do GnRH tem extensivas aplicações clínicas e o conhecimento sobre a fisiologia do GnRH é importante para aplicação mais racional destes análogos. Este artigo visa a descrever os mecanismos de ação destes análogos do GnRH, bem como revisar as diferentes indicações clínicas de seu uso em reprodução assistida

Administração do Lapachol durante o período de fetogênese em ratas: desenvolvimento pós-natal das crias / Lapachol administration during fetogenesis in rats: postnatal development of the pups

O Lapachol é um fármaco com largo espectro de atividade, que tem demonstrado efeito embriotóxico e fetotóxico. No presente trabalho, estuda-se o desenvolvimento de crias de ratas, cujas mães foram tratadas, durante o período de fetogênese, com 2,5mg de Lapachol/kg de peso corporal. O grupos controle e veículo receberam, respectivamente, 1ml de água destilada e 1ml de solução hidroalcoólica(v:v). Após o nascimento as crias foram reduzidas ao máximo de oito por mãe, foram pesadas e observadas diariamente, para a identificação de canibalismo e morte. Aos 4, 14 e 25 dias(desmame), as crias foram pesadas e registrado o número de sobreviventes. As mães foram submetidas a controle de peso corporal, consumo de ração e observadas para verificação de indícios clínicos de toxicidade. Os recém-nascidos do grupo de mães tratadas com Lapachol nasceram com peso corporal inferior aos grupos controle e veículo (C: 5,57+-0,57(79); V: 5,74+-0,77(79); T:5,3+-0,53(80) p<0.005). Posteriormente ganharam peso, não diferindo dos demais grupos. Não foram observadas quaisquer alterações nos índices de viabilidade, de crescimento e de lactação entre os grupos analisados. As mães não apresentaram indícios clínicos de toxicidade, não tiveram redução de peso corporal, nem alteraram o consumo de ração. Tais dadoos levam a conclusão de que o Lepachol, na dose usada, embora tenha causado retardo de crescimento fetal, não interferiu com o desenvolvimento posterior das crias.

Toxic effect of Solanum Lycocarpum in the male Wistar rats and swiss mice reproductive organs

Uma suspensäo de Solanum Lycocarpum foi administrada a ratos Wistar e camundongos suiços adultos em duas doses diferentes (60 mg/15ml e 120 mg/15ml de água destilada para ratos e 30 mg/15ml e 60 mg/15ml de água destilada para camundongos) para avaliar seu efeito no peso corporal, de testículos, de epidídimo esquerdo, de glândulas sexuais acessórias, de hipófise e na concentraçäo de espermatozóides. O extrato foi administrado duas vezes ao dia durante 5 dias, com sacrifício dos animais 3 dias após o término do tratamento. Os resultados indicaram que, a princípio, Solanum Lycocarpum näo tem efeito tóxico no rato Wistar adulto mas causou perda significativa de peso corporal, de próstata ventral, vesícula seminal e hipófise de camundongo suiço adulto. A reduçäo de peso de hipófise poderia estar indicando alto nível de testosterona ou ausência de estímulo para sua secreçäo. Quanto ao baixo peso de próstata ventral e vesícula seminal, mais estudos säo necessários para explicar as causas dessa reduçäo.