Follicular Fluid Estradiol Concentrations Research Articles

GnRH agonist and GnRH antagonist protocols in ovarian stimulation: differential regulation pathway of aromatase expression in human granulosa cells

Gonadotrophin-releasing hormone (GnRH) agonists and antagonists have been widely used to prevent premature LH surge during ovarian stimulation. However, studies have shown a significantly lower serum oestradiol concentration on the day of human chorionic gonadotrophin administration for cycles using GnRH antagonist. This study compared aromatase gene expression in granulosa lutein cells from 50 women randomly assigned to receive either GnRH agonist (group 1, n=28) or GnRH antagonist (group 2, n=22). The cellular mechanism involved in the observed effects was also investigated. GnRH antagonist treatment significantly affected serum oestradiol concentration (1894+/-138 versus 1074+/-63 pg/ml; P < or = 0.001), follicular-fluid oestradiol concentration in large follicles (18,565+/-2467 versus 10,184+/-1993 pg/ml; P < or = 0.05), aromatase activity (9600+/-1179 versus 5376+/-997 fmol/10(6) cells/h; P < or = 0.05) and mRNA aromatase/mRNA glyceraldehyde 3-phosphate dehydrogenase (15+/-3 versus 6+/-1; P < 0.05). Protein kinase C (PKC) activity in granulosa lutein cells from the GnRH antagonist group was 2.5-fold higher than in the GnRH agonist group. In-vitro experiments showed that selective down-regulation of PKC was only observed in GnRH-desensitized granulosa lutein cells. This report suggests that, in granulosa lutein cells, the modulation of the FSH-induced protein kinase A pathway by PKC was different in agonist versus antagonist cycles.

Differential genome-wide gene expression profiling of bovine largest and second-largest follicles: identification of genes associated with growth of dominant follicles

BackgroundBovine follicular development is regulated by numerous molecular mechanisms and biological pathways. In this study, we tried to identify differentially expressed genes between largest (F1) and second-largest follicles (F2), and classify them by global gene expression profiling using a combination of microarray and quantitative real-time PCR (QPCR) analysis. The follicular status of F1 and F2 were further evaluated in terms of healthy and atretic conditions by investigating mRNA localization of identified genes.MethodsGlobal gene expression profiles of F1 (10.7 +/- 0.7 mm) and F2 (7.8 +/- 0.2 mm) were analyzed by hierarchical cluster analysis and expression profiles of 16 representative genes were confirmed by QPCR analysis. In addition, localization of six identified transcripts was investigated in healthy and atretic follicles using in situ hybridization. The healthy or atretic condition of examined follicles was classified by progesterone and estradiol concentrations in follicular fluid.ResultsHierarchical cluster analysis of microarray data classified the follicles into two clusters. Cluster A was composed of only F2 and was characterized by high expression of 31 genes including IGFBP5, whereas cluster B contained only F1 and predominantly expressed 45 genes including CYP19 and FSHR. QPCR analysis confirmed AMH, CYP19, FSHR, GPX3, PlGF, PLA2G1B, SCD and TRB2 were greater in F1 than F2, while CCL2, GADD45A, IGFBP5, PLAUR, SELP, SPP1, TIMP1 and TSP2 were greater in F2 than in F1. In situ hybridization showed that AMH and CYP19 were detected in granulosa cells (GC) of healthy as well as atretic follicles. PlGF was localized in GC and in the theca layer (TL) of healthy follicles. IGFBP5 was detected in both GC and TL of atretic follicles. GADD45A and TSP2 were localized in both GC and TL of atretic follicles, whereas healthy follicles expressed them only in GC.ConclusionWe demonstrated that global gene expression profiling of F1 and F2 clearly reflected a difference in their follicular status. Expression of stage-specific genes in follicles may be closely associated with their growth or atresia. Several genes identified in this study will provide intriguing candidates for the determination of follicular growth.

Changes in the gene expression of adiponectin and adiponectin receptors (AdipoR1 and AdipoR2) in ovarian follicular cells of dairy cow at different stages of development

Adiponectin is one of the most important, recently discovered adipocytokines that acts at various levels to control male and female fertility through central effects on the hypothalamus-pituitary axis or through peripheral effects on the ovary, uterus, and embryo. We studied simultaneous changes in the gene expression pattern of adiponectin and adiponectin receptors 1 and 2 (AdipoR1 and AdipoR2) in granulosa and theca cells, cumulus-oocyte complex, and in corpus luteum in healthy bovine (Bos tarus) follicles at different stages of development. The expression levels of adiponectin, AdipoR1, and AdipoR2 mRNA were lower (P < 0.05) in granulosa and cumulus cells in comparison with that in theca cells and oocyte. In contrast with the oocyte, AdipoR1 in granulosa, theca, and luteal cells was expressed (P < 0.05) more than AdipoR2. Adiponectin expression increased (P < 0.05) in granulosa cells and in cumulus-oocyte complex during follicular development from small to large follicles. Opposite results were observed in theca cells. Expression of adiponectin was highest in the late stages of corpus luteum (CL) regression, whereas lower expression was recorded in active CL (P < 0.05). AdipoR1 and AdipoR2 expression increased during the terminal follicular growth in granulosa and theca cells (P < 0.05) and during the luteal phase progress in CL. There was positive correlation between adiponectin mRNA level in granulosa cells from large follicles and follicular fluid estradiol concentration (r = 0.48, P < 0.05) and negative correlation between adiponectin mRNA abundance in theca cells and follicular fluid progesterone concentration (r = –0.44, P < 0.05). In conclusion, we found that the physiologic status of the ovary has significant effects on the natural expression patterns of adiponectin and its receptors in follicular and luteal cells of bovine ovary.

Follicular-fluid anti-Mullerian hormone concentrations are predictive of assisted reproduction outcome in PCOS patients

Serum anti-Mullerian hormone (AMH) concentrations constitute a sensitive marker for ovarian ageing. In addition, concentrations of AMH in the follicular fluid constitute a useful marker of embryo implantation in assisted reproduction cycles. The present study measured serum and follicular-fluid AMH concentrations on the day of oocyte retrieval. These data showed that clinical pregnancy rates (25.0, 34.1 and 42.1%, respectively, P < 0.001), embryo implantation rates (24.3, 35.0 and 44.4%, respectively, P < 0.001) and fertilization rates (59.2, 70.9 and 79.5%, respectively, P < 0.001) were markedly different among the low, moderate and high follicular-fluid AMH groups but not among the different serum AMH concentration groups. Follicular-fluid AMH concentrations were negatively correlated with follicular-fluid oestradiol concentrations. The results of this study suggest that follicular-fluid AMH concentration on the day of oocyte retrieval would appear to better reflect the reproductive outcome in PCOS patients undergoing assisted reproduction.

Effect of Angiotensin II on Bovine Follicular Growth and mRNA Encoding Steroidogenic Enzymes, Gonadotrophin Receptors, and Tissue Development Genes.

Angiotensin II (AngII) has been shown to be involved in the regulatory mechanism of ovulation and oocyte nuclear and cytoplasmic maturation in cattle. In rats, AngII induces apoptosis in granulosa cells and atresia of antral follicles during follicular development. However, the angiotensin converting enzyme (ACE) mRNA expression increases at the end of follicular maturation, suggesting a possible modulatory role of AngII for ovulation in cows. In addition, the expression of AngII type 2 receptors (AGTR2) is higher in healthy than in atretic follicles and, in vitro, is stimulated by FSH in bovine granulosa cell culture. Based on this evidence, this study was carried out to establish the role of AngII during the follicular growth and the effect of AngII inhibition on steroid secretion and follicular cell gene expression profile. Cows were pre-synchronized to induce a new follicular wave and the largest follicle was injected with saralasin (AngII inhibitor; 10μM), AngII (10μM) or saline when it reached 7-8mm diameter. The intrafollicular injections were performed through a double-needle system and guided by a 7.5 MHz vaginal ultrasound probe. All follicles were monitored by ultrasonography for 96 hours after intrafollicular injection. Follicles injected with saline (control group) reached ovulatory size and ovulated after a challenge with the GnRH agonist gonadorelin, demonstrating that the intrafollicular injection did not affect follicular growth. Saralasin inhibited follicular growth in all treated cows (4/4), suggesting that AngII is essential for follicular growth in 7-8mm follicles. However, intrafollicular injection of AngII affected neither follicular growth nor the pattern of follicular dynamics, which were similar to control cows. These results imply that bovine ovarian follicles contain sufficient AngII for follicle development. In a second experiment, a single follicle of each cow was injected with saralasin or saline and cows were ovariectomized 24h later. The follicular fluid was aspirated to determine steroid concentrations, and granulosa and theca cells were recovered for gene expression measurement. Estradiol concentration in follicular fluid was determined by radioimmunoassay. Messenger RNA levels were analyzed by real-time RT-PCR and are expressed relative to cyclophilin. The inhibition of AngII action decreased abundance of mRNA encoding aromatase (CYP19), 3β-hydroxysteroid dehydrogenase (3β HSD), LH receptor, SerpinE2 and cyclinD2 in granulosa cells but not StAR, 17β HSD, FSH receptor, growth arrest and DNA damage inducible (GADD45b) or X-linked inhibitor of apoptosis protein (XIAP). In theca cells, the inhibition of AngII decreased the expression of AGTR2 but not the expression of steroidogenic enzymes genes. Taken together, these results show that AngII is essential for follicular growth, and plays important role in regulating genes involved in granulosa cell proliferation and differentiation, which are necessary for development of the dominant follicle. These data suggest that AngII signaling is involved in follicle growth and dominance in cattle. (poster)

Evidence Supporting a Role for Cocaine- and Amphetamine-Regulated Transcript (CARTPT) in Control of Granulosa Cell Estradiol Production Associated with Dominant Follicle Selection in Cattle

We demonstrated previously a negative association of granulosa cell cocaine- and amphetamine-regulated transcript (CARTPT) expression with follicle health status and inhibitory effects of the mature CARTPT peptide (CART) on follicle-stimulating hormone (FSH) signal transduction in vitro, resulting in reduced bovine granulosa cell CYP19A1 mRNA and estradiol production. The objectives of this study were to investigate temporal regulation of granulosa cell CARTPT expression (granulosa cell mRNA and follicular fluid CART peptide concentrations) during follicular waves, CART regulation of androstenedione production (precursor for estradiol biosynthesis) by thecal tissue collected at specific stages of a follicular wave, FSH regulation of granulosa cell CARTPT mRNA expression, and the ability of CART to inhibit granulosa cell estradiol production and CYP19A1 mRNA expression when administered in vivo. CART concentrations in healthy, estrogen-active follicles (estradiol greater than progesterone in follicular fluid) decreased after dominant follicle selection, and CARTPT mRNA was lower in healthy, estrogen-active versus estrogen-inactive atretic follicles (progesterone greater than estradiol) collected at the predeviation and early dominance stages. CART treatment reduced luteinizing hormone-induced androstenedione production by thecal tissue collected at predeviation and early dominance stages but not at later stages of a follicular wave. The FSH or insulin-like growth factor 1 treatment in vitro reduced granulosa cell CARTPT mRNA in a dose-dependent fashion. Administration of CART in vivo into follicles at the early dominance stage reduced follicular fluid estradiol concentrations and granulosa cell CYP19A1 mRNA. Collectively, results support a potential stage-specific regulatory role for CART in negative regulation of estradiol production associated with selection of the dominant follicle.

534. CO-ORDINATED GENE EXPRESSION IN BOVINE GRANULOSA CELLS PRECEDES FOLLICULAR DOMINANCE

During growth of bovine follicles, one emerges as the largest and dominant follicle. What regulates dominance remains unknown, but candidates include oestradiol, TGFB1, and recently CYP11A1 and focal intra-epithelial matrix (focimatrix). The two to four largest follicles were dissected from pairs of bovine ovaries and follicular fluid collected. A portion of the follicle wall was histologically classified for follicle health or atretia, and granulosa cells harvested for quantitative RT-PCR. Messenger RNA levels of focimatrix (COL4A1, LAMB2, HSPG2), steroidogenic enzymes (CYP11A1, CYP19A1) and TGFB1 genes were measured. Follicular fluid progesterone and oestradiol concentrations were measured by RIA. Follicles were identified as pre-deviated (before size-deviation) if the largest two or more healthy follicles were of equal size (6.7±0.1 mm, n = 14 animals, 35 follicles), and as post-deviated (after size-deviation) if they differed in size by 0.5–1.0 mm (7.2±0.2 mm; n = 11 animals, 26 follicles). For analyses, pre-deviated follicles were grouped into either the highest (oestradiol, CYP11A1) or lowest (TGFB1) expression (n = 14) and compared to the remaining follicles (n = 21). Deviated follicles were classified into dominant (n = 12) and subordinate (n = 14) based on diameter. Dominant follicles did not differ from subordinate follicles in any parameters measured, but were significantly larger than subordinate or pre-deviated follicles (P&lt;0.01). For pre-deviated follicles grouped on oestradiol no parameters differed significantly, and when grouped on TGFB1, LAMB2 (P&lt;0.05), HSPG (P&lt;0.05), CYP19A1 (P&lt;0.05) and TGFB1 (P&lt;0.01) differed but levels were lower, not higher as expected. When grouped on CYP11A1, COL4A1 (P&lt;0.05), LAMB2 (P&lt;0.01), HSPG2 (P&lt;0.01) and CYP19A1 (P&lt;0.001) were significantly elevated in the high CYP11A1 group. This suggests that CYP11A1 and focimatrix might be important in follicle dominance.

170 FOLLICULAR FLUID CONCENTRATION AND OOCYTE QUALITY FROM TOGGENBURG GOATS FED WITH UREA

Protein and urea concentrations impair oocyte and embryo development in vivo and in vitro through an unclear mechanism. A possible way to understand this process is to determine the concentration of hormones and metabolites in follicular fluid associated with normal development. The objective of this study was to determine the effect of dietary urea levels on follicular fluid concentration of hormones and metabolites and oocyte quality. A trial was conducted with 9 nonpregnant and nonlactating Saanen goats, which had been distributed in a randomized design and fed with diets with 0 (n = 4) and 2.4% of urea in the total dry matter (DM) of the diet (n = 5). Before follicle aspiration by laparotomy, the goats were synchronized by inserting intravaginal sponges containing 60 mg of acetate medroxyprogesterone (Progespon®, Sintex) for 10 days followed by 125 μg of cloprostenol (Ciosin® Coopers) 48 h before the removal of the sponge. The sponge was removed immediately before the follicular aspiration. The follicular development was stimulated with 70 mg of NIH-FSH-P1 (Folltropin V® Vetrepharm) i.m., and 300 IU of eCG i.m., (Novormon® Sintex) given 36 h before the follicular aspiration. Fluid from the 2 lartest follicles of each ovary were analyzed to determine the concentration of estradiol, progesterone, and testosterone by quimioluminesence, and glucose and urea concentrations were measured by enzymatic kit. The other follicles in each ovary were aspired with new needles and syringes and the oocyte quality was recorded. Oocytes were classified according to cytoplasma aspect and number of granulosa cells: Class A (dark cytoplasm and uniform aspect) with 3 (AMG) and &lt;3 layers of cumulus cells (AmG); class B (cytoplasm with color alterations, desuniform aspect and vacuoles) with 3 (BMG) and &lt;3 layers of cumulus cells (BmG); number of partially denuded oocytes (PD) and number of denuded oocytes (DO). Data were analyzed by ANOVA and treatment difference separated by SNK test. Follicular fluid estradiol concentration was lower in goats fed with urea (4.02 ± 0.16; 4.97 ± 0.18 ng mL–1; P &lt; 0.05), progesterone concentration did not differ between treatments (2.48 ± 0.58; 3.37 ± 0.52 ng mL–1; P &gt; 0.05), testosterone concentration was lower in the control animals (1.17 ± 0.48; 3.20 ± 0.43 ng mL–1; P &lt; 0.05). The glucose (91.44 ± 3.60; 84.78 ± 5.58 mg dL–1) and urea concentration (23.04 ± 1.06; 18.00 ± 2.35) were greater in the animals treated with 2.4% compared with 0% of urea (P &lt; 0.05), respectively. The number of oocytes in the different categories was not affected by treatment (P &gt; 0.05): AMG 1.20 ± 1.09 v. 0.50 ± 0.57, AmG 4.20 ± 2.16 v. 3.50 ± 3.10, BMG 0.40 ± 0.54 v. 0.25 ± 0.50, BmG 1.40 ± 0.54 v. 1.75 ± 1.25, DO 10.20 ± 3.76 v. 11.50 ± 5.44, in the 0 and 2.4% of urea groups respectively. Only the number of PD (1.60 ± 0.54 v. 3.50 ± 1.91) recovered from animals treated with 2.4% was greater than in controls (P &lt; 0.05). The hormone and metabolites concentration in follicular fluid as well as the oocyte quality was affected by the urea concentration of the diet. Supported by grant from: CNPq, FAPEMIG, Shering Plough®, Tecnopec®, Carbogel®.

Standardization and validation of an induced ovulation model system in buffalo cows: Characterization of gene expression changes in the periovulatory follicle

In bovines characterization of biochemical and molecular determinants of the dominant follicle before and during different time intervals after gonadotrophin surge requires precise identification of the dominant follicle from a follicular wave. The objectives of the present study were to standardize an experimental model in buffalo cows for accurately identifying the dominant follicle of the first wave of follicular growth and characterize changes in follicular fluid hormone concentrations as well as expression patterns of various genes associated with the process of ovulation. From the day of estrus (day 0), animals were subjected to blood sampling and ultrasonography for monitoring circulating progesterone levels and follicular growth. On day 7 of the cycle, animals were administered a PGF 2α analogue (Tiaprost Trometamol, 750 μg i.m.) followed by an injection of hCG (2000 IU i.m.) 36 h later. Circulating progesterone levels progressively increased from day 1 of the cycle to 2.26 ± 0.17 ng/ml on day 7 of the cycle, but declined significantly after PGF 2α injection. A progressive increase in the size of the dominant follicle was observed by ultrasonography. The follicular fluid estradiol and progesterone concentrations in the dominant follicle were 600 ± 16.7 and 38 ± 7.6 ng/ml, respectively, before hCG injection and the concentration of estradiol decreased to 125.8 ± 25.26 ng/ml, but concentration of progesterone increased to 195 ± 24.6 ng/ml, 24 h post-hCG injection. Inh-α and Cyp19A1 expressions in granulosa cells were maximal in the dominant follicle and declined in response to hCG treatment. Progesterone receptor, oxytocin and cycloxygenase-2 expressions in granulosa cells, regarded as markers of ovulation, were maximal at 24 h post-hCG. The expressions of genes belonging to the super family of proteases were also examined; Cathepsin L expression decreased, while ADAMTS 3 and 5 expressions increased 24 h post-hCG treatment. The results of the current study indicate that sequential treatments of PGF 2α and hCG during early estrous cycle in the buffalo cow leads to follicular growth that culminates in ovulation. The model system reported in the present study would be valuable for examining temporo-spatial changes in the periovulatory follicle immediately before and after the onset of gonadotrophin surge.

SPATIAL AND TEMPORAL REGULATION OF BMP-2, -6, -15 AND GDF-9 mRNA EXPRESSION IN DEVELOPING PORCINE OVARIAN FOLLICLES

Members of the transforming growth factor beta (TGF-beta) superfamily, namely the bone morphogenetic proteins (BMPs) and growth differentiation factor 9 (GDF-9), have been shown to play important roles during follicular development and ovulation. However, despite the wealth of information available in rodent and ruminant species, very little is known about the role of these genes in the porcine ovary. Therefore, the aims of the present study were to investigate the expression of BMP-2, -6, -15 and GDF-9 mRNA in the oocyte, granulosa cells and theca cells of porcine follicles and to determine if changes occur in the levels of expression of these genes during follicular growth. Oocyte, granulosa cells and theca cells were recovered from antral follicles (2-10 mm in diameter) of first parity sows at day 1, 2 and 4 after weaning or at day 14, 16 and 20 of their subsequent estrous cycle. These time points correspond to specific phases of follicular growth; the recruitment of a growing cohort of follicles, the mid-selection phase and the final selection of the pre-ovulatory follicle population. Plasma estrogen and progesterone analysis was used to confirm the expected physiological status of each animal. Follicular fluid estradiol concentration in day 4 and day 20 animals was also used to determine whether the follicles had been exposed to the LH surge. Total RNA was extracted, reverse transcribed and the levels of expression for these genes were determined using real-time PCR. The delta Ct values were calculated using cyclophilin as the internal control and the data were analysed using the general linear model procedure of SAS (SAS Institute Inc., Cary, NC). It is particularly interesting to notice that both BMP-2 and BMP-15 were expressed in all three cell types but were expressed at considerably higher levels in the oocyte (p < 0.001). BMP-2 mRNA levels increased in granulosa cells (P = 0.018) and theca cells (P < 0.001) from the recruitment phase to the mid-selection phase, remained constant until the final selection phase and decreased after the LH surge. BMP-15 mRNA levels also increased in both cell types from the recruitment phase to the mid-selection phase, however the levels decreased in the granulosa cells of the final pre-ovulatory follicle population while remaining constant during this phase in the theca cells (P < 0.05). As opposed to BMP-2, the level of BMP-15 mRNA did not change in response to the LH surge. Finally, BMP-15 mRNA levels showed a strong tendency to decrease in the oocytes from recruitment to the final selection phase (P = 0.054). These results confirm that the oocyte is the main source of BMP-2 and BMP-15 but contrary to other studies, their expression was also found in granulosa cells and theca cells. These differences suggest that follicular growth and development may be regulated differently in swine. (poster)

Superovulatory Ovarian Response in Mangalica Gilts is Not Influenced by Feeding Level

The aim of the study was to compare how different feeding levels affect the ovarian potential of follicular development and oocyte maturation in response to superovulatory treatment in native Mangalica (M, n = 17) compared with Landrace (L, n = 20) pigs. Gilts of both breeds were fed high-energy (HI-2.5 kg) or low-energy (LO - 1.25 kg) feed during oestrus synchronization (15 days of Regumate feeding) till the time of oocyte aspiration (Day 6 after Regumate). Follicular growth was stimulated by the administration of 1000 IU equiue choriou gonadotropiu (eCG) 24 h after Regumate treatment, and ovulation was induced by injection of 750 IU human choriou gonadotropiu (hCG) 80 h after eCG administration. Ultrasound (US) investigation was done three times (4-10 h before, and 40-44 and 72-74 h after eCG administration) for the observation of follicular development. Oocyte and follicular fluid (FF) were collected endoscopically 34 h after hCG injection. Cumulus-oocyte complexes were evaluated, their morphology determined, and thereafter fixed and stained for chromatin evaluation. Oocytes were classified as meiosis-resumed (germinal vesicle breakdown, diakinesis, metaphase I to anaphase I) or matured (telophase I and metaphase II). FF concentrations of oestradiol and progesterone were measured by validated radioimmunoassays. In L gilts, differences were observed between HI and LO in the number of preovulatory follicles (32.3 +/- 10.5 vs 17.1 +/- 12.3, p < 0.05), but not in M (25.3 +/- 2.9 vs 28.8 +/- 7.3, p > 0.05). Initial follicular growth was not affected by feeding levels; however, preovulatory follicle size was larger in M (7.1 +/- 0.9 and 6.9 +/- 1.1 mm vs 5.7 +/- 0.7 and 5.5 +/- 0.8 mm; p < 0.05). No differences were obtained with relation to mature chromatin configuration in both breeds (L gilts: HI - 70% and LO-67% vs M gilts: HI - 67% and LO - 63%). A twofold higher oestradiol concentration was detected in FF of HI-M and LO-M (29.6 +/- 6.8 and 30.9 +/- 10.3 ng/ml respectively) compared with that of L (16.9 +/- 9.7 and 17.9 +/- 3.6 ng/ml, respectively; p < 0.05). The mean FF progesterone level was nearly fivefold higher in M (2020.4 +/- 1056 and 1512.2 +/- 1121.8 ng/ml) compared with L (386.2 +/- 113.7 and 298.8 +/- 125.9 ng/ml, p < 0.05). The results indicate an influence of the feeding of altered energy on the number of recruitable preovulatory follicles in modern Landrace but not in native Mangalica breed. Moreover, the follicular steroid hormone milieu differs between Landrace and Mangalica gilts but not depending on feeding levels. Oocyte maturation was not affected by diet.

Concentrações de FSH, LH, estradiol, progesterona e histamina no soro, no fluido peritoneal e no fluido folicular de mulheres com e sem endometriose

PURPOSE: literature reports show that there are no conclusive data about the association between endometriosis and the concentrations of hormones involved in the control of reproduction. Thus, the present study was undertaken to determine FSH, LH, estradiol (E), progesterone (P), and histamine (Hi) concentrations in serum, peritoneal fluid and follicular fluid of women with and without endometriosis. METHODS: the extent of the disease was staged according to the revised American Fertility Society classification (1997). For the collection of serum and peritoneal fluid, 28 women with endometriosis undergoing diagnostic laparoscopy were selected (18 infertile women with endometriosis I-II and ten infertile women with endometriosis III-IV). For the control group, 21 fertile women undergoing laparoscopy for tubal sterilization were selected. Follicular fluid was obtained from 39 infertile women undergoing in vitro fertilization (21 women with endometriosis and 18 women without endometriosis). RESULTS: FSH and LH levels in serum, peritoneal fluid and follicular fluid did not differ significantly between groups. On the other hand, E and P concentrations in the peritoneal fluid were significantly lower in infertile women with endometriosis (E: 154.2±15.3 for stages I-II and 89.3 ng/mL±9.8 ng/mL for stages III-IV; P: 11.2±1.5 for stages I-II and 7.6 ng/mL±0.8 for stages III-IV) in comparison with control women (E: 289.1 ng/mL±30.1; P: 32.8±4.1 ng/mL) (Kruskal-Wallis/Dunn tests; p<0.05). In serum, estradiol and progesterone concentrations followed the same pattern. In the follicular fluid, E and Hi concentrations were significantly lower in women with endometriosis (E: 97.4±11.1 pg/mL; Hi: 6.6±0.9 ng/mL) in comparison to women without endometriosis (E: 237.5±28.5 pg/mL; Hi: 13.8±1.3 ng/mL) (Student t-test; p<0.05), while progesterone levels revealed no significant difference between groups. CONCLUSIONS: our results indicate ovary dysfunction in women with endometriosis, with reduction on E, P and Hi concentrations, which may contribute to the subfertility often associated with the disease.

Perifollicular vascularity in poor ovarian responders during IVF

Normal ovarian responders who have follicles with good vascularity shown by power Doppler scanning are associated with a better pregnancy rate following IVF treatment. This study evaluated the significance of perifollicular vascularity and follicular fluid hormonal profile in poor responders who developed < or = 3 dominant follicles. Before oocyte retrieval, they underwent power Doppler examination for perifollicular vascularity. Patients who had all follicles with low-grade vascularity were classified as Group A, whereas those with at least one follicle with high-grade vascularity were Group B. Their follicular fluid concentrations of estradiol (E2), progesterone, HCG, vascular endothelial growth factor (VEGF) and inhibin B were measured. A total of 58 consecutive patients were recruited: 38 in Group A and 20 in Group B. Implantation rate, clinical pregnancy rate and follicular fluid hormonal concentrations were comparable for Groups A and B. Multiple pregnancy and live birth rates appeared higher, whereas miscarriage rate were lower in Group B than Group A, but these differences did not reach statistical significance. There were no significant differences in the implantation, clinical pregnancy and live birth rates among poor responders with and without high-grade perifollicular vascularity.

Estradiol and testosterone concentrations in follicular fluid as criteria to discriminate between mature and immature oocytes

The objective of the present study was to examine the association between follicular fluid (FF) steroid concentration and oocyte maturity and fertilization rates. Seventeen infertile patients were submitted to ovulation induction with urinary human follicle-stimulating hormone, human menopausal gonadotropin and human chorionic gonadotropin (hCG). A total of 107 follicles were aspirated after hCG administration, the oocytes were analyzed for maturity and 81 of them were incubated and inseminated in vitro. Progesterone, estradiol (E2), estrone, androstenedione, and testosterone were measured in the FF. E2 and testosterone levels were significantly higher in FF containing immature oocytes (median = 618.2 and 16 ng/ml, respectively) than in FF containing mature oocytes (median = 368 and 5.7 ng/ml, respectively; P < 0.05). Progesterone, androstenedione and estrone levels were not significantly different between mature and immature oocytes. The application of the receiver-operating characteristic curve statistical approach to determine the best cut-off point for the discrimination between mature and immature oocytes indicated levels of 505.8 ng/ml for E2 (81.0% sensitivity and 81.8% specificity) and of 10.4 ng/ml for testosterone (90.9% sensitivity and 82.4% specificity). Follicular diameter was associated negatively with E2 and testosterone levels in FF. There was a significant increase in progesterone/testosterone, progesterone/E2 and E2/testosterone ratios in FF containing mature oocytes, suggesting a reduction in conversion of C21 to C19, but not in aromatase activity. The overall fertility rate was 61% but there was no correlation between the steroid levels or their ratios and the fertilization rates. E2 and testosterone levels in FF may be used as a predictive parameter of oocyte maturity, but not for the in vitro fertilization rate.

Cytochrome p450-dependent lipid metabolism in preovulatory follicles.

Estrogen biosynthesis and proteolysis are both important processes involved in ovarian follicular development, which may be influenced by cytochrome P450 (CYP)-dependent fatty acid metabolites. However, CYP-dependent lipid metabolism has not been characterized with respect to follicular maturation in vivo. Therefore, follicular fluid was collected in the hours before and after the LH surge in pigs, and concentrations of epoxy, hydroxy, and dihydroxy lipids were measured by liquid chromatography tandem mass spectrometry. Arachidonate oxidation and epoxyeicosatrienoic acid hydrolysis to dihydroxyeicosatrienoic acids (DHETs) were also assessed in thecal and granulosa tissue fractions, and the expression of CYP epoxygenases was evaluated by immunoblots using available antisera. To evaluate soluble epoxide hydrolase (sEH) expression, the porcine sEH was cloned from ovarian tissue, expressed and purified for antibody generation. The follicular fluid oxylipin concentrations ranged from 1-150 nm depending on the compound and estrous stage. The follicular fluid concentrations of CYP-dependent oxylipins increased at estrus, as did sEH expression; however, significant changes in epoxides were not observed, and the 11,12-DHET peak was delayed. The ratio of 14,15-DHET:11,12-DHET across all samples correlated with the log of follicular fluid estradiol concentrations (P < 0.01). Epoxygenase activities were similar in theca and granulosa, varying little with follicular development, whereas the decline of a single CYP2J isoform at ovulation was observed by immunoblots. The sEH activity was higher in granulosa than in theca. Finally, the dynamic changes in follicular CYP-dependent arachidonic acid metabolites and their modulatory function in vascular models suggest roles for these metabolites in follicular maturation, which may include regulation of estradiol biosynthesis and preovulatory remodeling of the follicular wall that should be fully explored in future studies.

Increased ovarian follicular angiogenesis and dynamic changes of follicular vascular plexuses induced by equine chorionic gonadotropin in the gilt.

Follicular angiogenesis and capillary degeneration are crucial ovarian processes in folliculogenesis. The present study was conducted to assess the changes in population of follicular vascular plexuses with different capillary status in prepubertal gilts 72 h after equine chorionic gonadotropin (eCG) (1,250 IU) treatment, using combined vascular corrosion casting and scanning electron microscopy. Follicular fluid concentrations of estradiol, progesterone and vascular endothelial growth factor (VEGF) were determined to confirm the follicular status. Based on the proliferative or degenerative characteristics of their capillaries, follicles were classified into three categories: active angiogenesis, low angiogenesis and degeneration. Irrespective of exogenous gonadotropin treatment in vivo, small follicular vascular plexuses (<4 mm in diameter) exhibited all three conditions in casted ovaries, while medium (4-5 mm) and large (>5 mm) plexuses showed only active angiogenesis or degeneration. eCG treatment significantly increased the population of large, but decreased that of small follicular plexuses. Most large follicular vascular plexuses showed active angiogenesis with higher follicular fluid estradiol: progesterone ratios and VEGF concentration. eCG also increased the percentage of medium follicular plexuses with active angiogenesis. The populations of small follicular plexuses with active angiogenesis were higher in controls, but decreased after eCG treatment. However, treatment of gilts with the gonadotropin increased the percentage of small plexuses (<1.0 mm) with low angiogenesis and those (1-3.9 mm) with extensive capillary degeneration. These findings are consistent with the hypothesis that angiogenesis is involved in selection and growth of small follicles in gilts under the regulation of gonadotropin.

Aberrant blood flow area and plasma gonadotropin concentrations during the development of dominant-sized transitional anovulatory follicles in mares.

Color Doppler transrectal ultrasound was used to evaluate blood flow area in the wall of dominant anovulatory follicles versus ovulatory follicles in mares during the transition between anovulatory and ovulatory seasons. Daily examinations were done in 11 control mares toward the end of the anovulatory season. In 13 separate mares, follicular fluid was collected from 30-mm follicles, and blood flow areas from control mares were used as a basis for designating the sampled follicle as either anovulatory or ovulatory. Blood flow area in the controls ranged from 0.18 to 0.35 cm(2) in six mares on the day of a 30-mm anovulatory follicle and from 0.25 to 0.86 cm(2) in 11 mares on the day of a 30-mm ovulatory follicle; the ranges did not overlap except for one follicle. In the controls, mean blood flow area was lower (P < 0.05) in the anovulatory group than in the ovulatory group for each day beginning with the first Doppler examination at 25 mm. For plasma LH in controls, an effect of follicle group (P < 0.0001) and an interaction (P < 0.0001) of group by day reflected lower (P < 0.05) concentrations in the anovulatory group on Days -6, -2, and 5-8 (Day 0 = 30-mm follicle). For plasma FSH, an interaction (P < 0.0001) reflected higher (P < 0.05) concentrations in the anovulatory group on Days -3 and 1-4. More (P < 0.05) statistically identified FSH surges occurred in the anovulatory group during Days -7 to 8. In the sampled mares, follicular-fluid concentrations of estradiol, free insulin-like growth factor-1, inhibin-A, and vascular endothelial growth factor were lower (P < 0.05) in 30-mm designated anovulatory follicles than in 30-mm designated ovulatory follicles. Results were interpreted as follows: 1) The future anovulatory dominant-sized follicle developed under an LH deficiency, 2) the LH deficiency led to reductions in blood flow area and in concentrations of follicular-fluid factors, and 3) the reduction in follicle production of FSH suppressors resulted in higher plasma FSH concentrations.

Ovarian follicular development in cattle selected for twin ovulations and births.

Comparisons of numbers of antral ovarian follicles and corpora lutea (CL), of blood hormone concentrations, and of follicular fluid steroid concentrations and IGFBP activity were conducted between cows selected (twinner) and unselected (control) for twin births to elucidate genetic differences in the regulation of ovarian follicular development. Ovarian follicular development was synchronized among cows by a single i.m. injection of PGF2alpha on d 18 of the estrous cycle; six cows per population were slaughtered at 0, 24, 48, and 72 h after PGF2alpha. Jugular vein blood was collected from each animal at PGF2alpha injection and at 24-h intervals until slaughter. Ovaries of twinner cows contained more small (< or = 5 mm in diameter, P < 0.05), medium (5.1 to 9.9 mm, P < 0.05), and large (> or = 10.0 mm, P < 0.01) follicles and more (P < 0.01) CL than ovaries of controls. Follicular fluid concentrations of estradiol, androstenedione, testosterone, and progesterone reflected the stage of follicular development and were similar for twinner and control follicles at the same stage. Earlier initiation of follicular development and/or selection of twin-dominant follicles in some twinner cows resulted in greater concentrations of estradiol in plasma at 0, 24, and 48 h and of estradiol, androstenedione, and testosterone in follicular fluid of large follicles at 0 h after PGF2alpha for twinner vs. control cows (follicular status x time x population, P < 0.01). Binding activities of IGFBP-5 and -4 were absent or reduced (P < 0.01) in follicular fluid of developing medium and large estro-gen-active (estradiol:progesterone ratio > 1) follicles but increased with atresia. Only preovulatory Graafian follicles lacked IGFBP-2 binding, suggesting a possible role for IGFBP-2 in selection of the dominant follicle. Concentrations of IGF-I were twofold greater (P < 0.01), but GH (P = 0.10) and cholesterol (P < 0.05) were less in blood of twinners. Three generations of selection of cattle for twin ovulations and births enhanced ovarian follicular development as manifested by increased numbers of follicles within a follicular wave and subsequent selection of twin dominant follicles. Because gonadotropin secretion and ovarian steroidogenesis were similar for control and twinner cattle, enhanced follicular development in twinners may result from decreased inhibition by the dominant follicle(s), increased ovarian sensitivity to gonadotropins, and/or increased intragonadal stimulation, possibly by increased IGF-I.

Correlations between steroids concentration in follicular fluid, pronuclear morphology and embryo qualities in in vitro fertilization.

Background: Several parameters of early embryo development are known as predictors of implantation success. Recently, zygote or embryo morphological assessments are thought to be a major method of selection in embryo transfer. We expected that the concentrations of the steroids in follicular fluid (FF) were associated with oocyte maturation and embryo quality. In the present paper, we evaluated the relationship of several parameters. Methods: We investigated 105 samples of FF from 22 subjects by in vitro fertilization (IVF). We evaluated the correlations between the FF concentrations of estradiol (E2) and progesterone (P4), the diameter of the ovarian follicles, fertilization, and zygote assessment based on pronuclear morphology and day 3 embryo qualities (i.e. number of blastomeres and fragmentation rate). Results: There was a positive correlation between the E2 concentrations in FF and serum (r=0.273, P<0.01), but there was no correlation between follicular diameter and the FF concentration of each steroid. The concentration of E2 in FF containing fertilized oocytes was not significantly different from that in FF containing unfertilized oocytes. At the pronuclear stage, the concentration of either steroid in FF did not differ among the morphological groups. The concentration of P4 in FF was significantly lower in the group in which pronuclei were detected at 20h after insemination than in the group in which pronuclei were not detected. The concentration of E2 in FF was significantly related to the number of blastomeres (r=0.271, P<0.05) and furthermore, was significantly higher in FF from which morphologically good embryos were obtained at day 3 (P<0.05). Conclusions: The FF concentrations of the steroids did not affect the pronuclear pattern, but P4 production may play a role in reducing the potential of the oocyte to develop pronuclei and the concentration of E2 may predict the cleavage capability of the oocyte. (Reprod Med Biol 2003; 2: 171-176).

Parturition body size and body protein loss during lactation influence performance during lactation and ovarian function at weaning in first-parity sows.

We investigated the effect of body protein mass at parturition and different degrees of body protein loss in lactation on sow performance. In a 2 x 2 factorial arrangement, 77 Genex gilts were fed to achieve either a standard or high body mass at parturition and to lose either a moderate (MPL) or high (HPL) amount of protein in lactation. Pregnant gilts were fed either 24.4 MJ of ME, 266 g of CP, and 11 g of lysine/d or 34.0 MJ of ME, 436 g of CP, and 20 g of lysine/d resulting in divergent (P < 0.01) live weights (165 vs. 193 kg) and calculated protein masses (24.3 vs. 30.0 kg) and slightly different backfat depths (20.0 vs. 22.8 mm; P < 0.05) at parturition. Diets fed during lactation were formulated to deliver 731 g of CP and 37 g of lysine/d or 416 g of CP and 22 g of lysine/d to induce differential body protein mobilization. Sows were slaughtered at weaning (d 26), and the weight of the organs and the lean, fat, and bone in five primal cuts was measured. The external diameter of the eight largest follicles on each ovary was recorded, and the follicular fluid from these follicles was collected, weighed, and analyzed for estradiol. Losses in lactational live weight (26 vs. 20 kg; P < 0.01) and calculated protein mass (17.8 vs. 10.7%; P < 0.001) were greater, and the carcass lean mass at weaning was 10% lighter (P < 0.05) in HPL sows. Backfat (5.1 +/- 0.8 mm; P = 0.29) and calculated fat mass (25.8 +/- 1.5%; P = 0.84) losses did not differ between treatments. Both sow body mass (P < 0.05) and lactation protein loss (P < 0.01) affected litter growth rate. Litter growth rate decreased (P < 0.05) at the end of lactation in HPL sows once these sows had lost 10 to 12% of their calculated protein mass. Ovarian follicular development was most advanced in high body mass sows that lost the least protein; these sows had the heaviest (P < 0.05) uterine weight and highest (P < 0.05) follicular fluid estradiol concentration. Follicular development was least advanced in standard body mass sows that lost the most protein. These sows had the lowest (P < 0.05) muscle:bone ratio at weaning and likely lost the largest proportion of their muscle mass compared with the other treatments. In conclusion, ovarian function at weaning and litter performance was higher in high body mass sows and in sows that lost the least protein in lactation, suggesting that a larger lean mass may delay the onset of a decrease in performance in sows that lose protein in lactation.