Follicular Fluid Androstenedione Research Articles

Sheep with ovarian androgen excess have fibrosis and follicular arrest with increased mRNA abundance for steroidogenic enzymes and gonadotropin receptors.

An androgen excess ovarian micro-environment may limit follicle progression in sheep. Two populations of ewes with divergent follicular fluid androstenedione (A4) were identified in a flock in Jordan: High A4; (A4) ≥ 30 ng/mL, (N = 12) or Control A4 (Control); A4 ≤ 15 ng/mL; (N = 12). We hypothesized High A4 ewes would have increased steroidogenic enzyme mRNA abundance, inflammation, and follicular arrest. Messenger RNA abundance for steroidogenic enzymes StAR, CYP17A1, CYP11A1, and HSD3B1 were increased in theca cells while CYP17A1, CYP19A1, and HSD3B1 were increased in granulosa cells in High A4 ewes compared to Control. Gonadotropin receptor mRNA expression for LHCGR was increased in theca and FSHR in granulosa in High A4 ewes. Messenger RNA expression of FOS when reduced, increases expression of CYP17A1 which was observed in High A4 granulosa cells compared to Control. Furthermore, High A4 ewes had greater numbers of primordial follicles (P < 0.001) and fewer developing follicles compared to Control before, and after 7 d of culture, indicating follicular arrest was not alleviated by cortex culture. Increased fibrosis in the ovarian cortex was detected in High A4 ewes relative to Control (P < 0.001) suggesting increased inflammation and altered extracellular matrix deposition. Thus, this High A4 ewes population has similar characteristics to High A4 cows and women with polycystic ovary syndrome suggesting that naturally occurring androgen excess occurs in multiple species and may be a causative factor in follicular arrest and subsequent female sub- or infertility.

A high-androgen microenvironment inhibits granulosa cell proliferation and alters cell identity

A naturally occurring bovine model with excess follicular fluid androstenedione (High A4), reduced fertility, and polycystic ovary syndrome (PCOS)-like characteristics has been identified. We hypothesized High A4 granulosa cells (GCs) would exhibit altered cell proliferation and/or steroidogenesis. Microarrays of Control and High A4 GCs combined with Ingenuity Pathway Analysis indicated that High A4 GCs had cell cycle inhibition and increased expression of microRNAs that inhibit cell cycle genes. Granulosa cell culture confirmed that A4 treatment decreased GC proliferation, increased anti-Müllerian hormone, and increased mRNA for CTNNBIP1. Increased CTNNBIP1 prevents CTNNB1 from interacting with members of the WNT signaling pathway thereby inhibiting the cell cycle. Expression of CYP17A1 was upregulated in High A4 GCs presumably due to reduced FOS mRNA expression compared to Control granulosa cells. Furthermore, comparisons of High A4 GC with thecal and luteal cell transcriptomes indicated an altered cellular identity and function contributing to a PCOS-like phenotype.

The endocrine milieu in naturally matured follicles is different in women with high serum anti-Müllerian hormone concentrations

Research questionIs the endocrine milieu different in women with low serum anti-Müllerian hormone (AMH) concentration compared with women with high concentration? DesignCohort study of 84 women (four groups) classified according to AMH concentration and age undergoing natural cycle IVF treatment. Concentrations of LH, oestradiol, testosterone, androstenedione and AMH were determined in follicular fluid (FF), associations analysed and clinical outcome parameters evaluated. ResultsA positive correlation between serum and FF AMH concentrations was confirmed. Follicular fluid androstenedione concentration was positively correlated with serum AMH concentration (P < 0.0001, r2 = 0.197). The correlation between FF LH and FF testosterone concentration in all women was not significant (P = 0.050, r2 = 0.046); however, the correlation between FF androstenedione in women with high serum AMH concentration was significant (P = 0.032, r2 = 0.220). Follicular fluid testosterone and androstenedione were positively correlated with FF oestradiol overall and in some individual groups. The high serum AMH concentration group showed the highest FF AMH and androstenedione concentrations and lowest oestradiol–testosterone and oestradiol–androstenedione ratios. High FF AMH concentration was associated with a higher clinical pregnancy rate and high FF oestradiol concentration with a slightly better embryo quality. ConclusionsDifferences in the endocrine milieu in women with high serum AMH concentration seem to be caused by increased follicular LH concentration. In women with high serum AMH concentration, FF androstenedione is increased and ratios of oestradiol–testosterone and oestradiol–androstenedione are decreased, suggesting a disturbed endocrine milieu caused by reduced metabolization of FF androgens into oestrogens. In natural cycles, FF AMH concentrations are positively associated with higher clinical pregnancy rates and oestradiol concentrations with a higher embryo score.

Metabolic and Endocrine Differences Between Dairy Cows That Do or Do Not Ovulate First Postpartum Dominant Follicles.

Most dairy cows develop the first dominant follicle postpartum within 2 wk after calving, but only about 40% of these follicles produce sufficient estradiol to stimulate ovulation despite having normal ultrasound appearance and growth. This study aimed to characterize metabolic, endocrine, and follicular fluid profiles of cows in which the first dominant follicle postpartum will become ovulatory and those with nonovulatory follicles. Luteinizing hormone pulse frequency, follicular fluid androstenedione, and follicular fluid estradiol concentrations were lower in nonovulatory cows suggesting that the function of theca cells is impaired. In addition, nonovulatory cows had more severe negative energy balance and greater insulin resistance postpartum. This study describes for the first time the steroid hormone profile of early postpartum follicles and shows that a steroidogenic defect most likely occurs in theca cells limiting the amount of androgen precursor available for estradiol production that impairs their ovulatory outcome.

Evidence That Increased Ovarian Aromatase Activity and Expression Account for Higher Estradiol Levels in African American Compared With Caucasian Women

Serum estradiol levels are significantly higher across the menstrual cycle in African American (AAW) compared with Caucasian women (CW) in the presence of similar FSH levels, yet the mechanism underlying this disparity is unknown. The objective of the study was to determine whether higher estradiol levels in AAW are due to increased granulosa cell aromatase mRNA expression and activity. The design of the study included daily blood sampling and dominant follicle aspirations at an academic medical center during a natural menstrual cycle. Healthy, normal cycling AAW (n = 15) and CW (n = 14) aged 19-34 years participated in the study. Hormone levels in peripheral blood and follicular fluid (FF) aspirates and aromatase and FSH receptor mRNA expression in granulosa cells were measured. AAW had higher FF estradiol [1713.0 (1144.5-2032.5) vs 994.5 (647.3-1426.5) ng/mL; median (interquartile range); P < .001] and estrone [76.9 (36.6-173.4) vs 28.8 (22.5-42.1) ng/mL; P < .001] levels than CW, independent of follicle size. AAW also had lower FF androstenedione to estrone (7 ± 1.8 vs 15.8 ± 4.1; mean ± SE; P = .04) and T to estradiol (0.01 ± 0.002 vs 0.02 ± 0.005; P = .03) ratios, indicating enhanced ovarian aromatase activity. There was a 5-fold increase in granulosa cell aromatase mRNA expression in AAW compared with CW (P < .001) with no difference in expression of FSH receptor. FSH, inhibin A, inhibin B, and AMH levels were not different in AAW and CW. Increased ovarian aromatase mRNA expression, higher FF estradiol levels, and decreased FF androgen to estrogen ratios in AAW compared with CW provide compelling evidence that racial differences in ovarian aromatase activity contribute to higher levels of estradiol in AAW across the menstrual cycle. The absence of differences in FSH, FSH receptor expression, and AMH suggest that population-specific genetic variation in CYP19, the gene encoding aromatase, or in factors affecting its expression should be sought.

Females with Reduced Fertility Have Excess Androstenedione in Follicular Fluid, Altered Theca Gene Expression and Increased VEGFA164b, Maternal Effect, and microRNA Processing mRNA Levels in Cumulus-Oocyte Complexes.

Ovarian dysfunction contributes significantly to female infertility. However, the intrinsic and exogenous factors that result in abnormal ovarian function are poorly defined. Thus, we have established a cow model of fertility to identify mechanisms regulating follicular growth, steroidogenesis and oocyte growth and maturation. Culling age due to failure to establish pregnancy was used to classify animals with low (>/= 2 years-of-age), moderate (> 2 - < 6 years-of-age), and high (>/= 6 years-of-age) fertility. Previous experiments using this model identified class-dependent changes in mural granulosa cell gene expression. The objective of this study was to identify the effect of ovarian and follicular environment on the oocyte molecular phenotype. To attain this objective, females from each fertility group were synchronized using a modified Co-Sync (CIDR) protocol and ovariectomies were performed 36 h after PGF2-alpha injection and CIDR removal. Surface antral follicle counts (AFC) for each ovary pair and the follicular fluid, theca cells, mural granulosa cells and cumulus-oocyte complex (COC) from each dominant follicle were collected. Dominant follicles were confirmed based on size (i.e. largest follicle on the ovaries) and an estrogen- to-progesterone ratio greater than 1.0 in the follicular fluid. Antral follicle counts were lower in low fertility (n = 14) than moderate (n = 17) or high fertility (n = 17) cows. Furthermore, androstenedione (A4) concentrations were 4 fold (P < 0.002) greater in follicular fluid from low compared to high fertility group cows. Given that increased androgens is a hallmark characteristic of the PCOS phenotype, gene expression in theca cells from the different fertility groups was compared. The affect of increased intrafollicular androgen levels on cumulus-oocyte gene expression in the dominant follicle was also examined. Quantitative, real-time RT-PCR demonstrated that mRNA abundance of maternal effect genes NLRP5 (8-fold P < 0.0001), Zar1 (2-fold; P < 0.0001) and Dnmt1 (P < 0.006) and miRNA processing genes DGCR8 (P < 0.005) and RNAsen (P < 0.005) were increased in low compared to high fertility cows. Increased mRNA abundance in low fertility animals is consistent with previous findings in PCOS oocytes and suggests that abnormal endocrine profiles of follicular fluid has a global effect on transcriptional and post-transcriptional regulation of mRNAs in the oocyte. Previous studies identified differences in the expression profile of vascular endothelial growth factor A (VEGFA) isoforms in mural granulosa cells between low, moderate, and high fertility group cows. In the current study, VEGFA_164B mRNA abundance was also increased in cumulus granulosa cells of low fertility cows (P < 0.005). Taken together, these data indicate that females with low fertility have intrinsic differences in ovarian morphology and steroidogenesis. Furthermore, the data suggest that increased androgen production in low fertility animals alters gene expression and/or mRNA stability during oocyte growth and maturation. These changes in oocyte gene expression may reduce the developmental competence of the oocyte and contribute to the reduced pregnancy rates in low fertility cows. USDA is an equal opportunity employer. (poster)

New insights into the pathogenesis of cystic follicles in cattle: Microarray analysis of gene expression in granulosa cells1

Ovarian follicular growth and development are regulated by extraovarian and intraovarian factors, which influence granulosa cell proliferation and differentiation. However, the molecular mechanisms that drive follicular growth are not completely understood. Ovarian follicular cysts are one of the most common causes of reproductive failure in dairy cattle. Nevertheless, the primary cause of cyst formation has not been clearly established. A gene expression comparison may aid in elucidating the causes of ovarian cyst disease. Our objective was to identify differentially expressed genes in ovarian granulosa cells between normal dominant and cystic follicles of cattle. Granulosa cells and follicular fluid were isolated from dominant and cystic follicles collected via either ultrasound-guided aspiration from dairy cows (n = 24) or slaughterhouse ovaries from beef cows (n = 23). Hormonal analysis for progesterone, estradiol, and androstenedione in follicular fluid was performed by RIA. Total RNA was extracted and hybridized to 6 Affymetrix GeneChip Bovine Genome Arrays (Affymetrix, Santa Clara, CA). Abundance of mRNA for differentially expressed selected genes was determined through quantitative real-time reverse-transcription PCR. Follicular cysts showed greater (P < 0.05) progesterone, lesser (P < 0.05) estradiol, and no differences (P > 0.10) in androstenedione concentrations compared with noncystic follicles. A total of 163 gene sequences were differentially expressed (P < 0.01), with 19 upregulated and 144 downregulated. From selected target genes, quantitative real-time reverse-transcription PCR confirmed angiogenin, PGE(2) receptor 4, and G-protein coupled receptor 34 genes as upregulated in cystic follicles, and Indian hedgehog protein precursor and secreted frizzled-related protein 4 genes as downregulated in cystic follicles. Further research is required to elucidate the role of these factors in follicular development and cyst formation.

Effect of days after calving on insulin-like growth factor-I, insulin-like growth factor binding proteins, progesterone, androstenedione, estradiol, and aromatase mRNA in dominant follicles of postpartum beef cows

The effect of days after calving on IGF-I, IGFBP, progesterone, androstenedione, estradiol, and aromatase mRNA in dominant ovarian follicles (DF) was evaluated in Angus × Hereford cows. Growth of DF (>9 mm) was monitored daily by ultrasonography and fluid from DF was collected in vivo at either 30 ± 2 d or 47 ± 2 d postpartum. Follicular fluid (FF) was also aspirated from DF of contemporary ovulatory cows at proestrus. Estrous behavior was monitored continuously using the HeatWatch system, and progesterone in plasma collected twice weekly was used to assess luteal activity. Anovulatory DF aspirated 30 and 47 d postpartum had similar concentrations of IGF-I, IGFBP, progesterone, estradiol and androstenedione in FF and IGF-I and IGFBP in plasma. The intervals from aspiration to estrus were similar for cows aspirated 30 and 47 d postpartum. Proestrous follicles had greater ( P < 0.01) estradiol (435 ± 79 ng/mL) than DF at 30 d (107 ± 63 ng/mL) or 47 d (68 ± 53 ng/mL) after calving. Concentrations of androstenedione in FF were also greater ( P < 0.01) in proestrous follicles than in DF aspirated at 30 or 47 d after calving. Concentrations of insulin-like growth factor-1 (IGF-I) and insulin-like growth factor binding proteins (IGFBP) in FF and plasma, and aromatase mRNA in granulosa cells were similar for anovulatory and proestrous cows. In conclusion, estradiol production by DF of postpartum anovulatory cows may be limited by inadequate production of androstenedione during the postpartum anovulatory interval and this may influence follicular maturation. Concentrations of IGF-I and IGFBP were similar in anovulatory and proestrous cows, an indication that alterations in the IGF-I system in the DF at 30–47 d after calving are not associated with delayed follicular development in postpartum beef cows.

Gonadotropin-releasing hormone antagonists increase follicular fluid insulin-like growth factor-I and vascular endothelial growth factor during ovarian stimulation cycles

The aim of the present study was to investigate the effect of gonadotropin-releasing hormone (GnRH) antagonists (GnRH-ant) on follicular fluid (FF) insulin-like growth factor-I (IGF-I) and FF vascular endothelial growth factor (VEGF) levels. Sixty women undergoing assisted reproduction were randomized and assigned to two different GnRH analog regimens: GnRH agonist (GnRH-a) and GnRH-ant. FF VEGF and FF IGF-I concentrations were significantly increased in the patients treated with GnRH-ant (p < 0.001). In the same patients we observed a statistically significant reduction in serum luteinizing hormone (LH) and estradiol (E2) levels (p < 0.001 and p < 0.05, respectively), FF E2 and FF androstenedione levels (p < 0.05 and p < 0.001, respectively), as well as a reduction in the number of pregnancies although this was not statistically significant. In the GnRH-ant group, FF VEGF levels were positively correlated with FF IGF-I levels, and both were negatively correlated with serum LH levels. The increase in FF IGF-I and FF VEGF levels in women treated with GnRH-ant could be explained by a deleterious follicular environment in response to profound suppression of LH and E2 levels.

Concentrations of gonadotrophins and steroids in pre-ovulatory follicular fluid and serum in relation to stimulation protocol and outcome of assisted reproduction treatment

In this prospective, randomized study, concentrations of gonadotrophins and steroids in pre-ovulatory follicular fluid (FF) and serum were related to type of stimulation protocol as well as to the outcome of assisted reproduction in 280 women subjected to the long protocol gonadotrophin-releasing hormone (GnRH) agonist pituitary down-regulation and ovarian stimulation with either human menopausal gonadotrophin (HMG) or recombinant FSH. In the women treated with HMG, concentrations of LH, FSH, oestradiol and androstenedione in FF were significantly higher, and those of human chorionic gonadotrophin (HCG) and progesterone significantly lower, than in the women treated with recombinant FSH (rFSH). More women became pregnant and delivered in the HMG than in the rFSH group. These differences, however, were not statistically significant. Concentrations of FSH in serum and of FSH and LH in FF were significantly higher in conception than in non-conception cycles, whereas all other hormone concentrations in FF and serum were similar. The present study demonstrates that the pre-ovulatory follicular fluid hormone profile is significantly influenced by the gonadotrophin preparation used for ovarian stimulation, and suggests that ovarian stimulation with HMG results in an intra-follicular hormone profile more similar to that characterizing conception cycles than stimulation with rFSH. However, as the present data represent means of FF hormone profiles, they do not allow the conclusion of a direct correlation between the intra-follicular concentration of a certain hormone and the ability of the corresponding embryo to implant and establish an ongoing pregnancy.

Overexpression of theca-cell messenger RNA in polycystic ovary syndrome does not correlate with polymorphisms in the cholesterol side-chain cleavage and 17α-hydroxylase/C 17-20 lyase promoters

Objective: To determine whether overexpression of CYP17 or CYP11A messenger (m)RNA in theca cells from polycystic ovaries is related to polymorphic regions in the gene promoters that may increase transcription. Design: Case-control study. Setting: Research institute. Patient(s): Fifty-one women with PCOS and 280 regularly cycling controls underwent genotyping. Thecal cells were obtained from 23 women with PCOS and 51 controls. Main Outcome Measure(s): Ovarian tissue was obtained from women with PCOS undergoing wedge resection for treatment of their infertility and from controls undergoing ovariectomy for indications unrelated to the study. Expression of mRNA in theca cells was measured by using competitive reverse transcriptase polymerase chain reaction. Genotype analysis for polymorphisms in the CYP11A and CYP17 promoters was performed by using polymerase chain reaction. Result(s): Although expression of CYP11A and CYP17 mRNA was higher in women with PCOS, no significant dose effects of CYP11A or CYP17 alleles were observed with respect to serum testosterone; follicular fluid androstenedione, estradiol, and androstenedione-to-estradiol ratio; or CYP11A or CYP17 mRNA expression. Conclusion(s): Overexpression of CYP17 and CYP11A mRNA in theca cells from polycystic ovaries is explained by polymorphic differences in the gene promoters.

In vitro fertilization following laparoscopic ovarian diathermy in patients with polycystic ovarian syndrome.

To evaluate the in vitro fertilization outcome in patients with polycystic ovarian syndrome previously treated by laparoscopic ovarian diathermy. We designed a prospective study to compare in vitro fertilization stimulation parameters and pregnancy rate for two groups of women with polycystic ovarian syndrome. In the first group, we included 23 patients previously treated by laparoscopic ovarian electrodiathermy (group A), in the second group we included 36 women who did not undergo surgical treatment (group B). All patients underwent the same ovarian hyperstimulation protocol and a standard in vitro fertilization and embryo transfer technique. In a limited number of women (five in group A and eight in group B) we measured the levels of sex steroids in follicular fluid. Statistical analysis was performed by means of chi-square test or Fisher's exact test to compare frequencies and by means of the Student's t-test or ANOVA test to compare means. Significance was set at p < 0.05. In group A we found a significantly higher ongoing pregnancy rate (28.6% in group A vs 7.3% in group B), a significantly lower estradiol peak level (1722.73 +/- 522.24 in group A vs 2314.48 +/- 692.60 in group B), a trend for lower abortion rates and an improvement in the number of patients administered human chorionic gonadotropin than in group B. The levels of estradiol, testosterone and androstenedione in follicular fluid were significantly lower from small to intermediate and to large follicles in group A, while in group B only estradiol and androstenedione were different. Lower levels of androstenedione and testosterone were found in each follicular pool of group A, compared to the respective pool in group B. We report a significantly better ongoing pregnancy rate after in vitro fertilization and embryo transfer procedures in women pretreated by means of laparoscopic ovarian electrocautery. These results may be related to lower androgen levels in the follicular fluid.

Seasonal and acute heat stress effects on steroid production by dominant follicles in cows.

The present study concerned the seasonal and acute effects of heat stress on steroid concentrations in follicular fluid and on steroid production by granulosa and theca interna cells, in bovine dominant follicles. Three groups of cows were studied: summer (n = 5), autumn (n = 5) and winter (n = 9) cows. During the winter season, another group of cows was acutely heat-stressed from days 3 through 5 of the estrous cycle (n = 5). On day 7 of the estrous cycle, follicular fluid from first-wave dominant follicles was aspirated, and dispersed granulosa and theca cells from each seasonal group were incubated for 18 h at normothermic (37.5 degrees C) or high (40.5 degrees C) temperatures. Cells were incubated in media only or in media containing testosterone (300 ng ml-1, for granulosa cells) or forskolin (4 micrograms ml-1, for theca cells). In follicular fluid the 17 beta-estradiol concentration was high (P < 0.05) in winter and low in autumn, and summer, the androstenedione concentration was high in summer (P < 0.05), low in autumn, and intermediate in winter. During the winter season, acute in vivo heat stress increased follicular fluid androstenedione and decreased estradiol to levels comparable with those prevailing in summer. Basal and forskolin-stimulated androstenedione production by theca cells was higher (P < 0.05) in the winter group than in the summer and autumn groups, and also higher than in the cows that were heat-stressed during winter, which suggests that theca cell function is susceptible to chronic (summer), short-term (winter) and delayed (autumn) heat stresses. In vitro incubation at high temperature (40.5 degrees C) reduced the high, forskolin-stimulated androstenedione production in winter (P < 0.05). Estradiol production by granulosa cells was high in winter and autumn, and low in summer (P < 0.05). Acute heat stress in winter did not alter estradiol production relative to winter controls, whereas a high incubation temperature (40.5 degrees C) reduced (P < 0.05) estradiol production only in the autumn, when the highest production rate was recorded. The results indicate a differential effect of heat stress on the functions of granulosa and theca cells. Both concurrent and delayed effects of heat stress on the steroidogenic capacity of ovarian follicles in cattle are presented.

Recombinant human follicle-stimulating hormone stimulates multiple follicular growth, but minimal estrogen production in gonadotropin-releasing hormone antagonist-treated monkeys: examining the role of luteinizing hormone in follicular development and steroidogenesis.

The two-cell theory predicts that follicular steroidogenesis requires the coordinate actions of both FSH and LH; however, the role of LH in follicular growth is less clear. The present study was designed to investigate the relative importance of LH and FSH in follicular growth and steroidogenesis. Cynomolgus monkeys were treated with a GnRH antagonist (antide; 3 mg/kg.day) for 20 days beginning in the midluteal phase of the menstrual cycle. After 10 days of antide administration, monkeys were injected with recombinant human FSH (rhFSH; 10 IU; n = 3), human menopausal gonadotropin (hMG; 10 IU; n = 3), or FSH plus 0.5 IU LH (n = 3) twice daily for 10 days. rhFSH stimulated multiple follicular development; however, peak serum estradiol levels were only 943 +/- 195 pmol/L. In contrast, monkeys treated with the same dose of hMG had significantly higher (P < 0.05) peak estradiol levels (6013 +/- 1322 pmol/L). The addition of 0.5 IU LH to the rhFSH treatment resulted in serum estradiol levels similar to those in monkeys treated with rhFSH only. Importantly, no differences in follicle number or size were evident among these treatment groups. Follicular fluid estradiol levels were consistent with serum levels (rhFSH, 187 +/- 11 nmol/L; hMG, 1531 +/- 173 nmol/L). Even larger proportional differences in follicular fluid androstenedione (rhFSH 13.6 +/- 1.4 nmol/L; hMG, 307 +/- 97.7 nmol/L) levels were found. The results in this LH-deficient primate model suggest that FSH alone is capable of stimulating ovarian follicular growth; however, the resulting follicles manifest minimal estradiol production, probably due to deficiencies in the LH-induced precursors to estradiol.

Effects of growth hormone administration in addition to gonadotrophins in normally ovulating women and polycystic ovary syndrome (PCO) patients.

Follicular fluid sex-steroids, insulin-like growth factor-I (IGF-I), IGF-I binding protein (IGF-I-BP) and epidermal growth factor were investigated in patients with polycystic ovaries and normally ovulating women, following ovulation induction with gonadotrophins or growth hormone plus gonadotrophins. Growth hormone supplementation enhanced the ovarian response to gonadotrophins, and significantly increased follicular fluid IGF-I in both groups, without affecting follicular fluid epidermal growth factor; growth hormone supplementation significantly decreased follicular fluid androstenedione in both groups.

Follicular fluid steroid and epidermal growth factor content, and in vitro estrogen release by granulosa-luteal cells from patients with polycystic ovaries in an IVF/ET program

The follicular fluid (FF) content of androgens, estrogens and epidermal growth factor (EGF) has been evaluated in a group of patients with policystic ovary disease (PCO) and in one of normally-ovulating infertile women (NOW) in an IVF/ET program. The in vitro response to follicle-stimulating hormone (FSH) has been also evaluated in granulosa luteal cells from the same patients. PCO patients showed significantly higher FF androstenedione ( Δ 4) and testosterone (T) and similar FF estrone (E 1) and 17β-estradiol (E 2) levels compared to controls. In vitro production of E 1 and E 2 by granulosa luteal cells from PCO patients and from controls were overlapping and their response to FSH was similar. These data indicate a normal intrinsic potential aromatase activity in ovaries from PCO patients stimulated with gonadotropins and suggest that PCOs do not derive from inherent ovarian aromatase deficiency. Increased FF androgen content following gonadotropin stimulation may result from theca cell hyperactivity and androgen accumulation in the follicular antrum of rescued hyperandrogenic follicles as well as from inhibitory factors that may inhibit aromatase activation in vivo, partially counteracting the effect of gonadotropins. FF EGF levels were significantly higher in the group of PCO patients compared to those of NOW. EGF may play a role in blunting the in vivo response of granulosa cells to gonadotropins.

Diet-Induced Hyperlipidemia in Cattle Modifies the Intrafollicular Cholesterol Environment, Modulates Ovarian Follicular Dynamics, and Hastens the Onset of Postpartum Luteal Activity1

Three experiments were conducted with female cattle during the postpartum period and during the estrous cycle to examine the effects of diet-induced hyperlipidemia on lipoprotein-cholesterol and triglyceride (TG) metabolism, ovarian follicular dynamics, and incidence of postpartum luteal activity. Dietary-lipid effects were examined independently of metabolizable energy intake. Feeding a high-lipid (HL) diet (8% total lipid) for the first 3 wk of the puerperium (Experiment 1) increased (p less than 0.0001) the concentration of total cholesterol and high-density lipoprotein-cholesterol (HDL-CH), but not TG, within follicular fluid (FF) of estrogen-active (E-A) and inactive (E-I) follicles. Increases (1.4- to 1.7-fold) were similar in proportion to those observed in peripheral blood serum, but absolute concentrations were about 45% of that in serum. Greater than 95% of the cholesterol in FF was HDL-CH, with a greater (p less than 0.06) proportion of HDL-CH sequestered by E-A compared to E-I follicles. The HL diet increased (p less than 0.06) the number of medium-sized (3.1-9.9 mm) follicles present at ovariectomy 19-21 days postcalving and increased (p less than 0.03) concentrations of FF androstenedione in E-I follicles 5-fold. Granulosa cells from preovulatory follicles (Experiment 2) of heifers fed HL diets secreted 2.1- to 3.5-fold greater (p less than 0.03) quantities of pregnenolone and progesterone in vitro. Finally, feeding HL supplements to postpartum range cattle for 30 days increased (p less than 0.05) the incidence of ovarian luteal activity by 18% (Experiment 3). Shifts in lipid metabolic status modify reproductive potential in cattle, independently of dietary energy intake.

Concentrations of insulin-like growth factor-I in follicular fluid and blood plasma of mares during early and late oestrus

To assess the relationship (s) among concentrations of insulin-like growth factor-I (IGF-I) in blood plasma and ovarian follicular fluid (FFL), and concentrations of steroids in FFL of individual preovulatory equine follicles, FFL and blood plasma were collected simultaneously from mares between Days 1 and 6 of oestrus. Follicles were placed into two groups: early oestrus (EE) (Day 1 or 2 of oestrus, n = 12) and late oestrus (LE) (Day 3–6 of oestrus, n = 10). LE follicles were larger and had greater ( P < 0.05) concentrations of oestradiol, progesterone and testosterone than EE follicles. Androstenedione and IGF-I did not differ between EE and LE follicles. Concentrations of IGF-I were greater in plasma than FFL (579 vs. 429 ng ml −1) and did not differ in plasma of EE and LE mares. The ratio of FFL: plasma IGF-I tended ( P < 0.15) to be greater in LE than EE follicles (86 vs. 69%). IGF-I levels in FFL correlated with follicular size ( r = 0.51, P < 0.02) but were not correlated ( P > 0.10) to any steroid in FFL. However, the ratio of FFL: plasma IGF-I was correlated with FFL oestradiol ( r = 0.75, P < 0.001) and FFL androstenedione ( r = 0.43, P < 0.05). These results support the hypothesis that changing permeability of follicles to plasma IGF-I may allow for increased IGF-I levels in follicles which, in turn, may enhance follicular differentiation.

Lack of effect of high-dose antioestrogen on the maturation and in-vitro fertilization of human oocytes.

Thirty-four women requesting laparoscopic sterilization underwent a fixed schedule regimen for multiple follicular development which included norethisterone and clomiphene citrate. Follicle aspiration for oocyte recovery was attempted laparoscopically 34 h after administration of 5000 IU human chorionic gonadotrophin (HCG). Nineteen women were given 80 mg tamoxifen orally 4 h prior to HCG injection, while 15 acted as controls. There was no statistical difference in fertilization rates in vitro between tamoxifen-treated patients and controls (80 and 68% respectively). In addition, the morphological characteristics of the oocytes, the rates of cleavage, and the concentrations of oestradiol, progesterone and androstenedione in follicular fluid were similar in the two groups. Tamoxifen was detected in substantial amounts in follicular fluids of patients given tamoxifen. These results suggest that high-dose tamoxifen, in clinically used doses, does not adversely affect the final stages of maturation or the fertilization and early cleavage of human oocytes.

Relationship between Concentrations of Immunoreactive Insulin-Like Growth Factor-I in Follicular Fluid and Various Biochemical Markers of Differentiation in Bovine Antral Follicles

Three experiments were conducted to determine the relationship between concentrations of insulin-like growth factor-I (IGF-I) in ovarian follicular fluid and various biochemical markers of follicular differentiation in bovine follicles. In Experiment I, ovaries were removed on Days 7, 14, 28, 42, or 56 after parturition from a total of 21 cows. In Experiment II, ovaries of 31 cows were removed between Days 20 and 30 postpartum after 48 or 96 h of either saline (0.9% NaCl, 5 ml) or luteinizing hormone-releasing hormone (LHRH, 500 ng/5 ml saline) injections given every 2 h via jugular cannulae. In Experiment III, ovaries of six cows were removed 48-50 h after a 35-mg injection of prostaglandin F2 alpha during the midluteal phase of an estrous cycle. In Experiments I and II, all follicles greater than or equal to 8.0 mm in diameter were removed from each ovary (n = 33 and 46, respectively). In Experiment III, fluid from all follicles greater than 4 mm in diameter were removed individually (n = 10), and fluid from follicles 1-4 mm in diameter were pooled for each cow. Follicles for each experiment were further categorized as either estrogen-active (E-A, concentration of estradiol greater than progesterone in follicular fluid) or estrogen-inactive (E-I, concentration of progesterone greater than estradiol in follicular fluid). Measurements of immunoreactive IGF-I (i-IGF-I) were made after separating IGFs from their binding proteins with an acid-ethanol extraction.(ABSTRACT TRUNCATED AT 250 WORDS)