Effects of Multivitamin/Mineral Supplementation on Trace Element Levels in Serum and Follicular Fluid of Women Undergoing in Vitro Fertilization (IVF)

We investigated effects of multivitamin/mineral supplementation on element levels in serum and follicular fluid of women undergoing IVF. We used three groups in this study. The first group was used as an age-matched and nonpregnant control (n = 13). Group 2 (n = 30) constituted the IVF group and women in the third group who were undergoing IVF also received a multivitamin/mineral tablet daily for 45 days. Follicular fluid and serum selenium and zinc levels and follicular fluid copper levels were lower in IVF patients than in controls although follicular fluid aluminum and iron levels were higher in IVF patients than in controls. However, follicular fluid and serum aluminum, copper, zinc and selenium levels, and serum magnesium levels were higher in the multivitamin/mineral group than in the IVF group although follicular fluid iron levels were lower in the multivitamin/mineral group than in the IVF group. In conclusion, we observed that copper, zinc, and selenium in serum and follicular fluid decreased in women undergoing IVF. Multivitamin/mineral supplementation in serum and follicular fluid of women undergoing IVF normalized the trace element levels.     

Localization and hormonal regulation of ovarian production of histamine in the sheep

Concentrations of histamine were measured within the follicular wall, follicular fluid and ovarian interstitium throughout the periovulatory period in sheep. Histamine within follicular tissue declined after the onset of the preovulatory surge of luteinizing hormone (LH) and remained low until after ovulation, when levels then increased markedly. Alterations in histamine within the follicular wall were not reflected by corresponding changes within follicular fluid or ovarian interstitium. Release of histamine from tissue during short-term incubation was greatest for follicles obtained after ovulation, which was not influenced by presence of LH in the incubation medium. Luteinizing hormone caused depletion of stores of histamine from the wall of follicles collected before the preovulatory surge of LH. Histamine could act as a paracrine mediator in the follicular mechanisms of ovulation and(or) luteinization.     

Cell-secreted vesicles in equine ovarian follicular fluid contain miRNAs and proteins: a possible new form of cell communication within the ovarian follicle

Proper cell communication within the ovarian follicle is critical for the growth and maturation of a healthy oocyte that can be fertilized and develop into an embryo. Cell communication within the follicle involves many signaling molecules and is affected by maternal age. Recent studies indicate that cell communication can be mediated through secretion and uptake of small membrane-enclosed vesicles. The goals of this study were to 1) identify cell-secreted vesicles (microvesicles and exosomes) containing miRNAs and proteins within ovarian follicular fluid and 2) determine if miRNA level differs in exosomes isolated from follicular fluid in young compared to old mares. We demonstrate the presence of vesicles resembling microvesicles and exosomes in ovarian follicular fluid using transmission electron microscopy and CD63-positive and RNA containing vesicles using flow cytometry. Moreover, proteomics analysis reveals that follicular fluid-isolated exosomes contain both known exosomal proteins and proteins not previously reported in isolated exosomes. MicroRNAs were detected in microvesicle and exosomes preparations isolated from follicular fluid by real-time PCR analysis. Uptake of fluorescent-labeled microvesicles by granulosa cells was examined using in vitro and in vivo approaches. MicroRNA expression profiling reveals that miRNAs in microvesicle and exosome preparations isolated from follicular fluid also are present within surrounding granulosa and cumulus cells. These studies revealed that cell communication within the mammalian ovarian follicle may involve transfer of bioactive material by microvesicles and exosomes. Finally, miRNAs present in exosomes from ovarian follicular fluid varied with the age of the mare, and a number of different miRNAs were detected in young vs. old mare follicular fluid.     

Hormone-Dependent Bacterial Growth,Persistence and Biofilm Formation – A Pilot Study Investigating Human Follicular Fluid Collected during IVF Cycles

Human follicular fluid, considered sterile, is aspirated as part of an in vitro fertilization (IVF) cycle. However, it is easily contaminated by the trans-vaginal collection route and little information exists in its potential to support the growth of microorganisms. The objectives of this study were to determine whether human follicular fluid can support bacterial growth over time, whether the steroid hormones estradiol and progesterone (present at high levels within follicular fluid) contribute to the in vitro growth of bacterial species, and whether species isolated from follicular fluid form biofilms. We found that bacteria in follicular fluid could persist for at least 28 weeks in vitro and that the steroid hormones stimulated the growth of some bacterial species, specifically Lactobacillus spp., Bifidobacterium spp. Streptococcus spp. and E. coli. Several species, Lactobacillus spp., Propionibacterium spp., and Streptococcus spp., formed biofilms when incubated in native follicular fluids in vitro (18/24, 75%). We conclude that bacteria aspirated along with follicular fluid during IVF cycles demonstrate a persistent pattern of growth. This discovery is important since it can offer a new avenue for investigation in infertile couples.     

Follicular fluid adrenomedullin concentrations in spontaneous and stimulated cycles: relationship to ovarian function and endothelin-1 and nitric oxide

The objective of this study was to determine concentration of adrenomedullin (AM) in follicular fluid and whether a correlation exists between AM and nitric oxide (NO) or endothelin-1 (ET-1) levels in follicular fluid, serum 17beta-estradiol or other parameters of ovarian function in spontaneous and gonadotrophin stimulated ovarian cycles. Follicular fluid samples were obtained at oocyte retrieval from 50 women who underwent an in vitro fertilization (IVF) program: 40 undergoing ovarian hyperstimulation with recombinant FSH and 10 had spontaneous ovarian cycles. AM, ET-1, and NO were detected in all of the follicular fluid samples and their concentrations were similar in spontaneous and stimulated cycles. In patients undergoing ovarian stimulation, follicular fluid AM levels correlated with serum 17beta-estradiol concentration. No correlation was found between follicular AM concentration and parameters of ovarian function. Similarly, no relationship was observed between ET-1, NO, and AM follicular fluid concentrations in either spontaneous or stimulated cycles.This study suggests a possible regulatory effect of the sexual hormones on AM production by the ovary during the ovulatory process. The site of AM secretion and its function (if any), however, remain to be established.     

Exosomal and Non-Exosomal Transport of Extra-Cellular microRNAs in Follicular Fluid: Implications for Bovine Oocyte Developmental Competence

Cell-cell communication within the follicle involves many signaling molecules, and this process may be mediated by secretion and uptake of exosomes that contain several bioactive molecules including extra-cellular miRNAs. Follicular fluid and cells from individual follicles of cattle were grouped based on Brilliant Cresyl Blue (BCB) staining of the corresponding oocytes. Both Exoquick precipitation and differential ultracentrifugation were used to separate the exosome and non-exosomal fraction of follicular fluid. Following miRNA isolation from both fractions, the human miRCURY LNA™ Universal RT miRNA PCR array system was used to profile miRNA expression. This analysis found that miRNAs were present in both exosomal and non-exosomal fraction of bovine follicular fluid. We found 25 miRNAs differentially expressed (16 up and 9 down) in exosomes and 30 miRNAs differentially expressed (21 up and 9 down) in non-exosomal fraction of follicular fluid in comparison of BCB- versus BCB+ oocyte groups. Expression of selected miRNAs was detected in theca, granulosa and cumulus oocyte complex. To further explore the potential roles of these follicular fluid derived extra-cellular miRNAs, the potential target genes were predicted, and functional annotation and pathway analysis revealed most of these pathways are known regulators of follicular development and oocyte growth. In order to validate exosome mediated cell-cell communication within follicular microenvironment, we demonstrated uptake of exosomes and resulting increase of endogenous miRNA level and subsequent alteration of mRNA levels in follicular cells in vitro. This study demonstrates for the first time, the presence of exosome or non-exosome mediated transfer of miRNA in the bovine follicular fluid, and oocyte growth dependent variation in extra-cellular miRNA signatures in the follicular environment.     

Hypocoagulable state of human preovulatory ovarian follicular fluid: role of sulfated proteoglycan and tissue factor pathway inhibitor in the fluid

Ovulation accompanied by tissue damage can cause an increase in the level of tissue factor (TF) in the follicular fluid, triggering the extrinsic coagulation pathway. However, follicular fluid must block fibrin formation and maintain fluidity until the release of the oocyte at ovulation. The combination of sulfated proteoglycan, antithrombin, and TF pathway inhibitor (TFPI) appears to play a critical role in the hypocoagulability of human follicular fluid. When compared with plasma, folicular fluid differs markedly in the levels of a number of important coagulation proteins. Principal among these are 15-fold, 13-fold, and 3.7-fold increases in free TFPI, thrombin-antithrombin complex, and TF, respectively. The excessively prolonged activated partial thromboplastin time (APTT) and prothrombin time (PT) of human ovarian follicular fluid appear to be primarily due to high concentrations of sulfated proteoglycans, which accelerate the inactivation of thrombin and the anti-Xa activity of TFPI. Thus, heparitinase treatment shortened the clotting times of follicular fluid and reduced the inhibition of thrombin by the proteoglycan fraction combined with a fraction containing antithrombin. The remaining prolongation of APTT and PT may be caused by high levels of free TFPI in follicular fluid, which were confirmed by Northern blotting analysis, demonstrating TFPI mRNA expression by granulosa cells.     

Effect of changes in FSH induced by bovine follicular fluid and FSH infusion in the preovulatory phase on subsequent ovulation rate and corpus luteum function in the ewe

Treatment of Damline ewes with i.v. injections of various doses (2, 5 or 10 ml) of bovine follicular fluid for 72 h after prostaglandin-induced luteal regression resulted in a significant decrease in plasma concentrations of FSH after a 1.5-2 h delay but did not affect LH. The half life of this decrease in plasma FSH levels (156 min) after injection of follicular fluid was similar to that for clearance (159 min) of ovine FSH after infusion. A significant rebound increase in plasma FSH levels occurred by 13 h after all follicular fluid injections, and the magnitude of this rebound was inversely related to the dose of follicular fluid injected. A significant delay in the onset of oestrus occurred only with 5 and 10 ml bovine follicular fluid. There was no significant effect on ovulation rate or subsequent corpus luteum function as measured by plasma concentrations of progesterone. Infusion of ovine FSH (50 micrograms/h for 48 h) during the period of follicular fluid treatment prevented the delay in onset of oestrus and resulted in a substantial (2-10-fold) increase in ovulation rate. Corpus luteum function in terms of progesterone secretion was also enhanced. These results show that (1) intermittent suppression of FSH during the preovulatory period in the ewe does not affect subsequent ovulation rate or corpus luteum function and (2) the delay in the onset of oestrus induced by bovine follicular fluid can be prevented by exogenous FSH.     

Microorganisms within Human Follicular Fluid: Effects on IVF

Our previous study reported microorganisms in human follicular fluid. The objective of this study was to test human follicular fluid for the presence of microorganisms and to correlate these findings with the in vitro fertilization (IVF) outcomes. In this study, 263 paired follicular fluids and vaginal swabs were collected from women undergoing IVF cycles, with various causes for infertility, and were cultured to detect microorganisms. The cause of infertility and the IVF outcomes for each woman were correlated with the microorganisms detected within follicular fluid collected at the time of trans-vaginal oocyte retrieval. Microorganisms isolated from follicular fluids were classified as: (1) ‘colonizers’ if microorganisms were detected within the follicular fluid, but not within the vaginal swab (at the time of oocyte retrieval); or (2) ‘contaminants’ if microorganisms detected in the vagina at the time of oocyte retrieval were also detected within the follicular fluid. The presence of Lactobacillus spp. in ovarian follicular fluids was associated with embryo maturation and transfer. This study revealed microorganisms in follicular fluid itself and that the presence of particular microorganisms has an adverse affect on IVF outcomes as seen by an overall decrease in embryo transfer rates and pregnancy rates in both fertile and infertile women, and live birth rates in women with idiopathic infertility. Follicular fluid microorganisms are a potential cause of adverse pregnancy outcomes in IVF in both infertile women and in fertile women with infertile male partners.     

Polycystic ovary syndrome: brain-derived neurotrophic factor (BDNF) plasma and follicular fluid levels

Polycystic ovary syndrome is one of the most common endocrine disorders in women of reproductive age. Features of PCOS are hyperandrogenism, chronic anovulation and polycystic ovaries on ultrasonography. Follicle development is a complex and carefully orchestrated phenomenon, involving gonadotropins and a rapidly expanding list of other intraovarian regulators, such as brain-derived neurotrophic factor (BDNF). The aim of this study is to evaluate BDNF in plasma and in follicular fluid in women affected by PCOS and in normal menstruating women. In PCOS patients the BDNF levels in plasma and in follicular fluid are higher than values obtained in healthy controls. Therefore we can hypothsize that high levels of luteinizing hormone, probably increase the secretion of BDNF in PCOS patients.     

Prostaglandin levels in preovulatory follicles from rabbit ovaries perfused

Prostaglandin (PG) levels in follicular fluid from preovulatory follicles of rabbit ovaries perfused were measured in order to compare PG changes in this model system with those that occur and in isolated, LH-treated follicles . One ovary from each rabbit was perfused without further treatment (control). The other ovary was exposed to LH (0.1 or 1 ug/ml) beginning 1 hour (h) after initiation of perfusion. Samples of perfusion medium were taken at frequent intervals for measurement of PGE, PGF, progesterone and estradiol 17β. The perfusions were terminated when the first ovulation occurred or appeared imminent as judged by changes in the size and shape of the follicles. Follicular fluid was then rapidly aspirated from all large follicles on both ovaries for PGE and PGF measurement.Ovulations occurred only in the LH-treated ovaries. Progesterone and estradiol levels were significantly elevated in the perfusion medium within 1 h of LH treatment in comparison to controls. PG levels in perfusion medium from the control and LH-treated ovaries were not different throughout perfusion and increased in both groups. In contrast, PG levels measured in follicular fluid from LH-treated ovaries were 4- to 5-fold greater than in fluid from control ovaries. It is concluded that ovulation induced by LH in this experimental model is accompanied by an increase in follicular PG levels similar to that seen in other and models. This difference in follicular PG levels between the LH-treated and control ovaries is, however, not reflected in the perfusion medium.     

Granulosa cell steroidogenesis before in vitro fertilization

Endocrine and gametogenic functions of the ovulatory follicle may be linked. To verify this, we studied granulosa cell steroidogenesis in relation to oocyte fertilization and preimplantation embryo development in vitro. Multiple follicles were stimulated in in vitro fertilization patients with clomiphene citrate and ovulation was induced with human chorionic gonadotropin (hCG). Oocytes were fertilized with husband's sperm and normal embryos were replaced 48 h later. Granulosa cells were separated from follicular fluid from 64 follicles and incubated for 3 h with and without aromatase substrate (1 microM testosterone). Progesterone and estradiol levels were measured in follicular fluid and incubation medium. Follicular fluid steroid levels and granulosa cell steroidogenesis showed no significant differences for oocytes which cleaved normally and those which did not. Granulosa cell aromatase activity was high in all follicles, suggesting that the low periovulatory follicular fluid estradiol level is not explained by a fall in granulosa cell aromatase after hCG. High granulosa cell progesterone production and follicular fluid progesterone were consistent with advanced granulosa cell luteinization. Oocytes undergoing polyspermic activation were from larger follicles with elevated follicular fluid progesterone levels, suggesting that follicular size and follicular fluid progesterone are correlated with "over-ripeness" and polyspermy. No simple relationship exists between oocyte function and the present indices of granulosa cell steroid metabolism.     

Prostaglandin levels in preovulatory follicles from rabbit ovaries perfused in vitro

Prostaglandin (PG) levels in follicular fluid from preovulatory follicles of rabbit ovaries perfused in vitro were measured in order to compare PG changes in this model system with those that occur in vivo and in isolated, LH-treated follicles in vitro. One ovary from each rabbit was perfused without further treatment (control). The other ovary was exposed to LH (0.1 or 1 microgram/ml) beginning 1 hour (h) after initiation of perfusion. Samples of perfusion medium were taken at frequent intervals for measurement of PGE, PGF, progesterone and estradiol 17 beta. The perfusions were terminated when the first ovulation occurred or appeared imminent as judged by changes in the size and shape of the follicles. Follicular fluid was then rapidly aspirated from all large follicles on both ovaries for PGE and PGF measurement. Ovulations occurred only in the LH-treated ovaries. Progesterone and estradiol levels were significantly elevated in the perfusion medium within 1 h of LH treatment in comparison to controls. PG levels in perfusion medium from the control and LH-treated ovaries were not different throughout perfusion and increased in both groups. In contrast, PG levels measured in follicular fluid from LH-treated ovaries were 4- to 5-fold greater than in fluid from control ovaries. It is concluded that ovulation induced by LH in this experimental model is accompanied by an increase in follicular PG levels similar to that seen in other in vivo and in vitro models. This difference in follicular PG levels between the LH-treated and control ovaries is, however, not reflected in the perfusion medium.     

Metabolite and ionic composition of follicular fluid from different-sized follicles and their relationship to serum concentrations in dairy cows

Metabolic changes in blood serum may be reflected in the biochemical composition of follicular fluid and could indirectly influence oocyte quality. The purpose of this study was to examine the biochemical composition of follicular fluid harvested from different-sized follicles and its relationship with that of blood serum in dairy cattle. Following slaughter, blood samples were collected from dairy cows (n=30) and follicular fluid aspirated from three size classes of non-atretic follicles (<4 mm, 6–8 mm and >10 mm diameter). Samples remained independent between cows and between size classes within cows. Serum and follicular fluid samples were assayed using commercial clinical and photometric chemistry assays for ions (sodium, potassium and chloride) and metabolites (glucose, β-hydroxybutyrate (β-OHB), lactate, urea, total protein, triglycerides, non-esterified fatty acids (NEFA) and total cholesterol). Results showed that follicular fluid concentrations of glucose, β-OHB and total cholesterol increased from small to large follicles and decreased for potassium, chloride, lactate, urea and triglycerides. There was a significant concentration gradient for all variables between their levels in serum and follicular fluid (P<0.05). Significant correlations were observed for chloride (r=0.40), glucose (r=0.56), β-OHB (r=0.85), urea (r=0.95) and total protein (r=0.60) for all three follicle size classes and for triglycerides (r=0.43), NEFA (r=0.50) and total cholesterol (r=0.42) for large follicles (P<0.05). The results from the present study suggest that the oocyte and the granulosa cells of dairy cows grow and mature in a biochemical environment that changes from small to large follicles. Furthermore, the significant correlation between the composition of serum and follicular fluid for the above-mentioned metabolites suggests that metabolic changes in serum levels will be reflected in the follicular fluid and, therefore, may affect the quality of both the oocyte and the granulosa cells.     

Exosomal miR-27 negatively regulates ROS production and promotes granulosa cells apoptosis by targeting SPRY2 in OHSS

Ovarian hyperstimulation syndrome (OHSS) is one of the most dangerous iatrogenic complications in controlled ovarian hyperstimulation (COH). The exact molecular mechanism that induces OHSS remains unclear. In recent years, accumulating evidence found that exosomal miRNAs participate in many diseases of reproductive system. However, the specific role of miRNAs, particularly the follicular fluid-derived exosomal miRNAs in OHSS remains controversial. To identify differentially expressed follicular fluid exosomal miRNAs from OHSS and non-OHSS patients, the analysis based on miRNA-sequence was conducted. The levels of 291 miRNAs were significantly differed in exosomes from OHSS patients compared with normal control, and exosomal miR-27 was one of the most significantly down-regulated miRNAs in the OHSS group. By using MiR-27 mimic, we found it could increase ROS stress and apoptosis by down-regulating the expression of p-ERK/Nrf2 pathway by negatively regulating SPRY2. These data demonstrate that exosomal miRNAs are differentially expressed in follicular fluid between patients with and without OHSS, and follicular fluid exosomal miR-27 may involve in the pathological process of OHSS development.     

Proteomic analysis of human follicular fluid from fertile women

Background

Follicular fluid is a unique biological fluid in which the critical events of oocyte and follicular maturation and somatic cell-germ cell communication occur. Because of the intimate proximity of follicular fluid to the maturing oocyte, this fluid provides a unique window into the processes occurring during follicular maturation. A thorough identification of the specific components within follicular fluid may provide a better understanding of intrafollicular signaling, as well as reveal potential biomarkers of oocyte health for women undergoing assisted reproductive treatment. In this study, we used high and low pH HPLC peptide separations followed by mass spectrometry to perform a comprehensive proteomic analysis of human follicular fluid from healthy ovum donors. Next, using samples from a second set of patients, an isobaric mass tagging strategy for quantitative analysis was used to identify proteins with altered abundances after hCG treatment.

Results

A total of 742 follicular fluid proteins were identified in healthy ovum donors, including 413 that have not been previously reported. The proteins belong to diverse functional groups including insulin growth factor and insulin growth factor binding protein families, growth factor and related proteins, receptor signaling, defense/immunity, anti-apoptotic proteins, matrix metalloprotease related proteins, and complement activity. In a quantitative analysis, follicular fluid samples from age-matched women undergoing in vitro fertilization oocyte retrieval were compared and 17 follicular fluid proteins were found at significantly altered levels (p < 0.05) between pre-hCG and post-hCG samples. These proteins belong to a variety of functional processes, including protease inhibition, inflammation, and cell adhesion.

Conclusions

This database of FF proteins significantly extends the known protein components present during the peri-ovulatory period and provides a useful basis for future studies comparing follicular fluid proteomes in various fertility, disease, and environmental exposure conditions. We identified 17 differentially expressed proteins after hCG treatment and together these data showed the feasibility for defining biomarkers that illuminate how the ovarian follicle microenvironment is altered in various infertility-related conditions.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9077-6) contains supplementary material, which is available to authorized users.     

Increase in ovulation rate after treatment of ewes with bovine follicular fluid in the luteal phase of the oestrous cycle

Administration of charcoal-treated bovine follicular fluid to Damline ewes twice daily (i.v.) from Days 1 to 11 of the luteal phase (Day 0 = oestrus) resulted in a delay in the onset of oestrous behaviour and a significant increase in ovulation rate following cloprostenol-induced luteolysis on Day 12. During follicular fluid treatment plasma levels of FSH in samples withdrawn just before injection of follicular fluid at 09:00 h (i.e. 16 h after previous injection of follicular fluid) were initially suppressed, but by Day 8 of treatment had returned to those of controls. However, the injection of follicular fluid at 09:00 h on Day 8 still caused a significant suppression of FSH as measured during a 6-h sampling period. Basal LH levels were higher throughout treatment due to a significant increase in amplitude and frequency of pulsatile secretion. After cloprostenol-induced luteal regression at the end of treatment on Day 12, plasma levels of FSH increased 4-fold over those of controls and remained higher until the preovulatory LH surge. While LH concentrations were initially higher relative to those of controls, there was no significant difference in the amount of LH released immediately before or during the preovulatory surge. These results suggest that the increase in ovulation rate observed during treatment with bovine follicular fluid is associated with the change in the pattern of gonadotrophin secretion in the luteal and follicular phases of the cycle.     

Modelling Thrombin Generation in Human Ovarian Follicular Fluid

A mathematical model is constructed to study thrombin production in human ovarian follicular fluid. The model results show that the amount of thrombin that can be produced in ovarian follicular fluid is much lower than that in blood plasma, failing to reach the level required for fibrin formation, and thereby supporting the hypothesis that in follicular fluid thrombin functions to initiate cellular activities via intracellular signalling receptors. It is also concluded that the absence of the amplification pathway to thrombin production in follicular fluid is a major factor in restricting the amount of thrombin that can be produced. Titration of the initial concentrations of the various reactants in the model lead to predictions for the amount of tissue factor and phospholipid that is required to maintain thrombin production in the follicle, as well as to the conclusion that tissue factor pathway inhibitor has little effect on the time that thrombin generation is sustained. Numerical experiments to determine the effect of factor V, which is at a much reduced level in follicular fluid compared to plasma, and thrombomodulin, illustrate the importance for further experimental work to determine values for several parameters that have yet to be reported in the literature.     

Localization and detection of ovarian follicular fluid protein in follicles of human ovaries.

Human ovarian follicular fluid protein has been partially purified and the active fraction designated as hGF2. Using specific polyclonal antiserum to hGF2, it was observed to be localized immunohistochemically in the granulosa cells of medium but not large follicles of human ovary. The hGF2 levels were estimated by ELISA in serum and follicular fluid of 10 gonadotropin-stimulated women recruited for IVF-ET programme. The results revealed a 3-fold increase in the concentration of hGF2 in follicular fluid compared to that in serum of these patients. These data indicate that the protein is secreted by granulosa cells and plays an important role in the regulation of follicular maturation and ovulation.     

Changes in the levels of pituitary and steroid hormones in ovine and human ovarian follicular fluid

Changes in the protein and steroid hormones of follicular fluid, aspirated from different follicles of sheep and human ovaries, have been measured and correlated with the size of the follicles. As the fluid contains a number of proteins, steroids have been measured directly and after ether extraction. The follicular fluid concentrations of progesterone and 17 beta-oestradiol measured directly in the fluid increased with the size of the follicles. The levels of free testosterone remained constant in all sizes of follicles, while those of bound hormone showed a 10- to 15-fold increase over the free testosterone concentrations in both the sheep and human follicular fluid. A decrease in the levels of bound testosterone in the fluid of large follicles (LFFL) coincided with the increase in bound 17 beta-oestradiol, suggesting the possible conversion of bound testosterone to oestrogen as the follicle attained maturity. The ratio of follicle-stimulating hormone (FSH) to luteinizing hormone (LH) varied in the fluid obtained from different size follicles, being 1:7 in small (SFFL), 1.3.5 in medium (MFFL) and 1:2.3 in large (LFFL) follicles of sheep ovaries. The LH content of follicular fluid of different size follicles appeared to be the same, with LFFL showing a minor increase over SFFL. In the human, the fluid from medium follicles contained very little LH compared to LFFL. These differences in the pattern of LH levels present in the fluid from different size follicles between human and sheep ovaries presumably reflect species variations in the entry of LH into the follicles.