Composition Of Follicular Fluid Research Articles

Metabolomic Analysis of Follicular Fluid in Normal-Weight Patients with Polycystic Ovary Syndrome.

This study aimed to examine the differential variations in the metabolic composition of follicular fluid (FF) among normal-weight patients with polycystic ovary syndrome (PCOS) and controls and to identify potential biomarkers that may offer insights into the early identification and management of these patients. We collected FF samples from 45 normal-weight women with PCOS and 36 normal-weight controls without PCOS who were undergoing in vitro fertilization-embryo transfer. An untargeted metabolomic study of collected FF from infertile women was performed using high-performance liquid chromatography-tandem spectrometry (LC-MS). The tendency of the two groups to separate was demonstrated through multivariate analysis. Univariate analysis and variable importance in projection were used to screen out differential metabolites. Metabolic pathway analysis was conducted using the Kyoto Encyclopedia of Genes and Genomes (KEGG), and a diagnostic model was established using the random forest algorithm. The metabolomics analysis revealed an increase in the expression of 23 metabolites and a decrease in that of 10 metabolites in the FF of normal-weight women with PCOS. According to the KEGG pathway analysis, these differential metabolites primarily participated in the metabolism of glycerophospholipids and the biosynthesis of steroid hormones. Based on the biomarker combination of the top 10 metabolites, the area under the curve value was 0.805. The concentrations of prostaglandin E2 in the FF of individuals with PCOS exhibited an inverse association with the proportion of high-quality embryos (p < 0.05). Our research identified a distinct metabolic profile of the FF from normal-weight women with PCOS. The results offer a broader comprehension of the pathogenesis and advancement of PCOS, and the detected differential metabolites could be potential biomarkers and targets for the treatment of PCOS.

Impact of maternal age on oxidation-reduction potential in follicular fluid and assisted reproductive technology treatment outcomes

Background: Maternal age influenced the quantity and quality of oocytes. The composition of follicular fluid was changed in advanced maternal age. This study was conducted to evaluate changes in redox potential and blastocyst formation potential in older women. Methods and materials: Retrospective study collected data from December 2020 to October 2023 on patients treated with in vitro fertilization with follicular fluid surveyed at the Center for Reproductive Endocrinology and Infertility (HUECREI), Hue University of Medicine and Pharmacy, Hue. Results: Two cohorts comprised the 456 patients: Group 1, consisting of individuals younger than 35 years of age, and Group 2, comprising those 35 years or older. Anti-Müllerian hormone (AMH), initial follicle-stimulating hormone (FSH), and total FSH dose utilized differed significantly between Groups 1 and 2, with median values of 3.79 versus 2.10 ng/mL (P value &lt; 0.001), 225.00 versus 277.50 IU/L (P value &lt; 0.001), and 2025.00 versus 2325.00 IU/L (P value &lt; 0.001), respectively. However, there were no significant differences in ORP, maturation rate, fertilization rate, or blastocyst formation rate between the two age groups. Variations in ORP were only observed among subgroups of patients with the ability to achieve an expected fertilization rate of ≥ 70% in Group 1. Specifically, the subgroup meeting expectations had a lower ORP of 83.00 ± 83.00 mV, in contrast to the non-expectation group which had an ORP of 96.14 ± 83.00 mV, accompanied by a P value of 0.005. The difference of ORP was not observed in the group that reached and did not reach the benchmark embryo formation ≥ 60% in the two age groups. Conclusion: Advanced maternal age have declined in ovarian function but no statistically significant difference in redox potential in follicular fluid has been observed. The fertilization rate and blastocyst formation rate were equivalent across the two age groups of women. The redox potential of follicular fluid was not statistically significant difference between cycles that reached and did not reach the benchmark of blastocyst formation ≥ 60% in the two age groups of women. Key words: Blastocyst, advanced maternal age, oxidative-reduction potential (ORP), in vitro fertilization.

Characteristics of the Follicular Fluid Extracellular Vesicle Molecular Profile in Women in Different Age Groups in ART Programs.

The aim of this study was to investigate the molecular composition of follicular fluid (FF) extracellular vesicles (EVs) in women of different reproductive ages and its possible relationship to sperm fertilizing ability. FF EVs were obtained by differential centrifugation. The concentration and size distribution of FF EVs were analyzed by nanoparticle tracking analysis. The lipidome and proteome were analyzed by liquid chromatography-mass spectrometry. The isolated FF EVs had a variety of shapes and sizes; their concentration and size distribution did not differ significantly between the age groups. In women younger than 35 years, the concentration of vesicular progesterone was 6.6 times higher than in women older than 35 years, and the total levels of the main lipid classes were increased in younger women. A proteomic analysis revealed that not only FF EV-specific proteins, but also proteins involved in sperm activation were present. New data were obtained on the composition of FF EVs, confirming their importance as molecular indicators of age-related changes in the female reproductive system. In addition, these results shed light on the possible interaction between the FF EVs of women in different age groups and male germ cells. Therefore, studying the transcriptomic and metabolomic profile of FF EVs may be a crucial approach to evaluate the efficacy of ART.

Characterization of preovulatory follicular fluid secretome and its effects on equine oocytes during in vitro maturation

In vitro maturation (IVM) of oocytes is clinically used in horses to produce blastocysts but current conditions used for horses are suboptimal. We analyzed the composition of equine preovulatory follicular fluid (FF) secretome and tested its effects on meiotic competence and gene expression in oocytes subjected to IVM. Preovulatory FF was obtained, concentrated using ultrafiltration with cut-off of 10 kDa, and stored at −80 °C. The metabolic and proteomic composition was analyzed, and its ultrastructural composition was assessed by cryo-transmission microscopy. Oocytes obtained post-mortem or by ovum pick up (OPU) were subjected to IVM in the absence (control) or presence of 20 or 40 μg/ml (S20 or S40) of secretome. Oocytes were then analyzed for chromatin configuration or snap frozen for gene expression analysis. Proteomic analysis detected 255 proteins in the Equus caballus database, mostly related to the complement cascade and cholesterol metabolism. Metabolomic analysis yielded 14 metabolites and cryo-transmission electron microscopy analysis revealed the presence of extracellular vesicles (EVs). No significant differences were detected in maturation rates among treatments. However, the expression of GDF9 and BMP15 significantly increased in OPU-derived oocytes compared to post-mortem oocytes (fold increase ± SEM: 9.4 ± 0.1 vs. 1 ± 0.5 for BMP15 and 9.9 ± 0.3 vs. 1 ± 0.5 for GDF9, respectively; p < 0.05). Secretome addition increased the expression of TNFAIP6 in S40 regardless of the oocyte source. Further research is necessary to fully understand whether secretome addition influences the developmental competence of equine oocytes.

Correlation of Metabolic and Ionic Components of Follicular Fluid with Estrogen Concentration

This study was aimed to assess the correlation between the metabolic and ionic composition of ovarian follicular fluid and its relationship to the concentration of estrogen and prolactin hormones as the follicle size changes in the local female cows. Ovaries were collected (80 ovaries) from non- pregnant 40 adult females cow at Hilla slaughterhouse at Babil province. The gathered ovaries were transported to the laboratory within two hours. Follicular fluid was pulled from small (2-5 mm) follicles, medium (6-10 mm), and large (11-20 mm) follicles, then it stored at 5 ° C until assayed. Follicular fluid samples were analyzed for metabolites composition of (cholesterol, total protein, and glucose), and ionic composition (calcium, phosphorus, copper, magnesium, zinc) ,and hormone concentration ( estrogen and prolactin). The results of the study showed that the total protein and glucose concentrations were increased significantly(p&gt; 05.0) with increased of follicle size. The study also showed the calcium, copper, and phosphorus concentration were increased significantly(p&gt; 05.0) with changes of follicular size, as well as the estrogen concentration was significantly increased (p &gt; 05.0) With increased follicular size. A significantly (p&gt; 05.0) correlation was found between both magnesium, zinc ions, and estrogen hormone.

Disturbed Follicular Microenvironment in Polycystic Ovary Syndrome: Relationship to Oocyte Quality and Infertility.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder associated with infertility and poor reproductive outcomes. The follicular fluid (FF) microenvironment plays a crucial role in oocyte development. This review summarizes evidence elucidating the alterations in FF composition in PCOS. Various studies demonstrated a pronounced proinflammatory milieu in PCOS FF, characterized by increased levels of cytokines, including but not limited to interleukin-6 (IL-6), tumor necrosis factor α, C-reactive protein, and IL-1β, concomitant with a reduction in anti-inflammatory IL-10. T lymphocytes and antigen-presenting cells are dysregulated in PCOS FF. PCOS FF exhibit heightened reactive oxygen species production and the accumulation of lipid peroxidation byproducts, and impaired antioxidant defenses. Multiple microRNAs are dysregulated in PCOS FF, disrupting signaling critical to granulosa cell function. Proteomic analysis reveals changes in pathways related to immune responses, metabolic perturbations, angiogenesis, and hormone regulation. Metabolomics identify disturbances in glucose metabolism, amino acids, lipid profiles, and steroid levels with PCOS FF. Collectively, these pathological alterations may adversely affect oocyte quality, embryo development, and fertility outcomes. Further research on larger cohorts is needed to validate these findings and to forge the development of prognostic biomarkers of oocyte developmental competence within FF. Characterizing the follicular environment in PCOS is key to elucidating the mechanisms underlying subfertility in this challenging disorder.

Different Lengths of Diet Supplementation with 10% Flaxseed Alter the Hormonal Profile and the Follicular Fluid Fatty Acid Content of Fattening Gilts.

The effect of 10% dietary flaxseed fed for 3 and 6 weeks on serum hormone levels of fattening gilts, the fatty acid (FA) follicular fluid (FF) composition of small and large antral follicles, and the steroidogenesis and IGF-I secretion by isolated small antral follicles and their response to regulatory hormones (LH, FSH, IGF-I) was studied using immunoassay and gas chromatography analyses. Both supplemental periods increased levels of P4 and IGF-I in blood serum. A shorter period inhibited steroidogenesis (P4, T, E2) and IGF-I secretion by small antral follicles, which was associated with decreased levels of monounsaturated FAs (MUFA) and preferred n-6 polyunsaturated FA (PUFA) metabolism. A longer period stimulated hormone secretion at elevated levels of saturated FAs (SFA) at the expense of MUFAs and PUFAs preferring the n-3 PUFA metabolism. Out of ovarian regulators, only LH and IGF-I were able to alter the secretion of steroids and IGF-I by small follicles of fattening pigs fed a basal diet. The effect of flaxseed on the secretion of follicular hormones after both supplemental periods was altered by all regulatory hormones in a dose-dependent manner. The level of SFAs and PUFAs in FF of large follicles increased with the length of flaxseed feeding, suggesting the suppression of ovulation.

Follicular fluid C3a-peptide promotes oocyte maturation through F-actin aggregation

BackgroundImmature cumulus-oocyte complexes are retrieved to obtain mature oocytes by in vitro maturation (IVM), a laboratory tool in reproductive medicine to obtain mature oocytes. Unfortunately, the efficiency of IVM is not satisfactory. To circumvent this problem, we therefore intended to commence with the composition of ovarian follicular fluid (FF), an important microenvironment influencing oocyte growth. It is well known that FF has a critical role in oocyte development and maturation. However, the components in human FF remain largely unknown, particularly with regard to small molecular peptides.ResultsIn current study, the follicular fluid derived from human mature and immature follicles were harvested. The peptide profiles of FF were further investigated by using combined ultrafiltration and LC–MS/MS. The differential peptides were preliminary determined by performing differentially expressed analysis. Human and mouse oocyte culture were used to verify the influence of differential peptides on oocyte development. Constructing plasmids, cell transfecting, Co-IP, PLA etc. were used to reveal the detail molecular mechanism. The results from differentially expressed peptide as well as cultured human and mouse oocytes analyses showed that highly conserved C3a-peptide, a cleavage product of complement C3a, definitely affected oocytes development. Intriguingly, C3a-peptide possessed a novel function that promoted F-actin aggregation and spindle migration, raised the percentage of oocytes at the MII stage, without increasing the chromosome aneuploidy ratio, especially in poor-quality oocytes. These effects of C3a-peptide were attenuated by C3aR morpholino inhibition, suggesting that C3a-peptide affected oocytes development by collaborating with its classical receptor, C3aR. Specially, we found that C3aR co-localized to the spindle with β-tubulin to recruit F-actin toward the spindle and subcortical region of the oocytes through specific binding to MYO10, a key regulator for actin organization, spindle morphogenesis and positioning in oocytes.ConclusionsOur results provide a new perspective for improving IVM culture systems by applying FF components and also provide molecular insights into the physiological function of C3a-peptide, its interaction with C3aR, and their roles in enabling meiotic division of oocytes.

PSV-3 The Influence of Synovex-C Growth Implants at Weaning with Different Supplementation Levels on Ovarian Parameters in Developing Beef Heifers

Abstract Use of growth promoting implants in beef heifers in Southwestern rangelands has been reported to improve growth, however, the effect on ovarian parameters is unknown. Therefore, the objective of this study was to investigate the influence of level of supplementation when heifers were administered a second Synovex-C implant at weaning on ovarian parameters. At two months of age all heifers (n = 17) were administered a Synovex- C implant. At weaning (8-months old), heifers were stratified by body weight and antral follicle counts and assigned to receive one of two levels of supplementation (High or Low) and Implant (No Implant or Implant). Heifers assigned to Implant were administered a second Synovex-C implant at weaning, thereby creating four treatment groups: High Implant (n = 4), High No Implant (n = 4), Low Implant (n = 5), and Low No Implant (n = 4). Heifers were utilized in a complete randomized block design with a 2x2 factorial. Heifers were individually fed supplement three times a week for 26 weeks with refusals being weighed after each feeding to determine intake. For the first 4 weeks of the study, High and Low heifers were offered 4.54 kg/h/d and 2.72 kg/h/d, respectively, of 20% CP cube. After acclimation High and Low heifers were offered 2.95 kg/h/d and 1.59 kg/h/d, respectively, of 32% CP cube for the remainder of the study. At the end of the supplementation period heifers underwent bilateral ovariectomies. Weight, height, and length of each ovary was recorded. Surface antral follicles were counted on both ovaries and recorded. Height and length of the largest preovulatory follicle was recorded and follicular fluid was collected for analysis of estradiol and progesterone concentrations. Ovarian and follicular measurements were analyzed using MIXED procedures in SAS. Ovarian weight (P ≥ 0.16), ovarian length (P ≥ 0.46), or ovarian height (P ≥ 0.32) were not influenced by implant, supplement level, or their interaction. There was no difference among total surface antral follicle count between implant (No Implant: 34.2 ± 4.3 vs Implant: 40.0 ± 4.6), supplement level (High: 32.75 ± 4.6 vs Low: 41.43 ± 4.3), and their interaction (P ≥ 0.19). Preovulatory follicle diameter was not impacted by implant (No Implant: 10.82 ± 1.27 mm vs Implant: 9.42 ± 1.27 mm), supplement level (High: 10.48 ± 1.27 mm vs Low: 9.77 ± 1.27 mm), or their interaction (P ≥ 0.45). Implant, supplement level, or their interaction did not influence progesterone (P ≥ 0.43), estradiol (P ≥ 0.42), or the estradiol:progesterone ratio (P ≥ 0.41) in follicular fluid. In conclusion, administration of a second Synovex-C implant at weaning did not impact ovarian parameters or the composition of follicular fluid. USDA is an equal opportunity provider and employer.

Prolonged follicular dominance is associated with dysregulated proteomic profile of the follicular fluid in Bos indicus cows

Prolonged follicular dominance is one of the conditions associated with disconcerted follicular dynamics that result in substantial economic losses to the farmers through low reproductive efficiency in cattle. Hormonal aberrations associated with prolonged follicular dominance may affect the follicular microenvironment and composition of follicular fluid. The current study focused on proteome changes of follicular fluid in prolonged follicular dominance compared to physiological follicular dominance. Prolonged dominance was induced in Deoni cows (n = 6) by using CIDR (previously used for 7 days) from day 4–8 of estrus, with PGF2 injection on day 6 and day 7 at 12 h intervals. Follicular fluid was collected by ultrasound guided transvaginal follicular aspiration method. Global proteomic analysis of follicular fluid revealed 217 proteins in the Deoni cow, with the majority of proteins involved in 21 pathways, 42 molecular functions, and 106 biological processes. Complement and coagulation cascades (22.8%) and cholesterol metabolism (4.68%) were the major pathways in which identified proteins were involved. Comparison of physiological and prolonged dominant follicular fluid revealed differential expression of 26 proteins, of which 15 were upregulated and 11 were downregulated. Proteins involved in complement and coagulation cascades, and vitamin digestion and absorption were found to be dysregulated in PFD. The present study suggests that the expression of proteins involved in inflammation, oocyte metabolism, and ovulation cascade were found to be dysregulated in the follicular fluid of prolonged follicular dominance consequently resulting in delayed ovulation or anovulation.

Extracellular Vesicles Contribute to the Difference in Lipid Composition between Ovarian Follicles of Different Size Revealed by Mass Spectrometry Imaging.

Follicular fluid (FF) ensures a safe environment for oocyte growth and maturation inside the ovarian follicle in mammals. In each cycle, the large dominant follicle (LF) contains the oocyte designated to be ovulated, whereas the small subordinate follicles (SFs) of the same wave will die through atresia. In cows, the oocytes from the SF, being 2 mm in size, are suitable for in vitro reproduction biotechnologies, and their competence in developing an embryo depends on the size of the follicles. FF contains proteins, metabolites, fatty acids, and a multitude of extracellular vesicles (ffEVs) of different origins, which may influence oocyte competence through bidirectional exchanges of specific molecular cargo between follicular cells and enclosed oocytes. FF composition evolves along with follicle growth, and the abundance of different lipids varies between the LF and SF. Here, significant differences in FF lipid content between the LFs and SFs within the same ovary were demonstrated by MALD-TOF mass spectrometry imaging on bovine ovarian sections. We then aimed to enlighten the lipid composition of FF, and MALDI-TOF lipid profiling was performed on cellular, vesicular, and liquid fractions of FF. Differential analyses on the abundance of detected lipid features revealed specific enrichment of phospholipids in different ffEV types, such as microvesicles (MVs) and exosomes (Exo), compared to depleted FF. MALDI-TOF lipid profiling on MVs and Exo from the LF and SF samples (n = 24) revealed that more than 40% of detected features were differentially abundant between the groups of MVs and Exo from the different follicles (p < 0.01, fold change > 2). Glycerophospholipid and sphingolipid features were more abundant in ffEVs from the SFs, whereas different lysophospholipids, including phosphatidylinositols, were more abundant in the LFs. As determined by functional analysis, the specific lipid composition of ffEVs suggested the involvement of vesicular lipids in cell signaling pathways and largely contributed to the differentiation of the dominant and subordinate follicles.

Proteome of follicular fluid from Murrah buffaloes (Bubalus bubalis): Normal cycling versus repeat-breeder females

Clinical evaluations have shown that repeat breeding is a major cause of infertility in buffaloes. The follicular fluid (FF) composition reflects directly metabolic status and fertility in females. Given this scenario, this study aimed to perform quantitative proteomic analysis of the FF collected from normal cycling (NC) and compared to repeat-breeder females. According to farm records, buffaloes were divided into two groups: normal cycling (NC, n = 7) and repeat-breeder (RB, n = 8) females. After estrus synchronization and using ultrasound-guided ovum pick-up, FF was aspirated from large follicles (˃ 8 mm). Posteriorly, proteins were identified by the shotgun method. A total of 119 proteins were identified and, among theses, three were uncharacterized and a protein (LOC123334375) was identified only in the NC group. The protein HP-25 homolog 2 was expressed only in RB females. The LFQ (label-free quantitation)-intensity of the proteins afamin (AFM), transthyretin (TTR), clotting factor IX (F9) and Xaa-Pro dipeptidase (PEPD) was significantly (P < 0.05) higher in RB than in NC females. In conclusion, the use of quantitative proteomics proved to be an important tool for the study of RB in buffaloes. The identification of HP-25 homolog 2 protein only in the RB females suggests that it can be used as a biomarker for this reproductive disorder. Additionally, an uncharacterized (LOC123334375) protein was identified only in normal cycling females and compensatory oxidative stress proteins were more expressed in repeat-breeders.

O-233 Follicular fluid extracellular vesicles as a potential tool for human spermatozoa selection in vitro

Abstract Study question What characteristics and features of follicular fluid (FF) extracellular vesicles (EVs) of women of different ages can affect the functional characteristics of spermatozoa? Summary answer The size, lipid composition, and progesterone levels of the FF EVs change with a woman's age and affect the fertilizing ability of spermatozoa. What is known already Aging influences on molecular composition of the FF, reducing fertilization processes occurring in the fallopian tubes. FF is an important factor in activating spermatozoa during oocyte fertilization, enhancing the process of sperm hyperactivation and capacitation. FF progesterone is one of the main chemoattractants in the female genital tract, attracting sperm to the oocyte and starting the fertilization processes. Our previous studies have shown a significant effect of FF EVs of young women compared to advanced maternal age women (AMA) on the hyperactivation and motility of spermatozoa. The binding of FF EVs to the acrosoma of the sperm also was detected. Study design, size, duration FF EVs were obtained by sequential centrifugation at different rotational speeds. The sperm fraction was isolated, washed by differential centrifugation in a density gradient, suspended in the saline to a concentration of 10*6/ml and incubated with EVs (1:2) at 37 °C in CO2-incubator for 1 h. Next steps: immunofluorescent staining, test for acrosomal reaction of spermatozoa after incubation, NTA analysis, the lipid profile and progesterone measuring of FF EVs of women of different ages. Participants/materials, setting, methods All study participants signed a voluntary informed consent for the use of biological samples for research purposes. Sperm samples were isolated from seminal fluid (n = 21) aged 28-36. FF EVs were obtained from young (n = 14) aged 22-30 and AMA patients (n = 14) aged 42-47. The methods used in this work also include immunofluorescence microscopy, nanoparticle tracking analysis, flow cytometry, liquid chromatography–mass spectrometry, statistical data analysis. Lipids were extracted from EVs using a modified Folch extraction protocol. Main results and the role of chance Progesterone levels were significantly higher in FF EVs samples from young women compared to FF EVs samples from AMA women and controls (3951000 a.u., 598500 a.u., 6701 a.u.). This may explain the increase in sperm hyperactivation and motility after incubation with FF EVs. The difference in size (144.78 nm ± 9.92 VS 127.03 nm ± 17.17) and lipid composition of young women's FF EVs may explain the more significant functional and therapeutic effect on spermatozoa compared to AMA women FF EVs. A total of 389 vesicle lipids were identified. The level of cholesterol esters, cholesterol, lysophosphatidylcholines, phosphatidylcholines was 1,5 times higher in FF EVs of young women. The level of di- and triglycerides, ceramides, oxidized forms of phosphatidylcholines was higher in FF EVs of AMA women. The level of spontaneous acrosomal reaction was almost 3 times lower in sperm samples after incubation with FF EVs compared to the control (11,21% VS 32,88%). This supports the hypothesis that FF EVs may restrain premature acrosomal reaction in the fallopian tubes. Age-related changes are reflected in reproductive important biological processes, which leads to a change in the composition and functions of FF and affect the functional role of FF EVs. Limitations, reasons for caution Small amount of data. To be completely confident in the importance of the participation of FF EVs in the activation of spermatozoa, it is necessary to perform proteomic analyses of FF EVs to discuss the possible mechanisms underlying the effects of FF EVs of different age groups on sperm characteristics. Wider implications of the findings Further studies may help not only to complement the fundamental understanding of the processes of fertilization and the impact on spermatozoa morphofunctional characteristics, but also to develop a tool for improving ART outcomes in couples with male infertility. The solutions could be the use of FF EVs from young donors. Trial registration number not applicable

Evaluation of cortisol, thyroxine (T3) and triiodothyronine (T4) concentrations in preovulatory follicles in jennies.

Reference values of follicular fluid components at different stages of follicular development in jennies are unknown. It is important to estimate such values because the biochemical composition of follicular fluid provides a suitable microenvironment for oocyte development, growth and maturation. The experimental protocol was carried out in the Equine Reproduction Clinic of the FMVZ-UNAM and was approved by an Institutional Sub-committee for the Care and Use of Experimental Animals (SICUAE.MMVZ-2020/4-2). Seven healthy female donkeys “Mexican burro” with average body condition score of 3 on a scale of 1 to 5 (Svendsen et al. The professional Handbook of the Donkey. 2008), were monitored by transrectal ultrasonography through the Veterinary Digital Diagnostic Ultrasound System 5 MHz (Model, DUS 60 VET) with a multi-frequency linear transducer. The ovum pick up (OPU) by transvaginal aspiration (TVA) was performed in a total of 70 preovulatory follicles, 10 for each jenny using a real- time B- mode ultrasound vaginal 6.5 MHz (Sonovet 600) with curvilinear transducer and 12 gauge (12 G) needle. Follicular fluid components were measured and analyzed by Luminex Multiplex Assays at the Universidad Nacional Autónoma de México. The relationship between cortisol, thyroxine (T3) and triiodothyronine (T4) concentrations in preovulatory follicles and follicular size was assessed. The preovulatory follicles are classified in three groups accordingto the diameter: small (27-30 mm), medium (31-35 mm) and large (36-38 mm). Concentrations of cortisol were lower in the large follicles group (18.8 ng/ml) compared with small (721.44 ng/ml) and medium follicles (361 ng/ml). The concentrations of thyroxine were higher in the medium follicle group (49 ng/ml) than in the small (21ng/ml) and large follicle groups (15.9 ng/ml). Finally, the concentration of triiodothyronine was higher in the small follicles group (1043 ng/ml) followed by the medium (545 ng/ml) and the lowest concentration was in the large follicles group (22 ng/ml). It is concluded that thyroxine concentrations are lower than T4 concentrations in preovulatory follicles irrespective of size as reported for human follicular fluid (Wakim. et. al. Fertil Steril 1993; 59(6) 1187–1190). Furthermore, no relationship was found between preovulatory follicular size and thyroxine hormone concentrations. It has been suggested that cortisol and triiodothyronine concentrations are related to preovulatory follicle size. Cortisol concentrations are higher in preovulatory follicles that in subordinate follicles and cortisol is a mediator of apoptosis in granulosa cells, differentiation, development and maturation of tissues, specifically in the oocyte (Acosta & Miyamoto. Anim Reprod Sci 2004; 82:127-140, Scarlett. et al. Dom Anim Endocrinol 2016; 59:11-22). The role of cortisol within the different sizes of preovulatory follicles is unknown.

Metabolite abundance in bovine preovulatory follicular fluid is influenced by follicle developmental progression post estrous onset in cattle.

Introduction: Preovulatory follicle response to the luteinizing hormone (LH) surge leads to metabolic, molecular, and functional changes in the oocyte and somatic follicular cells from the onset of estrus to ovulation. Follicular fluid contains metabolites, miRNAs, proteins, and hormones that are byproducts of follicular metabolism and support cellular processes of oocyte, cumulus, and granulosa constituents. Numerous studies have highlighted the importance of follicular fluid composition to support fertility, but critical gaps exist toward understanding dynamic modifications in the follicular fluid metabolome from estrous onset to ovulation. The hypothesis was that abundance of follicular fluid metabolites is dependent on follicle progression post LH surge and variability in follicular fluid metabolome profiles indicate key processes required for preparation of the follicle and oocyte for optimal fertility. The objective was to generate preovulatory follicular fluid metabolome profiles and discern differences in the metabolome of preovulatory follicular fluid samples collected at onset of estrus, 11 h post estrous onset, and 18 h post estrous onset. Methods: Estrus was synchronized in non-lactating Jersey cows (n=40) and follicular fluid was collected immediately after the first observed standing mount (hr 0) or at approximately h 11 or 18 after the first standing mount. Ultra-High-Performance Liquid Chromatography-High Resolution Mass Spectrometry was performed on preovulatory follicular fluid samples (n = 9 collected at hr 0, 9 at h 11, and 10 at h 18) and a multiple linear model was performed to determine if time post estrous onset impacted metabolite abundance. Results: Metabolites influenced by time post estrous onset were tested for enrichment in KEGG pathways. Ninety metabolites were identified in follicular fluid samples. Twenty metabolites differed in abundance among timepoints post estrous onset (p ≤ 0.05). Pathways corresponding to amino acid and energy metabolism were enriched with metabolites impacted by time post estrous onset (FDR ≤ 0.10). Discussion: Results from the current study indicate early response to the LH surge to increase bioavailability of amino acids and metabolites used by the cumulus and granulosa cells for energy production and shuttled into the oocyte to support meiotic maturation. Such metabolites may later be used by the ovulatory follicle for protein production.

Comparative analysis of biochemical, hormonal, and mineral compositions of preovulatory and cystic ovarian follicles in buffalo during the non-breeding season.

This study is a comparative analysis of the biochemical, hormonal, and mineral compositions of follicular fluid in preovulatory and cystic follicles of water buffalo (Bubalus bubalis). In total, reproductive tracts from 215 buffalo along with intact ovaries were collected randomly from an abattoir. The incidence of cystic conditions found in this study was 3.72% (8/215), involving the right ovary in 62.5% of instances and the left ovary in 37.5% of instances during the non-breeding season. Follicular fluid was aspirated from preovulatory follicles (12-15 mm diameter, oestrogen-active, follicular phase or stage IV corpus luteum on one of the two ovaries, n = 10) and cystic follicles (at least 20 mm diameter, no corpus luteum on any one of the two ovaries, n = 8). The follicular fluid samples were assayed for biochemical components (uric acid, creatinine, blood urea nitrogen, cholesterol, total protein, glucose, ascorbic acid, and alkaline phosphatase), hormones (progesterone, estradiol, and insulin), and minerals (calcium, magnesium, phosphorus, copper, zinc, and cobalt). Cystic follicles had greater (P < 0.05) concentrations of creatinine, blood urea nitrogen, cholesterol, progesterone, copper, zinc, and cobalt, and lesser (P < 0.05) concentrations of uric acid, glucose, ascorbic acid, estradiol, insulin, calcium, magnesium, and phosphorus compared with preovulatory follicles. These results indicated the marked differences in follicular fluid composition between preovulatory and cystic follicles in buffalo. Some of the changes were indicative of oxidative stress and disturbed steroidogenesis, two important mechanisms shown to be associated with cystic ovarian disease in various species. Further studies are warranted to investigate whether these differences are directly or indirectly involved in the formation of cystic follicles or are mere manifestations of the condition.

The effect of growth hormone on the metabolome of follicular fluid in patients with diminished ovarian reserve

BackgroundIncreasing evidence supports that the co-treatment with growth hormone (GH) enhances ovarian response and oocyte quality during controlled ovarian stimulation (COS) in patients with diminished ovarian reserve (DOR). The composition of follicular fluid (FF) plays an essential role in oocyte development and mirrors the communication occurring between the oocyte and follicular microenvironment. However, the effect of GH on the FF metabolome remains unclear.MethodsThis prospective observational study recruited DOR patients undergoing in vitro fertilization (IVF) cycles with minimal stimulation protocol for COS. Each patient receiving GH co-treatment was matched to a patient without GH co-treatment by propensity score matching. The FF was collected after isolating oocytes and assayed by gas chromatograph-mass spectrometry (GC-MS) metabolomics. The Pearson correlation was performed to evaluate the relationship between the number of oocytes retrieved and the levels of differential metabolites. The KEGG database was used to map differential metabolites onto various metabolic pathways.ResultsOne hundred thirty-four FF metabolites were identified by GC-MS metabolomics. Twenty-four metabolites, including glutathione, itaconic acid and S-adenosylmethionin (SAM) showed significant differences between the GH and control groups (p-value < 0.05 and q-value < 0.1). In addition, the number of oocytes retrieved was significantly higher in the GH group compared to the control group (3 vs 2, p = 0.04) and correlated with the levels of five differential metabolites. Among them, the levels of antioxidant metabolite itaconic acid were upregulated by GH administration, while SAM levels were downregulated.ConclusionsThe co-treatment with GH during COS may improve oocyte development by altering FF metabolite profiles in DOR patients. However, given the downregulation of SAM, a regulator of genomic imprinting, the potential risk of imprinting disturbances should not be neglected.

Does Endometriosis Impact the Composition of Follicular Fluid in IL6 and AMH? A Case-Control Study

The aim of this study was to compare follicular liquid levels of IL6 and AMH in women with and without endometriosis and to evaluate their potential impact on ICSI outcomes. It is a prospective case-control study conducted on 25 women with proven endometriosis and 50 patients diagnosed with other causes of infertility. All these patients were candidates for ICSI cycles. Their follicular fluid was collected at the time of oocyte retrieval and used to evaluate IL-6 and AMH titers by electro-chemiluminescent immunoassay (Cobas e411-Roche). The IL-6 levels in follicular fluid were higher in the endometriosis group than in the control group (152.3 vs. 19.9 pg/mL; p = 0.02). The median level for AMH was 2.2 ± 1.88 ng/mL with no statistical difference between the two groups (2.2 vs. 2.7 ng/mL, p = 0.41). No significant correlation between the follicular IL6 and AMH levels was observed. The oocyte quality seems to be preserved in patients with endometriosis with the adequate response to ovarian stimulation. High levels of follicular IL6 are in accordance with the inflammatory phenomenon of the disease; however, this increase has no impact on ICSI outcomes.

Effect of PFOA exposure on diminished ovarian reserve and its metabolism

Owing to its difficulty in degrading and ease of accumulation in the body, perfluorooctanoic acid (PFOA) has a detrimental effect on reproduction. This study aimed to examine the effect of PFOA concentration in follicular fluid during ovulation stimulation on embryo quality and the impact of PFOA exposure on the metabolic components of follicular fluid. This was a single-center prospective study that included 25 patients with diminished ovarian reserve (DOR), 25 with normal ovarian reserve (NOR), and 25 with polycystic ovary syndrome (PCOS). Follicular fluid samples were analyzed using ultra-high performance liquid chromatography-tandem mass spectrometry. We demonstrated that the PFOA levels of follicular fluid in the DOR group were higher than those in the NOR group and PCOS group (P < 0.05). PFOA concentration in the PCOS group was negatively correlated with high-quality embryos (P < 0.05). To gain more insight into the impact of PFOA on the metabolic composition of follicular fluid, we classified the DOR group based on the PFOA concentration, for which metabolomic analysis was performed. In the high-concentration PFOA group, there was an increase and a decrease in three and nine metabolites, respectively, compared to that in the low-concentration group. These results suggest that PFOA may alter the metabolic composition of follicular fluid, thus, affecting ovarian reserve function.

Biochemical alterations in the follicular fluid of bovine peri-ovulatory follicles and association with final oocyte maturation.

Follicular fluid (FF), a product of vascular transudate and granulosa and thecal cell secretions, is the milieu that has evolved to support oocyte growth and maturation which plays a central role in oocyte quality determination. Therefore, a suboptimal FF composition may be reflected in compromised oocyte progression through maturation, fertilization or embryo development. To date, the composition of bovine FF remains understudied. To address this, we comprehensively characterized the metabolomic constituency of bovine FF in the period during which the oocyte undergoes meiotic maturation. More specifically, FF from pre (-24 h) and peri (-2 h) -ovulatory follicles was profiled by high-throughput untargeted ultra-high-performance liquid chromatography tandem mass spectroscopy. A total of 634 metabolites were identified, comprising: lipids (37.1%), amino acids (30.0%), xenobiotics (11.5%), nucleotides (6.8%), carbohydrates (4.4%), cofactors and vitamins (4.4%), peptides (3.6%) and energy substrates (2.1%). The concentrations of 67 metabolites were significantly affected by stage of follicle development, 33.3% (n=21) were reduced (P≤0.05) by a mean of 9.0-fold, whereas 46 were elevated (P≤0.05) by a mean of 1.7-fold in peri vs. pre -ovulatory FF. The most pronounced individual metabolite concentration decreases were hypoxanthine (98.9-fold), xanthine (65.7-fold), 17β-oestradiol (12.4-fold), and inosine (4.6-fold). In contrast, the greatest increases were in retinal (4.9-fold), 1-methyl-5-imidazoleacetate (2.7-fold), and isovalerylcarnitine (2.7-fold). This global metabolomic analysis of bovine FF temporal dynamics provides new information for understanding the environment supporting oocyte maturation and facilitating ovulation, that has the potential for improving oocyte quality both in vivo and in vitro.