Follicular fluid is an important external environment for the growth and development of oocytes. A thorough identification of specific components in follicular fluid can better the existing understand of intracellular signal transduction and reveal potential biomarkers of oocyte health in women undergoing assisted reproductive therapy. To study on follicular fluid metabolomics components at different ages based on lipid metabolism, we have adopted a new method of SWATH to MRM(the sequential window acquisition of all theoretical fragment-ion spectra to multiple reaction monitor)metabolomics to provide extensive coverage and excellent quantitative data. This was done to investigate the differences in follicular fluid of patients undergoing in vitro fertilization (IVF) and embryo transfer in different age groups and to further explore the relationship between follicular fluid, age and reproductive function. A combination of Ultra-high-performance liquid chromatography and high resolution mass spectrometry techniques were used to analyze the follicular fluid of 230 patients enrolled for the IVF cycle. The patients were of different ages grouped into two groups:the younger and older patients.The obtained multidimensional chromatographic data were processed by principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). The charge ratios and mass numbers enabled for the identification of different fragments in the samples. Matching information obtained through database search and the fragment information obtained by fragment ion scan structurally identified substances in the samples. This was used to determine the differential compounds. The quality of oocytes decline with age,and the lipid composition in follicular fluid also changes,The lipid metabolism that changes with age may be related to the quality of oocytes.The main differences were in lipid metabolites. Some were up-regulated: Arachidonate, LysoPC(16:1), LysoPC(20:4) and LysoPC(20:3) while others were down-regulated: LysoPC(18:3) and LysoPC(18:1). Metabolomic analysis of follicular fluid revealed that with the increase in age, several differential metabolites are at play. Among these metabolites, lipid metabolism undergoes significant changes that affect the development of oocytes thus causing reduced fertility in older women. These differential metabolites related to follicular development may provide possible detection and treatment targets for promoting oocyte health, and provide scientific basis for understanding the environment of oocyte development.

/pdf/study-on-follicular-fluid-metabolomics-components-at-36fdvmit5x.pdf

Study on follicular fluid metabolomics components at different ages based on lipid metabolism.

Follicular atresia is one of the main factors limiting the reproductive power of domestic animals. At present, the molecular mechanisms involved in porcine follicular atresia at the metabolic level remain unclear. In this study, we divided the follicles of Bama Xiang pigs into healthy follicles (HFs) and atretic follicles (AFs) based on the follicle morphology. The expression of genes related to atresia in granulosa cells (GCs) and the concentration of hormones in the follicular fluid (FF) from HFs and AFs were detected. We then used liquid chromatography-mass spectrometry-based non-targeted metabolomic approach to analyze the metabolites in the FF from HFs and AFs. The results showed that the content of estradiol was significantly lower in AFs than in HFs, whereas that of progesterone was significantly higher in AFs than that in HFs. The expression of BCL2, VEGFA, and CYP19A1 was significantly higher in HFs than in AFs. In contrast, the expression of BAX and CASPASE3 was significantly lower in HFs. A total of 18 differential metabolites (DMs) were identified, including phospholipids, bioactive substances, and amino acids. The DMs were involved in 12 metabolic pathways, including arginine biosynthesis and primary bile acid biosynthesis. The levels of eight DMs were higher in the HF group than those in the AF group (p < 0.01), and those of 10 DMs were higher in the AF group than those in the HF group (p < 0.01). These findings indicate that the metabolic characteristics of porcine AFs are lower levels of lipids such as phospholipids and higher levels of amino acids and bile acids than those in HFs. Disorders of amino acid metabolism and cholic acid metabolism may contribute to porcine follicular atresia.

/pdf/non-targeted-metabolomics-reveals-metabolic-characteristics-283nwt2vm8.pdf

Non-targeted Metabolomics Reveals Metabolic Characteristics of Porcine Atretic Follicles.

Ovarian follicle provides a favorable environment for enclosed oocytes, which acquire their competence in supporting embryo development in tight communications with somatic follicular cells and follicular fluid (FF). Although steroidogenesis in theca (TH) and granulosa cells (GC) is largely studied, and the molecular mechanisms of fatty acid (FA) metabolism in cumulus cells (CC) and oocytes are emerging, little data is available regarding lipid metabolism regulation within ovarian follicles. In this study, we investigated lipid composition and the transcriptional regulation of FA metabolism in 3–8 mm ovarian follicles in bovine. Using liquid chromatography and mass spectrometry (MS), 438 and 439 lipids were identified in FF and follicular cells, respectively. From the MALDI-TOF MS lipid fingerprints of FF, TH, GC, CC, and oocytes, and the MS imaging of ovarian sections, we identified 197 peaks and determined more abundant lipids in each compartment. Transcriptomics revealed lipid metabolism-related genes, which were expressed constitutively or more specifically in TH, GC, CC, or oocytes. Coupled with differential lipid composition, these data suggest that the ovarian follicle contains the metabolic machinery that is potentially capable of metabolizing FA from nutrient uptake, degrading and producing lipoproteins, performing de novo lipogenesis, and accumulating lipid reserves, thus assuring oocyte energy supply, membrane synthesis, and lipid-mediated signaling to maintain follicular homeostasis.

https://www.mdpi.com/1422-0067/19/10/3261/pdf

Lipid Identification and Transcriptional Analysis of Controlling Enzymes in Bovine Ovarian Follicle.

Lipid metabolism in ovarian follicular cells supports the preparation of an enclosed oocyte to ovulation. We aimed to compare lipid composition of a dominant large follicle (LF) and subordinated small follicles (SFs) within the same ovaries. Mass spectrometry imaging displayed the differences in the distribution of several lipid features between the different follicles. Comparison of lipid fingerprints between LF and SF by Matrix Assisted Laser Desorption/Ionisation Time-Of-Flight (MALDI-TOF) mass spectrometry revealed that in the oocytes, only 8 out of 468 detected lipids (1.7%) significantly changed their abundance (p 2). In contrast, follicular fluid (FF), granulosa, theca and cumulus cells demonstrated 55.5%, 14.9%, 5.3% and 9.8% of significantly varied features between LF and SF, respectively. In total, 25.2% of differential lipids were identified and indicated potential changes in membrane and signaling lipids. Tremendous changes in FF lipid composition were likely due to the stage specific secretions from somatic follicular cells that was in line with the differences observed from FF extracellular vesicles and gene expression of candidate genes in granulosa and theca cells between LF and SF. In addition, lipid storage in granulosa and theca cells varied in relation to follicular size and atresia. Differences in follicular cells lipid profiles between LF and SF may probably reflect follicle atresia degree and/or accumulation of appropriate lipids for post-ovulation processes as formation of corpus luteum. In contrast, the enclosed oocyte seems to be protected during final follicular growth, likely due in part to significant lipid transformations in surrounding cumulus cells. Therefore, the enclosed oocyte could likely keep lipid building blocks and energy resources to support further maturation and early embryo development.

https://www.mdpi.com/1422-0067/21/18/6661/pdf

MALDI-TOF Mass Spectrometry Revealed Significant Lipid Variations in Follicular Fluid and Somatic Follicular Cells but Not in Enclosed Oocytes between the Large Dominant and Small Subordinate Follicles in Bovine Ovary.

Abstract In order to investigate the metabolic characteristics of human follicular fluid (FF) and to reveal potential metabolic predictors of follicular development (FD) with clinical implications, we analyzed a total of 452 samples based on a two-stage study design. In the first stage, FF samples from both large follicles (LFs) and matched-small follicles (SFs) of 26 participants were analyzed with wide-spectrum targeted metabolomics. The metabolic signatures were described by multi-omics integration technology including metabolomic data and transcriptomic data. In the second stage, the potential biomarkers of FD were verified using enzyme-linked immunoassay with FF and blood serum from an independent 200 participants. We describe the FF metabolic signatures from ovarian follicles of different developmental stages. Lysophosphatidylcholine (LPC) can be used as a biomarker of FD and ovarian sensitivity, advancing the knowledge of metabolic regulation during FD and offering potential detection and therapeutic targets for follicle and oocyte health improvements in humans. 

/pdf/metabolic-signatures-in-human-follicular-fluid-identify-2bft4bpb.pdf

Metabolic signatures in human follicular fluid identify lysophosphatidylcholine as a predictor of follicular development

BACKGROUND Increasing the success rate of in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) is a duty of clinicians that has made many seek a variety of protocols. This study was undertaken to use a liquid chromatography-mass spectrometry (LC-MS) to define the alterations of follicular fluid (FF) lipid metabolites in patients undergoing progestin-primed ovarian stimulation (PPOS) compared with short-term protocol, revealing potential correlations between the differentially expressed lipids and ameliorative clinical outcomes. MATERIAL AND METHODS Ninety-three infertile women undergoing IVF/ICSI treatment with PPOS (n=62) or a short-term protocol (n=31) were prospectively enrolled in a randomized controlled trial. FF samples were obtained from dominant follicles at the time of oocyte retrieval. Lipid metabolism profiles were analyzed using LC-MS. RESULTS Twelve lipids were found to be higher in patients treated with the PPOS protocol than in those receiving the short-term protocol, including triacylglycerols (TAG-34: 1+NH4, TAG-58: 0+NH4, TAG-64: 3+NH4, and TAG-64: 8+NH4), diacylglycerol DAG-38: 6+NH4, phosphatidylglycerols (PG-26: 0, PG-30: 2, and PG-40: 5), phosphatidylethanolamine PE-32: 2, lysophosphatidylethanolamine LPE-14: 1, lysophosphatidylinositol LPI-12: 0, and lysophosphatidylcholine LPC-16: 0. CONCLUSIONS Our data demonstrate that the PPOS protocol increases the levels of 12 lipids in FF, which reveals a strong association between the differentially elevated lipids and better IVF/ICSI outcomes.

/pdf/lipidomic-components-alterations-of-human-follicular-fluid-26ovp3982u.pdf

Lipidomic Components Alterations of Human Follicular Fluid Reveal the Relevance of Improving Clinical Outcomes in Women Using Progestin-Primed Ovarian Stimulation Compared to Short-Term Protocol.

Medroxyprogesterone 17-acetate (MPA) combined with human menopausal gonadotropin (hMG) has been effectively used for ovarian stimulation in clinical practice. However, the molecular mechanism of MPA + hMG treatment in follicular development is poorly described. Here we performed a study to investigate the impact of MPA + hMG on ovarian stimulation utilizing a mouse model in vivo. Forty female BALB/C mice were randomly divided into four groups of 10 each and treated during ciestrus stage and continued for 5 days: control group, MPA group, hMG group, and MPA + hMG group. Morphological and molecular biology methods were used for detecting serum hormones and ovarian function. MPA + hMG group exhibited increasing follicle stimulating hormone (FSH), antral follicle, FSH receptor (FSHR) and phosphorylated mammal target of rapamycin (p-mTOR), and decreasing luteinizing hormone (LH), estradiol (E2), progesterone (P), corpus luteum, phosphoinositide 3-kinase (PI3K), Akt and mTOR compared with control group. In contrast, MPA + hMG group showed reduced FSH, LH, E2, P, corpus luteum, LH receptor (LHR), and activated PI3K,/Akt/mTOR pathway compared with hMG group (P < 0.05). Collectively, these data definitively established that MPA plus hMG may modulate the hormone, hormone receptor and PI3K/Akt/mTOR signaling pathway to influence follicular development in the mouse ovary. Our study provides overwhelming support for MPA + hMG as an effective treatment for infertility in women.

/pdf/the-role-of-combining-medroxyprogesterone-17-acetate-with-3qbwd7o6d2.pdf

The role of combining medroxyprogesterone 17-acetate with human menopausal gonadotropin in mouse ovarian follicular development

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Lipidomic Components Alterations of Human Follicular Fluid Reveal the Relevance of Improving Clinical Outcomes in Women Using Progestin-Primed Ovarian Stimulation Compared to Short-Term Protocol.

The role of combining medroxyprogesterone 17-acetate with human menopausal gonadotropin in mouse ovarian follicular development