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

Infertility is a worldwide problem affecting couples in their childbearing age. It is one of the three major diseases affecting human life and health. Development of social economy has led to a change in the concept of marriage and childbearing. The reproductive age of women has gone higher, and more older women are having pregnancy requirements. However, the reproductive capability of more older women is declining [1]. Finding solutions to fertility problems of aging women has become of paramount importance in reproductive medicine.However,the mechanism of age-related decline in reproductive function is still unclear. Numerous studies have shown that its mechanism may be related to a series of changes such as increased oocyte apoptosis, oxidative stress disorder, energy conversion disorder, and endocrine and metabolic abnormalities caused by age [2‐6].

Follicular fluid is the culture medium in which an oocyte grows and differentiates. It directly or indirectly influences the fertility and up growth potency of oocytes thus indirectly affecting women’s reproductive function.Changes in follicular microenvironment in older women may be responsible for altered protein function thus affecting the viability of oocytes. This undoubtedly causes adverse pregnancy outcomes [7, 8].

Metabolomics is an emerging omics technology developed after genomics and proteomics.It enables simultaneous qualitative and quantitative analysis of all metabolites of an organ, tissue or cells in a specific physiological period or condition in order to find target differential metabolites. It can be used for early diagnosis of disease, drug target discovery and disease mechanism research. Rapid development of metabolomics have enabled the use of follicular fluid as an objective evaluation index to reflect the quality of oocytes. This plays an important role in predicting the outcome of an in-vitro fertilization (IVF) cycle. Researchers in reproductive medicine have identified follicular fluid as a biomarker source for evaluating oocyte quality. Changes in its composition affect oocyte quality.Results obtained from the metabolomics analysis of follicular fluid have become an important indicator of clinical prediction of IVF outcomes [9].Previous studies have shown that the composition of the follicular fluid changes with age, which may be an important cause of the aging of oocytes [10]. These studies also revealed that the change process of female reproductive function may be the result of comprehensive regulation of multiple aspects and approaches. This study used metabolomics to analyze human follicular fluids (HFF) from patients of different ages.It opened up a new direction for exploring the mechanism of oocyte aging and provided new ideas and methods for treating senile infertility as well as improving IVF cycle outcomes.

In the present study, we differentiated the characteristics of metabolic ions in follicular fluid in the younger and older patients and identified several differential metabolites.

Metabonomic analysis of follicular fluid showed that several major metabolites changed with age, with some upregulated: arachidonic acid, LysoPC (16:1), LysoPC (20:4) and LysoPC (20:3), while others downregulated: LysoPC (18:3) and LysoPC (18:1). Among these metabolites, significant changes will take place in lipid metabolism, mainly characterized by the up-regulation of arachidonic acid, which affects oocyte development, while LPC participates in the regulation of follicular development and oocyte maturation, and its complex changes are closely related to follicular development. These differential metabolites related to follicular development may provide possible detection and therapeutic targets for promoting oocyte health. To provide scientific basis for understanding the environment of oocyte development.

Lipids is the general term used to refer to oils, fats, and lipids. They are important nutrients needed by the human body. They supply energy needed by the body as well as provide essential fatty acids for the body. Similarly, lipids are important energy support for oocyte growth and development; they participate in the construction of its membrane, regulate cell cycle and survival, malignant transformation and apoptosis [7‐9].. It can be assumed that as age increases, lipid metabolism in the follicular fluid may cause a change in the oocyte. However,no study has shown an existing relationship between lipid metabolism oocyte aging. This study aimed to explore the role of lipid metabolism in oocyte aging in two different age groups through follicular fluid metabolomics It at the same time focused on opening up new directions for the treatment of senile infertility and provide new ideas and methods to improve IVF outcome. This study suggests that a decline in female fertility due to age might be related to the change in lipid metabolism in the follicular fluid.

Arachidonate (AA) is an omega-6 polyunsaturated fatty acid similar to the saturated peanut acid in peanut oil [1]. AA is an all-cis-5,8,11,14-eicosatetraenoic acid with a chemical formula: CH3(CH2)4(CH=CH-CH2)4(CH2)2COOH. It is a saturated fatty acid containing four carbon-carbon double bonds and one carbon-oxygen double bond. It is widely distributed in the animal kingdom though it is found in small amounts in a variety of glycerides and glycerophospholipids. It has been found that AA and niclosamide potentially induce apoptosis through the common pathway of cytochrome c release from mitochondria.This is consistent with the view that niclosamide partially mediates cell death through arachidonic acid. Interestingly, AA has been found to increase mitochondrial permeability transition (PT) and cytochrome c release [13].

In addition to the environment in which lipid metabolism affect follicular development, lipid peroxidation is also closely related to follicular development [14]. One study found that the expressions of arachidonic acid 12-lipoxygenase (Alox12) and arachidonic acid 15-lipoxygenase (Alox15) were significantly up-regulated in an obese diet reflecting an increase in lipid peroxidation. Lipid peroxidation is associated with many disease states associated with oxidative stress, aging and metabolic diseases [14]. Understanding of the multiple roles of lipoxygenases (other than in the classic arachidonic acid cascade) is increasing [15], creating a clearer picture of their role in generating the cellular redox balance.Lipoxygenases are pro-oxidative enzymes that by forming hydroperoxy lipids,can alter the redox state and gene expression pattern within the cell [16]. Apart from the implications for cellular redox balance, an increase in lipoxygenase activity also leads to greater oxidation of membrane lipids which in turn impairs the normal functions of the cell membrane and membrane-bound enzymes [17].Such impairments at the tissue level early in reproductive life may lead to increasing dysregulation of normal ovarian function later in life. Furthermore, there may be direct effects of Alox12 expression on both ovulation and follicular reserve. As an example, Alox12 is expressed in granulosa cells, thecal cells and follicular fluid at the time of ovulation in rats [18, 19],while inhibition of lipoxygenase can impair ovulation [19].Intriguingly,polymorphisms in the Alox12 gene have been linked with early age natural menopause in various human populations [20, 21], implying that their expression may be key to follicular reserve later in life.Estrogen biosynthesis and proteolysis are both important processes involved in ovarian follicular development though they may be influenced by cytochrome P450 (CYP)-dependent fatty acid metabolites. However, CYP-dependent lipid metabolism has not been characterized with respect to follicular maturation in vivo. The dynamic changes in follicular CYP-dependent arachidonic acid metabolites and their modulatory function in vascular models suggest roles for these metabolites in follicular maturation. This may include regulation of estradiol biosynthesis and preovulatory remodeling of the follicular wall that should be fully explored in future studies [22]. Folliculogenesis is an ordered sequence of oocyte development and maturation;it involves the proliferation and differentiation of granulosa cells and is crucial for mammalian reproduction [23]. During follicular development, a series of changes occur. First, the primordial follicle grows into a growth follicle with somatic cells proliferation after which the follicular antrum is formed as the ovarian granulosa cell undergoes proliferation and differentiation. The majority of follicles then become atresia and only few grow dramatically to become dominant follicles and ovulation follows. Follicular fluid (FF) within the follicular antrum is the microenvironment of oocyte. It contains hormones, growth factors, proteins and phospholipids which are partially produced by Granulosa cells(GCs). Phospholipids are the most abundant lipids in the cell membrane and they include Lysophosphatidic Acid (LPA), LysophosphatidylCholine (LPC), Sphingosine-1 Phosphates and Sphingophoryl Choline [24]. Some studies have shown that LPC is metabolized into LPA by phospholipase [25, 26].

A potential biological mechanism by which LPC could influences mitochondrial oxidative capacity is through its role in the synthesis pathway of cardiolipin. Cardiolipin is a unique dimeric phospholipid containing four fatty acid chains that are specific to mitochondria. Cardiolipin is an essential constituent of mitochondrial membranes [27]. LPC in human plasma can be generated by three activities: phospholipase A2 (PLA2) on phosphatidylcholine, by the activity of endothelial lipase (EL) including phospholipase A1 (PLA1) on high-density lipoprotein [28] and from phosphatidylcholine during the formation of cholesteryl esters. LPC is also found in membranes both mitochondria and endoplasmic reticulum [29, 30]. Synthesis of cardiolipin involves LPA and phosphatidic acid (PA). LPC can be hydrolyzed to LPA by autotaxin, a secreted glycoprotein that is widely expressed in tissues [31, 32]. LPA can also be formed from the acylation of glycerol-3-phosphate on the outer mitochondrial membrane. Two isoforms of acylglycerol-3-phosphate acyltransferase (AGPAT); AGPAT 4 and 5 are located on the outer mitochondrial membrane and catalyze the acylation of LPA to PA [33]. PA is transferred to the inner mitochondrial membrane and is converted to nascent cardiolipin via CDP-glycerol, phosphatidylglycerophosphate, and phosphatidylglycerol. LPCs are highly mobile within intact cells and thus are good candidates for a cytoplasmic messenger that transduces signals to activate downstream processes and gene expression in the nucleus [34]. LPC can activate several second messengers; extracellular-signal-regulated kinases and protein kinase C, which have been shown to be involved in the regulation of follicular development and oocyte maturation [35]. In humans, blood triglyceride levels tend to increase, while blood lysophosphatidylcholine levels tend to decrease with age. Specific sphingolipid and phospholipid blood profiles have also been shown to change with age and are associated with exceptional human longevity. These data suggest that lipid-related interventions may improve human health span and that blood lipids likely represent a rich source of human aging biomarkers [36].

Using advanced SWATH™-based mass spectrometry, we investigated metabolic changes in the follicular fluid of the younger and older patients.Metabonomic analysis of follicular fluid showed that several major metabolites changed with age, with some upregulated: arachidonic acid, LysoPC (16:1), LysoPC (20:4) and LysoPC (20:3), while others downregulated: LysoPC (18:3) and LysoPC (18:1). Among these metabolites, significant changes will take place in lipid metabolism, mainly characterized by the up-regulation of arachidonic acid, which affects oocyte development, while LPC participates in the regulation of follicular development and oocyte maturation, and its complex changes are closely related to follicular development. These differential metabolites related to follicular development may provide possible detection and therapeutic targets for promoting oocyte health. To provide scientific basis for understanding the environment of oocyte development.