Fernanda Bertuccez Cordeiro

Proteomic analysis of follicular fluid from women with and without endometriosis: new therapeutic targets and biomarkers.

Endometriosis is a gynecological disease that affects women of reproductive age. The protein profiles of women with endometriosis who were able or unable to achieve pregnancy and women without endometriosis who did achieve pregnancy were compared in this study. The follicular fluid was collected from 21 patients undergoing in vitro‐fertilization treatment, according to the following groups: nine women in the control group (Group C), four women with endometriosis who achieved pregnancy (Group E.P), and eight women with endometriosis who did not achieve pregnancy (Group E.NP). Follicular fluid proteins were separated using 2D‐electrophoresis,and their spots were compared, excised, and submitted to LC–ESI‐MS/MS for proteins identification. The analysis showed 29 differentially expressed spots among the groups, and from these, 21 proteins were identified. Analysis showed some functional enrichment in the E.P group, including response to oxidative stress and apoptosis, while the E.NP group showed functions related to response to reactive oxygen species and positive regulation of apoptosis. These data suggest that endometriosis leads to differential protein expression in the follicular fluid, which can influences the outcome of pregnancy. These proteins may be potential targets for better diagnostics and new therapeutic intervention in affected women, as well as assisting in comprehending the physiopathologic mechanisms underlying endometriosis. Mol. Reprod. Dev. 80: 441–450, 2013.

Lipidomics analysis of follicular fluid by ESI-MS reveals potential biomarkers for ovarian endometriosis

PurposeThe aim of the present study was to analyze the lipid profile of follicular fluid from patients with endometriosis and endometrioma who underwent in vitro fertilization treatment (IVF).MethodsThe control group (n = 10) was composed of women with tubal factor or minimal male factor infertility who had positive pregnancy outcomes after IVF. The endometriosis group consisted of women with endometriosis diagnosed by videolaparoscopy (n = 10), and from the same patients, the endometriomas fluids were collected, which composed the endometrioma group (n = 10). From the follicular fluid and endometriomas, lipids were extracted by the Bligh and Dyer method, and the samples were analyzed by tandem mass spectrometry.ResultsWe observed phosphatidylglycerol phosphate, phosphatidylcholine, phosphatidylserine, and phosphatidylnositol bisphosphate in the control group. In the endometriosis group, sphingolipids and phosphatidylcholines were more abundant, while in the endometrioma group, sphingolipids and phosphatidylcholines with different m/z from the endometriosis group were found in high abundance.ConclusionThis analysis demonstrated that there is a differential representation of these lipids according to their respective groups. In addition, the lipids found are involved in important mechanisms related to endometriosis progress in the ovary. Thus, the metabolomic approach for the study of lipids may be helpful in potential biomarker discovery.

Lipid profiling of follicular fluid from women undergoing IVF: Young poor ovarian responders versus normal responders

Abstract This study identified possible lipid biomarkers in follicular fluid from women with poor ovarian response. These biomarkers indicate pathophysiological pathways and have potential diagnostic applications. An observational case-control study of young women undergoing ovarian stimulation for in-vitro fertilization was conducted. The participants were categorized into a poor ovarian response group and a normal ovarian response to stimulation group. All of the women underwent the same ovarian stimulation protocol, and follicular fluid was collected after ovarian aspiration. Analyses were performed using matrix-assisted laser desorption/ionization mass spectrometry. Principal component analysis and Volcano plots were used to describe follicular fluid classification models based on the lipid profiles. A total of 10 lipids were differentially expressed between the study and control groups. Of these lipid ions, three belonged to the phosphatidylcholine subclass and were present in higher concentrations in the control group. The other seven differential lipids were present in the study group and classified into four lipid subclasses: phosphatidylethanolamines, phosphatidylglycerols, phosphatidylinositols, and diacylglycerols. These distinctive lipids may be involved in hormonal responses and oocyte development processes and may be useful as biomarkers for therapeutic intervention in women with poor ovarian response.

Follicular fluid alterations in endometriosis: label-free proteomics by MSE as a functional tool for endometriosis

Abstract Endometriosis is a chronic gynecological condition that affects 10-32% of women of reproductive age and may lead to infertility. The study of protein profiles in follicular fluid may assist in elucidating possible biomarkers related to this disease. For this, follicular fluid samples were obtained from women with tubal factor or minimal male factor infertility who had pregnancy outcomes after in vitro fertilization (IVF) treatment (control group, n = 10), women with endometriosis (endometriosis group, n = 10), along with the endometrioma from these same patients were included (endometrioma group, n = 10). For proteomic analysis, samples were pooled according to their respective groups and normalized to protein content. Proteins were analyzed by in tandem mass spectrometry (MSE) Spectra processing and the ProteinLynx Global Server v.2.5. was used for database searching. Data was submitted to the biological network analysis using Cytoscape 2.8.2 with ClueGO plugin. As a result, 535 proteins were identified among all groups. The control group differentially or uniquely expressed 33 (6%) proteins and equal expression of 98 (18%) proteins was observed in the control and endometriosis groups of which 41 (7%) proteins were further identified and/or quantified. Six (1%) proteins were observed in both the endometriosis and endometrioma groups, but 212 (39%) proteins were exclusively identified and/or quantified in the endometrioma group. There were 9 (1%) proteins observed in both the control and endometrioma groups and there were 139 (25%) proteins common among all three groups. Distinct differences among the protein profiles in the follicular fluid of patients included in this study were found, identifying proteins related to the disease progression and IVF success. Thus, some pathways related to endometriosis are associated with the presence of specific proteins, as well as the absence of others. This study provides a first step to the development of more sensitive diagnostic tests and treatment.

Follicular fluid lipid fingerprinting from women with PCOS and hyper response during IVF treatment

PurposePolycystic ovary syndrome (PCOS) is an endocrine-metabolic disorder that leads to lower natural reproductive potential and presents a challenge for assisted reproductive medicine because patients may exhibit immature oocyte retrieval and a higher risk of ovarian hyper stimulation syndrome during in vitro fertilization (IVF) treatment. This study aimed to identify potential lipid biomarkers for women with PCOS and a hyper response to controlled ovarian stimulation.MethodsFollicular fluid samples were collected from patients who underwent IVF, including normal responder women who became pregnant (control group, n = 11), women with PCOS and a hyper response to gonadotropins (PCOS group, n = 7) and women with only hyper response to gonadotropins (HR group, n = 7). A lipidomic analysis was performed by electrospray ionization mass spectrometry, and candidate biomarkers were analyzed by tandem mass spectrometry experiment.ResultsThe lipid profiles indicated particularities related to differences in phosphatidylcholine (PCOS and HR), phosphatidylserine, phosphatydilinositol and phosphatidylglycerol (control), sphingolipids (PCOS) and phosphatidylethanolamine (control and HR).ConclusionsThese findings contribute to the understanding of the molecular mechanisms associated with lipid metabolism in the PCOS-related hyper response, and strongly suggest that these lipids may be useful as biomarkers, leading to the development of more individualized treatment for pregnancy outcome.

Protein expression in human cumulus cells as an indicator of blastocyst formation and pregnancy success

PurposeThe goal for the present study was to implement a technique for protein extraction and identification in human cumulus cells (CCs).MethodsForty samples of CCs were collected after ovum pick-up from patients undergoing intracytoplasmic sperm injection (ICSI). Samples were split into the blastocyst group (n = 10), including patients in which all embryos converted into blastocysts, and the non-blastocyst group (n = 10), including patients in which none of the embryos reached the blastocyst stage or the positive-pregnancy (n = 10) and negative-pregnancy group (n = 10). Proteins were extracted and injected into a liquid chromatography system coupled to a mass spectrometer. The spectra were processed and used to search a database.ResultsThere were 87 different proteins in samples from the blastocyst and non-blastocyst groups, in which 30 were exclusively expressed in the blastocyst group and 17 in the non-blastocyst group. Among the 72 proteins detected in the pregnancy groups, 19 were exclusively expressed in the positive, and 16 were exclusively expressed in the negative-pregnancy group.ConclusionsCC proteomics may be useful for predicting pregnancy success and the identification of patients that should be included in extended embryo culture programs.

Is Lipidomic the Answer to the Search of a Biomarker for Organ Preservation Protocol in Head and Neck Squamous Cell Carcinoma

In the last decade organ preservation protocols based on chemoradiotherapy (CRT) has been showing the possibility of preserving function without jeopardizing survival for locally advanced head and neck squamous cell carcinoma (HNSCC). Still, only a percentage of the patients will benefit from this approach and, to date, no biomarkers are known to correctly predict these patients. More recently, modern mass spectrometry method has been used to determine metabolic profiles, and lipidomics, in particular, emerged as a new field of study in oncology and other diseases. This study aimed to analyze the lipid profile on saliva from patients undergoing to a prospective, single center, open-label, non-randomized phase II trial for organ preservation on HNSCC. The lipid analysis was performed using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). Multivariate statistical analyses based on principal component analysis and orthogonal partial least square-discriminant analysis were applied to MALDI-TOF-MS data to visualize differences between the lipid profiles and identify potential biomarkers. The results assisted on distinguishing complete responders from non-responders to the treatment protocol. In conclusion, we demonstrated that a group of lipids is differentially abundant in saliva from HNSCC patients submitted to an organ preservation protocol, being able to differentiate responders from non-responders. These results suggest the potential use of lipid biomarkers to identify patients who may benefit from this treatment. Also, we showed that saliva testing might be routinely used in clinical practice, for being a non-invasive alternative to blood testing, besides inexpensive and easy to obtain.

Hyper response to ovarian stimulation affects the follicular fluid metabolomic profile of women undergoing IVF similarly to polycystic ovary syndrome

IntroductionDuring in vitro fertilization (IVF), the hyper response to controlled ovarian stimulation (COS) is a common characteristic among patients diagnosed with polycystic ovary syndrome (PCOS), although non-diagnosed patients may also demonstrate this response.ObjectivesIn an effort to investigate follicular metabolic characteristics associated with hyper response to COS, the present study analyzed follicular fluid (FF) samples from patients undergoing IVF.MethodsFF samples were obtained from patients with PCOS and hyper response during IVF (PCOS group, N = 15), patients without PCOS but with hyper response during IVF (HR group, N = 44), and normo-responder patients receiving IVF (control group, N = 22). FF samples underwent Bligh and Dyer extraction, followed by metabolomic analysis by ultra-performance liquid chromatography mass spectrometry, considering two technical replicates. Clinical data was analyzed by ANOVA and chi-square tests. The metabolomic dataset was analyzed by multivariate statistics, and the significance of biomarkers was confirmed by ANOVA.ResultsClinical data showed differences regarding follicles production, oocyte and embryo quality. From the 15 proposed biomarkers, 14 were of increased abundance in the control group and attributed as fatty acids, diacylglycerol, triacylglycerol, ceramide, ceramide-phosphate, phosphatidylcholine, and sphingomyelin. The PCOS patients showed increased abundance of a metabolite of m/z 144.0023 that was not attributed to a class.ConclusionThe clinical and metabolic similarities observed in the FF of hyper responders with and without PCOS diagnosis indicate common biomarkers that could assist on the development of accessory tools for assessment of IVF parameters.

Follicular fluid lipidomics reveals lipid alterations by LH addition during IVF cycles

IntroductionOvulation induction protocols are key components for performing assisted reproduction treatments successfully. The importance of adding exogenous LH to the controlled ovarian stimulation protocols in young women is still a matter of discussion in the clinical practice.ObjectiveTo estimate how LH addition to controlled ovarian stimulation protocols may affect the follicular fluid lipid profile of women undergoing in vitro fertilization treatment.MethodsWe conducted the study using 28 self-paired samples, 14 per group. The patients received FSH during their first cycle of ovarian stimulation (FSH group). If the treatment did not result in pregnancy, the same patients returned for a new cycle and received stimulus with the addition of LH to the previous protocol (Low-dose-LH group). Lipidomics analysis was performed by UPLC-MSE mass spectrometry. The software Progenesis QI was used to identify potential lipid biomarkers. Statistical analysis was performed using the SPSS 18.0 and MetaboAnalyst 2.0 software.ResultsThe analysis of clinical data showed no statistically significant differences between groups, in contrast to lipidomic analysis. Concerning lipidomic profile, the lipids differentially expressed in FSH group belong to the following subclasses:triacylglycerols; branched fatty acids and diacylglycerophosphoethanolamines; while in Low-dose-LH group the subclasses are gangliosides; acylglycerophosphoethanolamines; triacylglycerols; acylglycerophosphoserines; GalNAcβ1-3Galα1-4Galβ1-4Glc-(Globo series); amino fatty acids; triacylglycerols and prostaglandins.ConclusionThe differences found between the groups may contribute to the establishment of potential therapeutical targets based on LH-associated lipid biomarkers aiming to individualize treatments and obtain reproductive success.

Follicular fluid lipid peroxidation levels in women with endometriosis during controlled ovarian hyperstimulation

Abstract This observational study aimed to establishing a relationship between lipid peroxidation and endometriosis in women undergoing controlled ovarian hyperstimulation. A total of 79 women were divided into two groups: (i) controls (tubal or male factor); and (ii) endometriosis (stages III/IV). The endometriosis diagnosis was confirmed by videolaparoscopy and the controlled ovarian stimulation protocol was similar to all patients. Follicular fluid (FF) lipid peroxidation levels were determined through the quantification of malondialdehyde. Statistical analysis was performed using parametric and non-parametric tests, logistic regression was performed to estimate the chance of achieving a pregnancy in each group and a moving average was calculated for the endometriosis group. Peroxidation levels in the endometriosis group were significantly higher when compared to controls. The moving average showed a decrease of MDA levels in the endometriosis group with increasing female age. Moreover, women with endometriosis who were under 33 years of age were 4.3 times more likely to achieve a pregnancy than women above that age. In conclusion, endometriosis is associated with increased FF oxidative stress (OS) in patients undergoing in vitro fertilization (IVF). Also, increasing age is associated with a decrease in severity of the oxidative status, but a decreased chance of pregnancy.