Impact of Extraction Methods and Transportation Conditions on Lipid Profiles of Bovine Oocytes.

Lipids play numerous pivotal physiological roles in mammalian reproduction, being indispensable for oocyte competence acquisition and post-fertilization embryonic development. Profiling lipids in minute samples, such as oocytes, presents challenges but has been accomplished through mass spectrometry technologies like Multiple Reaction Monitoring (MRM) profiling. With the dual objectives of simplifying workflow and examining the influence of preanalytical conditions, we assessed whether transportation at room temperature affects the lipid profile of bovine oocytes. To this end, samples were prepared using either monophasic (methanol only) or biphasic liquid extraction protocols (Bligh & Dyer method) and transported either on dry ice or at room temperature inside sealed-vacuum packages to prevent lipid oxidation. Subsequently, employing a comprehensive method, we screened a list of 316 MRMs from 10 different lipid subclasses in oocyte lipid extracts. Principal Component Analysis (PCA) revealed similar lipid profiles concerning temperature during transportation, whereas clear differentiation among samples was observed based on the lipid extraction method. Univariate analysis indicated that the one-phase methanol extraction resulted in higher relative abundances of phospholipids, except for phosphatidylserines. Conversely, the Bligh & Dyer extraction favored the detection of neutral intracellular lipids (triacylglycerols, free fatty acids, cholesteryl esters, and acyl-carnitines). Consequently, lipid recovery was directly correlated with the polarity of lipid class and the extraction method. Regarding transportation temperature, phosphatidylethanolamine, triacylglycerol, and free fatty acids exhibited lower abundances when samples were transported at room temperature. Based on multivariate and univariate analyses, we conclude that if samples undergo the same lipid extraction protocol and are transported in the same batch at room temperature inside vacuum-sealed bags, it is feasible to analyze lipid extracts of bovine oocytes and still obtain informative lipid profiling results.

Contribution of lipids to the organelle differential profile of in vitro-produced bovine embryos.

Each living organism is unique because of the lipid identity of its organelles. The diverse distribution of these molecules also contributes to the role of each organelle in cellular activity. The lipid profiles of whole embryos are well documented in the literature. However, this approach can often lead to the loss of relevant information at the subcellular and consequently, metabolic levels, hindering a deeper understanding of key physiological processes during preimplantation development. Therefore, we aimed to characterize four organelles in vitro-produced bovine embryos: lipid droplets (LD), endoplasmic reticulum (ER), mitochondria (MIT), and nuclear membrane (NUC), and evaluate the contribution of the lipid species to each organelle evaluated. Expanded blastocysts were subjected to cell organelle isolation. Thereafter, lipid extraction from cell organelles and lipid analysis using the Multiple Reaction Monitoring (MRM) profiling method were performed. The LD and ER displayed a greater number of lipids (Phosphatidylcholine - PC, Ceramide - Cer, and Sphingomielin - SM) with high signal-to-noise intensities. This result is due to the high rate of biosynthesis, lipid distribution, and ability to store and recycle lipid species of these organelles. The NUC had a more distinct lipid profile than the other three organelles, with high relative intensities of PC, SM, and triacylglycerols (TG), which is consistent with its high nuclear activity. MIT had an intermediate profile that was close to that of LD and ER, which aligns with its autonomous metabolism for some classes of phospholipids (PL). Our study revealed the lipid composition of each organelle studied, and the roles of these lipids could be associated with the characteristic organellar activity. Our findings highlight the lipid species and classes that are relevant for the homeostasis and function of each associated organelle and provide tentative biomarkers for the determination of in vitro embryonic development and quality.

Effect of lipid extraction and room temperature transportation of bovine oocytes determined by MRM profiling.

Lipids play many important physiological roles in mammalian reproduction, being essential for the acquisition of oocyte competence and post-fertilization embryonic development. Lipid profiling in samples of minute size, such as oocytes, is challenging but has been achieved by mass spectrometry technologies such as multiple reaction monitoring (MRM) profiling. With the goals of further simplifying sample workflow and investigating the influence of pre-analytical conditions, we have evaluated how different extraction methods and transportation of lipid extracts in vacuum and at room temperature impacted the lipid profile of bovine oocytes. Using a comprehensive method, 316 MRMs associated with lipids of 10 different classes were screened in oocyte lipid extracts prepared by 2 extraction methods (one-step methanol addition or Bligh and Dyer) and transporting them in dry ice or at room temperature inside vacuum packages. No changes in the multivariate analysis (PCA) were noticeable due to transportation temperature, while lipid profiles were more affected by the lipid extraction protocol. Sample extraction using pure methanol favored the detection of phospholipids uniformly, while Bligh and Dyer favored the detection of neutral intracellular lipids. Triacylglycerol lipids and free fatty acids yielded decreased abundances when samples were transported at room temperature. We conclude that if samples are submitted to the same lipid extraction protocol and same transportation batch at room temperature coupled with vacuum conditions it is possible to analyze lipid extracts of bovine oocytes and still obtain informative lipid profiling results.

Proteomic profile of extracellular matrix from native and decellularized chorionic canine placenta.

Placental plasticity, employing rapid growth and remodeling to supply the growing fetus, is majorly related to its extracellular matrix (ECM) components. Thus, we studied the proteome profiled of canine native and decellularized placenta to characterize the proteome related to maintenance of a microenvironment and structure suitable for tissue engineering applications. Protein was profiled from native (n=3) and decellularized (n=3) 35-days old canine placenta using the mass spectrometer Orbitrap Fusion Lumos. A total of 52 proteins were filtered and revealed ontologies connected to skeleton structuration, collagen processing, germ layers formation, cell adhesion, response to amino acids, and others. Also, the major enriched pathways were ECM-receptor interaction, focal adhesion, PI3K-Akt signaling, protein digestion and absorption. Aside, proteins related to structure (collagens), cell adhesion (laminin and fibronectin), ECM remodeling (MMP2 and TIMP3) and vascularization (VEGF and RLN) were present in decellularized condition. Our findings support the requirement of a proteomic profile to visualize the maintenance of essential protein groups for ECM structuring and physiology, that should support functions related to cell adhesion, vasculogenesis and as a reservoir of soluble molecules. Altogether, the 35-days old decellularized canine placenta can provide an adequate microenvironment for cell anchoring for further regenerative medicine application.

Proteomic profile of extracellular matrix from native and decellularized chorionic canine placenta

Placental plasticity, employing rapid growth and remodeling to supply the growing fetus, is majorly related to its extracellular matrix (ECM) components. Thus, we studied the proteome profiled of canine native and decellularized placenta to characterize the proteome related to maintenance of a microenvironment and structure suitable for tissue engineering applications. Protein was profiled from native (n=3) and decellularized (n=3) 35-days old canine placenta using the mass spectrometer Orbitrap Fusion Lumos. A total of 52 proteins were filtered and revealed ontologies connected to skeleton structuration, collagen processing, germ layers formation, cell adhesion, response to amino acids, and others. Also, the major enriched pathways were ECM-receptor interaction, focal adhesion, PI3K-Akt signaling, protein digestion and absorption. Aside, proteins related to structure (collagens), cell adhesion (laminin and fibronectin), ECM remodeling (MMP2 and TIMP3) and vascularization (VEGF and RLN) were present in decellularized condition. Our findings support the requirement of a proteomic profile to visualize the maintenance of essential protein groups for ECM structuring and physiology, that should support functions related to cell adhesion, vasculogenesis and as a reservoir of soluble molecules. Altogether, the 35-days old decellularized canine placenta can provide an adequate microenvironment for cell anchoring for further regenerative medicine application.

Effects of paternal diet and antioxidant addition to the semen extender on bovine semen characteristics and on the phenotype of the resulting embryo.

The aim of this study was to examine the effects of long-term dietary supplementation of young Nellore bulls with rumen-protected polyunsaturated fatty acids (PUFAs) and of the inclusion of catalase in the semen extender on semen quality, in vitro sperm fertilizing ability, and intracytoplasmic lipid content in the resulting embryos. Twelve Nellore bulls were supplemented with rumen-protected PUFAs or with a basal diet from 14 to 24 months of age. The semen was collected at the end of supplementation. For cryopreservation, the ejaculate was divided into two equal volumes and catalase was added to the extender in one of the fractions. Thus, the experimental design consisted of a 2 × 2 factorial scheme with two diets (control and PUFA) and two extenders (Cat+ and Cat-). Total motility and the percentage of rapid cells in fresh semen were negatively affected by dietary supplementation with PUFAs (P < 0.05), but these effects did not persist after freezing. The frozen/thawed semen of animals fed PUFAs exhibited an increase in the percentages of damaged plasma and acrosomal membranes, as well as an increase in the proportion of lipids ions at m/z 578 and m/z 757 detected by MALDI-MS. Nevertheless, there was no effect of the treatments on in vitro embryo development. However, embryos derived from bulls supplemented with PUFAs exhibited higher lipid accumulation compared to control (P < 0.05). In conclusion, PUFA supplementation promoted worsening of semen quality without affecting the in vitro sperm fertilizing ability; however, the paternal diet affected the intracytoplasmic lipid content in the resulting embryos.

Mammalian ovarian lipid distributions by desorption electrospray ionization-mass spectrometry (DESI-MS) imaging.

Merging optical images of tissue sections with the spatial distributions of molecules seen by imaging mass spectrometry is a powerful approach to better understand the metabolic roles of the mapped molecules. Here, we use histologically friendly desorption electrospray ionization-mass spectrometry (DESI-MS) to map the lipid distribution in tissue sections of ovaries from cows (N = 8), sows (N = 3), and mice (N = 12). Morphologically friendly DESI-MS imaging allows the same sections to be examined for morphological information. Independent of the species, ovarian follicles, corpora lutea, and stroma could be differentiated by principal component analysis, showing that lipid profiles are well conserved among species. As examples of specific findings, arachidonic acid and the phosphatidylinositol PI(38:4), were both found concentrated in the follicles and corpora lutea, structures that promoted ovulation and implantation, respectively. Adrenic acid was spatially located in the corpora lutea, suggesting the importance of this fatty acid in the ovary luteal phase. In summary, lipid information captured by DESI-MS imaging could be related to ovarian structures and data were all conserved among cows, sows, and mice. Further application of DESI-MS imaging to either physiological or pathophysiological models of reproductive conditions will likely expand knowledge of the roles of specific lipids and pathways in ovarian activity and mammalian fertility. Graphical abstract Desorption electrospray ionization-mass spectrometry (DESI-MS) is performed directly from frozen ovarian tissue sections placed onto glass slides. Because the desorption and ionization process of small molecules is so gentle, the tissue architecture is preserved. The sample can then be stained and tissue morphology information can be overlaid with the chemical information obtained by DESI-MS.

Influence of cAMP modulator supplementation of in vitro culture medium on Bos taurus indicus embryos.

The effectiveness of the use of natriuretic peptide C (NPPC) in the blocking of meiosis has already been proven in several species. However, there are no reports on the use of NPPC in the activation of metabolic processes in embryos. Whereas modulations of cAMP concentrations alter the lipid metabolism of bovine oocytes, the present study aims to evaluate the effect of NPPC on the development, lipid content and transcript levels of genes related to lipid metabolism of IVP bovine embryos. For this purpose, ovaries were obtained from a slaughterhouse, and oocytes were fertilized in vitro (D0). From D5 of in vitro culture, embryos were treated with 100 nM NPPC (NPPC group) or with no NPPC (Control group) and evaluated in terms of Blastocyst (D7) and hatching rates (D10). For the assessment of the cytoplasmatic lipid amounts, blastocysts were stained with Sudan Black B dye. The embryonic lipid profile was investigated by electrospray ionization desorption-mass spectrometry (DESI-MS). The abundance of nine transcripts related to lipid metabolism were assessed using the Biomark HD system. For statistical analysis, blastocyst and hatching rates, lipid content by the Sudan Black B and variation of gene expression between groups were compared by Student t-test. For lipid profile analysis, principal component analysis (PCA) and fold-change were performed. The embryo lipid content was similar between NPPC (881 ±â€¯3.7) and Control (883 ±â€¯5.2) groups (p > 0.05). However, cholesteryl esters and TAGs were downregulated by NPPC at multiple levels according to the DESI-MS profiles. Of the analyzed genes, ELOVL6 and SREBF1 showed an up-regulation in the control group (p < 0.05), while CPT2 was observed to be up-regulated in the NPPC-treated embryos. There was no significant difference in the blastocyst production rate between NPPC (44.4%) and Control (42.4%), however the hatching rate at D10 was higher (p < 0.05) in the NPPC group (69.77%) when compared to the Control group (48.33%). These findings demonstrate that NPPC alters the mRNA expression of genes related to lipid metabolism and that it exerts a positive effect on the hatching rates of IVP Bos taurus indicus embryos.

Breed-specific factors influence embryonic lipid composition: comparison between Jersey and Holstein.

Some embryos exhibit better survival potential to cryopreservation than others. The cause of such a phenotype is still unclear and may be due to cell damage during cryopreservation, resulting from overaccumulation and composition of lipids. In cattle embryos, in vitro culture conditions have been shown to impact the number of lipid droplets within blastomeres. Thus far, the impact of breed on embryonic lipid content has not been studied. In the present study were compared the colour, lipid droplet abundance, lipid composition, mitochondrial activity and gene expression of in vivo-collected Jersey breed embryos, which are known to display poor performance post-freezing, with those of in vivo Holstein embryos, which have good cryotolerance. Even when housed and fed under the same conditions, Jersey embryos were found to be darker and contain more lipid droplets than Holstein embryos, and this was correlated with lower mitochondrial activity. Differential expression of genes associated with lipid metabolism and differences in lipid composition were found. These results show genetic background can impact embryonic lipid metabolism and storage.

Genetic influence on the reduction in bovine embryo lipid content by l-carnitine.

The decreased rate of pregnancy obtained in cattle using frozen in vitro embryos compared with in vivo embryos has been associated with over-accumulation of intracellular lipid, which causes cell damage during cryopreservation. It is believed that the higher lipid content of blastomeres of bovine embryos produced in vitro results in darker-coloured cytoplasm, which could be a consequence of impaired mitochondrial function. In this study, l-carnitine was used as a treatment to reduce embryonic lipid content by increasing metabolism in cultured bovine embryos. We have observed previously that in vivo embryos of different dairy breeds collected from cows housed and fed under the same conditions differed in lipid content and metabolism. As such, breed effects between Holstein and Jersey were also examined in terms of general appearance, lipid composition, mitochondrial activity and gene expression. Adding l-carnitine to the embryo culture medium reduced the lipid content in both breeds due to increased mitochondrial activity. The response to l-carnitine was weaker in Jersey than in Holstein embryos. Our results thus show that genetics influence the response of bovine embryos to stimulation of mitochondrial metabolism.

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

PURPOSE: Polycystic 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. METHODS: Follicular 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. RESULTS: The lipid profiles indicated particularities related to differences in phosphatidylcholine (PCOS and HR), phosphatidylserine, phosphatydilinositol and phosphatidylglycerol (control), sphingolipids (PCOS) and phosphatidylethanolamine (control and HR). CONCLUSIONS: These 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.

Lipid characterization of individual porcine oocytes by dual mode DESI-MS and data fusion.

The development of sensitive measurements to analyze individual cells is of relevance to elucidate specialized roles or metabolic functions of each cell under physiological and pathological conditions. Lipids play multiple and critical roles in cellular functions and the application of analytical methods in the lipidomics area is of increasing interest. In this work, in vitro maturation of porcine oocytes was studied. Two independent sources of chemical information (represented by mass spectra in the positive and negative ion modes) from single oocytes (immature oocytes, 24-h and 44-h in vitro matured oocytes) were acquired by using desorption electrospray ionization-mass spectrometry (DESI-MS). Low and mid-level data fusion strategies are presented with the aim of better exploring the large amount of chemical information contained in the two mass spectrometric lipid profiles. Data were explored by principal component analysis (PCA) within the two multi-block approaches to include information on free fatty acids, phospholipids, cholesterol-related molecules, di- and triacylglycerols. After data fusion, clearer differences among immature and in vitro matured porcine oocytes were observed, which provide novel information regarding lipid metabolism throughout oocyte maturation. In particular, changes in TAG composition, as well as increase in fatty acid metabolism and membrane complexity were evidenced during the in vitro maturation process. This information can assist the improvement of in vitro embryo production for porcine species.

Identification of Corynebacterium spp. isolated from bovine intramammary infections by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Corynebacterium species (spp.) are among the most frequently isolated pathogens associated with subclinical mastitis in dairy cows. However, simple, fast, and reliable methods for the identification of species of the genus Corynebacterium are not currently available. This study aimed to evaluate the usefulness of matrix-assisted laser desorption ionization/mass spectrometry (MALDI-TOF MS) for identifying Corynebacterium spp. isolated from the mammary glands of dairy cows. Corynebacterium spp. were isolated from milk samples via microbiological culture (n=180) and were analyzed by MALDI-TOF MS and 16S rRNA gene sequencing. Using MALDI-TOF MS methodology, 161 Corynebacterium spp. isolates (89.4%) were correctly identified at the species level, whereas 12 isolates (6.7%) were identified at the genus level. Most isolates that were identified at the species level with 16 S rRNA gene sequencing were identified as Corynebacterium bovis (n=156; 86.7%) were also identified as C. bovis with MALDI-TOF MS. Five Corynebacterium spp. isolates (2.8%) were not correctly identified at the species level with MALDI-TOF MS and 2 isolates (1.1%) were considered unidentified because despite having MALDI-TOF MS scores >2, only the genus level was correctly identified. Therefore, MALDI-TOF MS could serve as an alternative method for species-level diagnoses of bovine intramammary infections caused by Corynebacterium spp.

Hyaluronidase alters the lipid profile of cumulus cells as detected by MALDI-TOF MS and multivariate analysis.

This research aimed to study the changes in lipid composition in cumulus cells using hyaluronidase according to the intracytoplasmic sperm injection protocol commonly used in human reproduction clinics. The lipid extraction was performed by the Blight-Dyer protocol and the lipid profiles were obtained by MALDI-TOF MS in positive and negative modes. The mass spectra data were processed with MassLynx and the statistical analysis was performed using MetaboAnalyst 2.0. Fifteen ions were selected for each mode as potential markers for differences between the groups. These ions were identified in the human metabolome database as phosphatidylserine with and without treatment, phosphatidylethanolamine in the after treatment group and phosphatidylinositol in the before treatment group, which are lipids that may be involved in cell apoptosis and signaling. We concluded that MALDI-TOF MS coupled with multivariate analysis can be utilized as a strategy to obtain and study the lipid profiles of cumulus cells and as a tool to study the metabolic state of cumulus cells.

Lipid structural information from a single equine embryo by MALDI-MS / Informação estrutural de lipídios a partir de um único embrião equino pela técnica de MALDI-MS

It was reported the potential of MALDI-MS for the characterization of lipid species present in a single equine embryo, and studied some lipid structures detected by collision induced dissociation (CID) experiments. In the positive ion mode spectrum, it were observed mostly protonated and sodiated species of sphingomyelins (SM), phosphatidylcholines (PC) and triacylglycerols (TAG). In the negative ion mode, it were observed phosphatidylethanolamines (PE) and phosphatidylinositols (PI). MS/MS spectrum of most intense lipid ions was performed to show MALDI-MS/MS structural information potential. MS/MS spectrum in the positive mode of m/z 760.6 (attributed as PC34:1) depicted characteristic PC fragments of m/z 184.1 (choline polar head), and the neutral loss (NL) of 183 (phosphorylcholine). For the ion of m/z 766.6 (attributed as PE 38:5), we observed the NL of 140, characteristic of PE. For the ion of m/z 808.7 (attributed as PC 38.5), besides the fragment at m/z 184.1 at the NL of 183, it was possible to observe the loss of trimethylamine (ion of m/z 749.6), and the cyclophosphane (ion of m/z 147.0). Finally, for the negative ion mode, we isolated and fragmented the ion at m/z 863.6, which was attributed as PI 36:1 due to the presence of m/z 153 (glycerol phosphate – H2 O-H), 223 (phospho inositol – 2H2 O-H), 241 (phospho inositol – H2 O-H), 281 (oleic acid), and 581.3 (lysophosphoinositol – H2 O-H). It was concluded that MALDI-MS allowed the detection of a broad range of PC, SM, PE, PI and TAG lipid species, as well as a fast and confident characterization of lipid structures from a single equine embryo.

É relatado o potencial da técnica de MALDI-MS para caracterizar espécies de lipídios presentes em um único embrião equino e estudadas algumas estruturas lipídicas detectadas por dissociação induzida por colisão (CID). No espectro de modo íon positivo, foram observadas espécies, principalmente, protonadas e sodiadas de esfingomielinas (SM), fosfatidileolinas (PC) e triacilgliceróis (TAG). No modo negativo, foram observadas fosfatidiletanolaminas (PE) e fosfatidilinositos (PI). Espectros de íons de lípidos com maior intensidade foram utilizados para demonstrar o potencial da informação estrutural por MALDI-MS/MS. O espectro no modo positivo de m/z (massa sobre carga) 760,6 (atribuída como PC34:1) apresentou características de fragmentos PC de m/z 184,1 (denominada cabeça polar de colina), além de perda neutral (NL) de m/z 183 (fosforilcolina). Para o íon de m/z 766,6 (atribuída como PE38:5), observou-se a NL de 140, característica do PE. Para o íon de m/z 808,7 (38,5 atribuído como PC), além do fragmento m/z 184,1 na NL de 183, foi possível observar a perda de trimetilamina (íon de m/z 749,6) e o ciclofosfano (íon de m/z 147,0). Finalmente, para o modo de íon negativo, foram isolados e fragmentados o íon de m/z 863,6 que foi atribuído como PI36:1, devido à presença de m/z 153 (fosfato de glicerol – H2 O-H ), 223 (inositol fosfo - 2H2 O-H) , 241 (fosfoinositol – H2 O-H), 281 (ácido oleico) e 581,3 (lisofosfoinositol – H2 O+H). Foi concluído que a MALDI - MS permite a detecção de uma ampla gama de espécies de PC, SM, PE, PI e TAG lipídicas, bem como a caracterização rápida e confiante de estruturas lipídicas a partir de um único embrião equino.

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

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.

Differential seminal plasma proteome according to semen retrieval in men with spinal cord injury.

OBJECTIVE: To evaluate protein expression profile and to quantify proteins present in seminal plasma from men with spinal cord injury (SCI) and healthy men without SCI. DESIGN: Experimental study. SETTING: University hospital. PATIENT(S): Twelve SCI patients divided into two groups, six who underwent electroejaculation (EEJ) and six who underwent penile vibratory stimulation (PVS); and ten control subjects presenting normal sperm motility and concentration. INTERVENTION(S): EEJ and PVS. MAIN OUTCOME MEASURE(S): The seminal plasma protein profile was analyzed by two proteomic strategies: data-independent label-free quantitative proteomics (MS(E)) and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D SDS-PAGE). RESULT(S): A total of 638 different proteins were identified by MS(E) and 18 by 2D SDS-PAGE followed by tandem mass spectrometry. Interactome analysis showed key reproductive biologic processes-insemination, sperm and oocyte fusion, and acrosome reaction-related to all groups, as were triglyceride stimuli. Processes related to actin and muscle function and to iron oxidation, transportation, and homeostasis were found only in the EEJ and PVS groups; response to hydrogen peroxide and increased immune response was found only in the PVS group. CONCLUSION(S): This study was able to demonstrate differential protein expression among control, PVS, and EEJ groups; SCI is responsible for alterations in seminal plasma protein profile leading to a deviation from homeostasis; proteins reported in both PVS and EEJ groups correlate with the pathophysiology of SCI-related infertility.

Prediction of embryo implantation potential by mass spectrometry fingerprinting of the culture medium.

This study has evaluated the performance of a multivariate statistical model to predict embryo implantation potential by processing data from the chemical fingerprinting of culture medium samples used for human embryo culture. The culture medium for 113 embryos from 55 patients undergoing ICSI was collected after embryo transfer. The samples were split into positive (n=29) and negative (n=84) implantation groups according their implantation outcomes (100% or 0% implantation). The samples were individually diluted and analyzed by electrospray ionization mass spectrometry (ESI-MS). The m/z ratios and relative abundances of the major ions in each spectrum were considered for partial least square discriminant analysis. Data were divided into two subsets (calibration and validation), and the models were evaluated and applied to the validation set. A total of 5987 ions were observed in the groups. The multivariate statistical model described more than 82% of the data variability. Samples of the positive group were correctly identified with 100% probability and negative samples with 70%. The culture media used for embryos that were positive or negative for successful implantation showed specific biochemical signatures that could be detected in a fast, simple, and noninvasive way by ESI-MS. To our knowledge, this is the first report that uses MS fingerprinting to predict human embryo implantation potential. This biochemical profile could help the selection of the most viable embryo, improving single-embryo transfer and thus eliminating the risk and undesirable outcomes of multiple pregnancies.

The follicular microenviroment as a predictor of pregnancy: MALDI-TOF MS lipid profile in cumulus cells.

PURPOSE: This research proposed to study the changes in lipid composition in cumulus cells (CCs) from women who achieved pregnancy compared with women who did not, after in vitro fertilization treatment. This approach has the potential to provide novel information on the lipid metabolism of the CCs and as an additional method to predict pregnancy. METHOD: Fifty-four samples from couples with tubal and male factor infertility and where the female partner was age 35 or younger were divided in two groups according to their level of hCG 14 days after embryo transfer as follows: (1) 23 samples in pregnant group and (2) 31 samples in non-pregnant group. Lipid extraction was performed by the Bligh-Dyer protocol, and lipid profiles were obtained by MALDI-TOF MS. Mass spectra data were processed with MassLynx, and statistical analysis was performed using MarkerLynx extended statistic. OPLS-DA model was built. RESULTS: S-plot Analysis revealed three ions as potential markers in the pregnant group, and five ions in the non-pregnant group. These ions were identified in the human metabolome database (HMDB) as phosphatidylcholine in the pregnant group and as phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol species in the non-pregnant group. These lipids might be involved in cell proliferation and differentiation, apoptosis and GAP junction regulation. CONCLUSION: We conclude that MALDI-TOF MS can be used as an informative and fast analytical strategy to obtain and study the lipid profile of cumulus cells and can potentially be used as a supporting tool to predict pregnancy based on the metabolic state of the CCs.