Analysis Of Human Follicular Fluid Research Articles

A Proteomic Analysis of Human Follicular Fluid: Proteomic Profile Associated with Embryo Quality.

Embryo selection is a key point of in vitro fertilization (IVF). The most commonly used method for embryo selection is morphological assessment. However, it is sometimes inaccurate. Follicular fluid (FF) contains a complex mixture of proteins that are essential for follicle development and oocyte maturation. Analyzing human FF proteomic profiles and identifying predictive biomarkers might be helpful for evaluating embryo quality. A total of 22 human FF samples were collected from 19 infertile women who underwent IVF/intracytoplasmic sperm injection (ICSI) treatment between October 2021 and November 2021. FFs were grouped into two categories on the basis of the day 3 embryo quality, grade I or II in the hqFF group and grade III in the nhqFF group. FF was analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). The key differentially expressed proteins (DEPs) were validated by parallel reaction monitoring (PRM) and enzyme-linked immunosorbent assay (ELISA). Differentially expressed proteins were further analyzed using DAVID software. A total of 558 proteins were identified, of which 50 proteins were differentially expressed in the hqFF versus nhqFF group, including 32 upregulated proteins (> 1.20-fold, P < 0.05) and 18 downregulated proteins (< 0.67-fold, P < 0.05). Bioinformatics analyses showed that the upregulated DEPs were enriched in components of the coagulation and complement systems and negative regulation of peptidase activity, while the downregulated DEPs were enriched in molecular function of extracellular matrix structural and constituent collagen binding. Our results suggested that a number of protein biomarkers in FF were associated with embryo quality. It may help develop an effective and noninvasive method for embryo selection.

Proteomic Analysis of Human Follicular Fluid Reveals the Pharmacological Mechanisms of the Chinese Patent Drug Kunling Pill for Improving Diminished Ovarian Reserve

Objective To explore the pharmacological mechanism of a Chinese patent drug (Kunling Pill (KLP)) on improving diminished ovarian reserve based on proteomic analysis. Methods A total of 18 patients divided into three groups (the normal ovary reserve (NOR), diminished ovary reserve (DOR), and KLP groups) undergoing assisted reproductive technology by standard ovarian stimulation protocols were recruited to collect follicular fluid. Data-independent acquisition mass spectrometry was used to identify differentially expressed proteins by nano-LC-MS/MS. Bioinformatic analysis was conducted to predict the functions and pathways of the identified proteins. Clinical, hormonal, and biochemical parameters were also analyzed in the three groups. Results A total of 144 differentially expressed proteins were screened out, including 56 proteins that were downregulated and 88 proteins that were upregulated in the DOR group compared with the NOR group, while 27 proteins were shared in the KLP-treated group. Among them, 10 proteins were upregulated and 17 proteins were downregulated in the KLP-treated group compared with the DOR group. The most enriched biological processes accounted for 28 GO terms, including cellular process, biological regulation, metabolic process, and regulation of biological process. Significant pathways were associated with fatty acid elongation, fatty acid degradation, fatty acid metabolism, nicotinate and nicotinamide metabolism, and valine, leucine, and isoleucine degradation. Conclusion Our study provides the proteome profiles of human follicular fluid from DOR patients treated by KLP. Functional analyses of proteome datasets revealed that core proteins (SAA1, MIF, and PRDX5) and related pathways (fatty acid metabolism, nicotinate and nicotinamide metabolism, and tyrosine and purine metabolism) are possible pharmacological mechanisms through which KLP improves DOR. Therefore, these findings may help better understand the complex mechanisms through which DOR is treated by the Chinese patent drug KLP.

Proteomic analysis of human follicular fluid from polycystic ovary syndrome patients.

Comparative proteomic profiling of human follicular fluid (HFF) from polycystic ovary syndrome (PCOS) and non-PCOS patients who displayed low levels of fertility was carried out via two-dimensional gel electrophoresis (2-DE) combined with mass spectrometry. HFF, an important reproductive fluid, was used for the proteomic analysis of PCOS patients to determine the effect of PCOS on folliculogenesis. HFF was obtained from 10 women (average age, 35 years) undergoing in vitro fertilization at two hospitals. The proteins of HFF were separated using 2-DE analysis and validated by Western blot assay. Approximately 250 protein spots were separated on the 2-DE gel. Among them, the expression levels of seven proteins were found to change at least 1.5-fold in the PCOS patient group. Three protein spots, albumin, uncharacterized protein 1, and uncharacterized protein 2, were downregulated in PCOS patients. However, four protein spots, gelsolin, vitamin D binding protein, serum albumin, and complement factor B, were upregulated in PCOS patient group. These proteins may serve as a panel of potential pathological biomarkers during fertilization and oocyte development.

TMT Based Proteomic Analysis of Human Follicular Fluid From Overweight/Obese and Normal-Weight Patients With Polycystic Ovary Syndrome.

Background: Polycystic ovary syndrome (PCOS) is a major endocrine and metabolic disorder with heterogeneous manifestations and complex etiology. As a leading cause of anovulatory infertility, the molecular diversity of the follicular microenvironment has not been fully elucidated. The aim of the present study was to investigate the follicular fluid proteomic profiles of overweight/obese and normal-weight women with PCOS, to identify novel molecular mechanisms underlying PCOS and to determine the effect of obesity on the follicular fluid protein profiles.Methods: Follicular fluid samples were collected from 3 different groups: overweight/obese PCOS patients (n = 29), normal-weight PCOS patients (n = 29), and normo-ovulatory controls (n = 29). We used a quantitative approach with tandem mass tag labeling and liquid chromatography tandem mass spectrometry to identify the differentially expressed proteins. Differential abundance of four selected proteins was confirmed by ELISA. Gene Set Enrichment Analysis was also conducted to further explore our findings. Furthermore, we compared the clinical, hormonal, and biochemical characteristics of overweight/obese and normal-weight patients with PCOS to determine the effects of obesity.Results: A total of 1,153 proteins were identified, of which 41 and 19 proteins were differentially expressed in the overweight/obese PCOS group vs. the control group, and in the normal-weight PCOS group vs. the control group, respectively. Bioinformatics analyses showed that the inflammatory, immunological, and metabolic-related biological processes were co-enriched in both subgroups of PCOS. Apolipoprotein A-II, complement C5, fetuin-B, and stromal cell-derived factor 1 were found to be involved in various processes and were validated using the ELISA analysis. From clinical features and proteomic data, obesity was found to worsen follicular development disturbances in PCOS.Conclusion: In this proteomic study, a panel of proteins were found differentially expressed in the follicular fluid of PCOS. Inflammatory, immunological, and metabolic abnormalities were identified inside the intra-follicular environment, which could be aggravated by obesity. The identified proteins were correlated with follicular growth and may be considered as candidate biomarkers as well as therapeutic targets of PCOS.

Relation between trace elements levels and oxidative status in follicular fluid and the number and maturity of oocytes aspirated in patients undergoing assisted reproduction techniques

Human follicular fluid is a complex body fluid that constitutes the microenvironment of developing follicles in the ovary. Follicular fluid contains a number of proteins that modulate oocyte maturation and ovulation. Information about the protein constituents of follicular fluid may provide a better understanding of ovarian physiology in addition to opening new avenues for investigating ovarian disorders. However, the composition of follicular fluid proteome remains poorly defined. In this study, we carried out SDS-PAGE, OFFGEL and SCX-based separation followed by LC–MS/MS analysis to characterize the proteome of human follicular fluid. We report high confidence identification of 480 proteins, of which 320 have not been described previously in the follicular fluid. The identified proteins belong to diverse functional categories including growth factor and hormones, receptor signaling, enzyme catalysis, defense/immunity and complement activity. Our dataset should serve as a resource for future studies aimed at developing biomarkers for monitoring oocyte and embryo quality, pregnancy outcomes and ovarian disorders.Proteome analysis of human follicular fluid by multi-pronged approach of protein peptide fractionation revealed 480 proteins with high confidence. The identified protein may facilitate the understanding of folliculogenesis. This protein dataset should serve as a useful resource for development of biomarkers for oocyte quality, in vitro fertilization techniques and female infertility.

Ultra-trace element analysis of human follicular fluid by ICP-MS/MS: pre-analytical challenges, contamination control, and matrix effects.

Follicular fluid (FF), which is the fluid that envelops the developing oocyte (egg cell) in the ovary, can be analyzed to assess trace element content as well as to determine potential exposure to toxic elements in women seeking in vitro fertilization (IVF) treatment. Such measurements may be useful in establishing associations with potential adverse effects on oocyte viability and subsequent pregnancy outcomes. The principal goal of this study was to leverage the next generation of inorganic mass spectrometry based on ICP-MS/MS to address the numerous analytical challenges of (ultra-)trace element analysis of human FF specimens. Ultra-trace element measurements are defined by the Clinical Laboratory Standards Institute as fluid concentrations below 10 μg L-1 or tissue mass fractions below 1 μg g-1. Stringent pre-analytical procedures were developed to minimize exogenous contamination during FF specimen collection and storage in a prospective study of 56 women seeking IVF treatment. ICP-MS/MS instrumental parameters were carefully optimized, and the method validated for 11 biologically important elements that included 4 at trace levels (Cu, Se, Sr, and Zn) and 7 at ultra-trace levels (As, Cd, Co, Mo, Mn, Hg, and Pb). Method limits of detection (LODs) for ultra-trace elements varied from 5.6 ng L-1 for Cd to 0.11 μg L-1 for Mo. A total of 197 human FF specimens were analyzed using the proposed ICP-MS/MS method with 84% of specimens detectable for Pb and 100% detectable for Co, Cu, Mn, Mo, Sr, and Zn. The method based on ICP-MS/MS was compared to a previous method developed for FF using SF-ICP-MS.

SWATHHM-Based Metabolomics of Follicular Fluid in Patients Shows That Progesterone Adversely Affects Oocyte Quality.

Objective We reveal the relationship between progesterone level in follicular fluid and oocyte quality based on sequential window acquisition of all theoretical fragment-ion spectra (SWATH™), a powerful high-resolution mass spectrometric data independent acquisition technique. Method Follicular fluid samples were collected from 22 subjects (the level of progesterone > 1.5 ng/mL) of progesterone group, as well as from 22 subjects (the level of progesterone < 1.5 ng/mL) of control group, and analyzed using UPLC-Q-TOF. All methods were performed in accordance with ISO 9001:2008. Novel SWATH acquisition mode on an ultra-high performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (with resolving power 20,000–40,000) was investigated for the analysis of human follicular fluid. The principal component variable grouping detects intersample variable correlation and groups variables with similar profiles which simplifies interpretation and highlights related ions and fragments. It can also extract product ion spectra from the data collected by fragmenting a wide precursor ion window. Results Follicular fluid from the two groups differed with respect to five metabolites. Follicular fluid from the progesterone group contained elevated levels of 8-hydroxyguanosine and 4-hydroxynonenal and reduced levels of ATP, estradiol, and L-carnitine. The increased progesterone level on the day of HCG injection could negatively impact oocyte quality, thus reducing the pregnancy rate of IVF patients.

Proteomic analysis of human follicular fluid associated with successful in vitro fertilization

BackgroundHuman follicular fluid (HFF) provides a key environment for follicle development and oocyte maturation, and contributes to oocyte quality and in vitro fertilization (IVF) outcome.MethodsTo better understand folliculogenesis in the ovary, a proteomic strategy based on dual reverse phase high performance liquid chromatography (RP-HPLC) coupled to matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (LC-MALDI TOF/TOF MS) was used to investigate the protein profile of HFF from women undergoing successful IVF.ResultsA total of 219 unique high-confidence (False Discovery Rate (FDR) < 0.01) HFF proteins were identified by searching the reviewed Swiss-Prot human database (20,183 sequences), and MS data were further verified by western blot. PANTHER showed HFF proteins were involved in complement and coagulation cascade, growth factor and hormone, immunity, and transportation, KEGG indicated their pathway, and STRING demonstrated their interaction networks. In comparison, 32% and 50% of proteins have not been reported in previous human follicular fluid and plasma.ConclusionsOur HFF proteome research provided a new complementary high-confidence dataset of folliculogenesis and oocyte maturation environment. Those proteins associated with innate immunity, complement cascade, blood coagulation, and angiogenesis might serve as the biomarkers of female infertility and IVF outcome, and their pathways facilitated a complete exhibition of reproductive process.

Back Cover: Proteomic analysis of human follicular fluid in poor ovarian responders during in vitro fertilization

DOI: 10.1002/pmic.201600333 Poor ovarian response (POR) observed in controlled ovarian stimulation (COS) is often observed during in vitro fertilization and embryo transfer (IVF-ET) cycles and is a major problem. It is difficult to predict POR, as there are no specific biomarkers known. In this study, we used quantitative proteomic analyses to investigate potential biomarkers that can predict poor response during IVF based on follicular fluid samples. Consequently, three proteins (PZP, RENI, and SRPX) were regarded as statistically significant. For more details, see the research article by Jae Won Oh et al., article number 1600333.

Proteomic analysis of human follicular fluid in poor ovarian responders during in vitro fertilization.

Poor ovarian response (POR) in controlled ovarian stimulation is often observed during in vitro fertilization and embryo transfer cycles and it is a major problem. A POR has been found to be related to several factors, including advanced age, high body mass index, and history of ovarian or pelvic surgery. However, it is difficult to predict POR, as there are no specific biomarkers known. In this study, we used quantitative proteomic analyses to investigate potential biomarkers that can predict poor response during in vitro fertilization based on follicular fluid samples. A total of 1079 proteins were identified using a high-resolution orbitrap mass spectrometer coupled online to a nanoflow-LC system. It is notable that 65 upregulated and 66 downregulated proteins were found to be functionally enriched in poor responders. We also validated these differentially expressed proteins using a triple-quadrupole mass spectrometer for quantification of targeted proteins. Of the differentially expressed proteins, three proteins (pregnancy zone protein, renin, and sushi repeat-containing protein SRPX) were regarded as statistically significant (p < 0.05).

N-glycoproteomic analysis of human follicular fluid during natural and stimulated cycles in patients undergoing in vitro fertilization.

ObjectiveHyperstimulation methods are broadly used for in vitro fertilization (IVF) in patients with infertility; however, the side effects associated with these therapies, such as ovarian hyperstimulation syndrome (OHSS), have not been well studied. N-glycoproteomes are subproteomes used for the remote sensing of ovarian stimulation in follicular growth. Glycoproteomic variation in human follicular fluid (hFF) has not been evaluated. In this study, we aimed to identify and quantify the glycoproteomes and N-glycoproteins (N-GPs) in natural and stimulated hFF using label-free nano-liquid chromatography/electrospray ionization-quad time-of-flight mass spectrometry.MethodsFor profiling of the total proteome and glycoproteome, pooled protein samples from natural and stimulated hFF samples were selectively isolated using hydrazide chemistry to obtain the total proteomes and glycoproteomes. N-GPs were validated by the consensus sequence N-X-S/T (92.2% specificity for the N-glycomotif at p<0.05). All data were compared between natural versus hyperstimulated hFF samples.ResultsWe detected 41 and 44 N-GPs in the natural and stimulated hFF samples, respectively. Importantly, we identified 11 N-GPs with greater than two-fold upregulation in stimulated hFF samples compared to natural hFF samples. We also validated the novel N-GPs thyroxine-binding globulin, vitamin D-binding protein, and complement proteins C3 and C9.ConclusionWe identified and classified N-GPs in hFF to improve our understanding of follicular physiology in patients requiring assisted reproduction. Our results provided important insights into the prevention of hyperstimulation side effects, such as OHSS.

Proteomic analysis of human follicular fluid from fertile women.

BackgroundFollicular fluid is a unique biological fluid in which the critical events of oocyte and follicular maturation and somatic cell-germ cell communication occur. Because of the intimate proximity of follicular fluid to the maturing oocyte, this fluid provides a unique window into the processes occurring during follicular maturation. A thorough identification of the specific components within follicular fluid may provide a better understanding of intrafollicular signaling, as well as reveal potential biomarkers of oocyte health for women undergoing assisted reproductive treatment. In this study, we used high and low pH HPLC peptide separations followed by mass spectrometry to perform a comprehensive proteomic analysis of human follicular fluid from healthy ovum donors. Next, using samples from a second set of patients, an isobaric mass tagging strategy for quantitative analysis was used to identify proteins with altered abundances after hCG treatment.ResultsA total of 742 follicular fluid proteins were identified in healthy ovum donors, including 413 that have not been previously reported. The proteins belong to diverse functional groups including insulin growth factor and insulin growth factor binding protein families, growth factor and related proteins, receptor signaling, defense/immunity, anti-apoptotic proteins, matrix metalloprotease related proteins, and complement activity. In a quantitative analysis, follicular fluid samples from age-matched women undergoing in vitro fertilization oocyte retrieval were compared and 17 follicular fluid proteins were found at significantly altered levels (p < 0.05) between pre-hCG and post-hCG samples. These proteins belong to a variety of functional processes, including protease inhibition, inflammation, and cell adhesion.ConclusionsThis database of FF proteins significantly extends the known protein components present during the peri-ovulatory period and provides a useful basis for future studies comparing follicular fluid proteomes in various fertility, disease, and environmental exposure conditions. We identified 17 differentially expressed proteins after hCG treatment and together these data showed the feasibility for defining biomarkers that illuminate how the ovarian follicle microenvironment is altered in various infertility-related conditions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12014-015-9077-6) contains supplementary material, which is available to authorized users.

A Proteomic Analysis of Human Follicular Fluid: Comparison between Younger and Older Women with Normal FSH Levels

The follicular fluid (FF) is produced during folliculogenesis and contains a variety of proteins that play important roles in follicle development and oocyte maturation. Age-related infertility is usually considered as a problem that can be solved by assisted reproduction technology. Therefore, the identification of novel biomarkers that are linked to reproductive aging is the subject of this study. FF was obtained from healthy younger (20–32 years old) and older (38–42 years old) women undergoing intracytoplasmic sperm injection (ICSI) due to male factor infertility. The FF was analyzed by two-dimensional gel electrophoresis (2-DE). The power of two-dimensional gel electrophoresis and the identification of proteins were exploited using matrix-assisted laser desorption-ionization time-of-flight/time-of-flight (MALDI-TOF-TOF) mass spectrometry. Twenty three protein spots showed reproducible and significant changes in the aged compared to the young group. Of these, 19 protein spots could be identified using MALDI-TOF-TOF-MS. As a result of MASCOT search, five unique downregulated proteins were identified in the older group. These were identified as serotransferrin, hemopexin precursor, complement C3, C4 and kininogen. A number of protein markers were found that may help develop diagnostic methods of infertility.

A proteomic analysis of human follicular fluid: comparison between fertilized oocytes and non-fertilized oocytes in the same patient

Human follicular fluid constitutes the microenvironment of follicles and includes various biological active proteins that can affect follicle growth and oocyte fertilization. Conducting proteomic evaluations of human follicular fluid may be helpful for identifying potential biomarkers possibly possessing a predictive value for oocyte quality and the success of in vitro fertilization. We performed proteomic profiling of human follicular fluids containing oocytes that were fertilized and resulted in pregnancy and follicular fluids containing oocytes that were not fertilized in the same patients undergoing intracytoplasmic sperm injection using the LTQ Orbitrap coupled with liquid chromatography-tandem mass spectrometry (LC/MS/MS) analyses. We identified a total of 503 proteins in human follicular fluids containing fertilized and non-fertilized oocytes obtained from 12 patients. We also found that 53 proteins exhibited significantly different spectral counts between the two groups, including heparan sulfate proteoglycan perlecan, which showed significant upregulation in the follicular fluids containing fertilized oocytes in comparison with that observed in the follicular fluids containing non-fertilized oocytes. Our results suggest a possibility that proteins identified by LC/MS/MS in follicular fluid might not only be involved in folliculogenesis, but also function as biomarkers possessing predictive potential for oocyte maturation and the success of IVF when their expression levels are significantly different between fertilized and non-fertilized oocytes, although no distinctive biomarkers were identified in the current study.

Proteomic analysis of human follicular fluid: A new perspective towards understanding folliculogenesis

Human follicular fluid is a complex body fluid that constitutes the microenvironment of developing follicles in the ovary. Follicular fluid contains a number of proteins that modulate oocyte maturation and ovulation. Information about the protein constituents of follicular fluid may provide a better understanding of ovarian physiology in addition to opening new avenues for investigating ovarian disorders. However, the composition of follicular fluid proteome remains poorly defined. In this study, we carried out SDS-PAGE, OFFGEL and SCX-based separation followed by LC-MS/MS analysis to characterize the proteome of human follicular fluid. We report high confidence identification of 480 proteins, of which 320 have not been described previously in the follicular fluid. The identified proteins belong to diverse functional categories including growth factor and hormones, receptor signaling, enzyme catalysis, defense/immunity and complement activity. Our dataset should serve as a resource for future studies aimed at developing biomarkers for monitoring oocyte and embryo quality, pregnancy outcomes and ovarian disorders. Proteome analysis of human follicular fluid by multi-pronged approach of protein peptide fractionation revealed 480 proteins with high confidence. The identified protein may facilitate the understanding of folliculogenesis. This protein dataset should serve as a useful resource for development of biomarkers for oocyte quality, in vitro fertilization techniques and female infertility.

Trace elements in human follicular fluid: development of a sensitive multielement ICP-MS method for use in biomonitoring studies

Little information is available in the literature regarding trace element analysis of human follicular fluid. Most peer-reviewed publications offer few details about sample preparation, method development, or method validation. We have attempted to fill this gap in current knowledge by providing comprehensive details of our method development and validation process for the measurement of 24 trace elements (As, Ba, Be, Cd, Co, Cr, Cs, Cu, Hg, Mo, Mg, Mn, Pb, Pt, Rb, Se, Sb, Sn, Sr, Tl, U, V, W, Zn) in follicular fluid. A number of different sample preparation procedures were explored, with a goal of optimizing method detection limits (MDLs), for both quadrupole-inductively coupled plasma-mass spectrometry (Q-ICP-MS) and sector field (SF)-ICP-MS. Human follicular fluid samples (n = 72) were obtained from 64 women undergoing in vitro fertilization (IVF) treatment, and were successfully analyzed for 24 trace elements using SF-ICP-MS.

A proteomic analysis of human follicular fluid in women undergoing IVF cycles

A proteomic analysis of human follicular fluid in women undergoing IVF cycles

Retinol binding protein is a major carrier of progesterone in vitro and in human follicular fluid

OBJECTIVE: The human retinol binding protein 4 (RBP) is a member of the lipocalin family of proteins and is the specific vitamin A carrier. It has diverse biological functions including intra- and extracellular transport of retinoids, regulation of reproduction and insulin resistance signaling. Our recent proteomic analysis of human follicular fluid (FF) demonstrate high level of RBP presence and nearly undetectable level of retinoids. This study was conducted to investigate the RBP function in human FF as a possible carrier of other than retinals hydrophobic ligands.DESIGN: Experimental study.MATERIALS AND METHODS: Recombinant human RBP (rhRBP) was isolated from bacteria as inclusion bodies and purified by HPLC. Renaturation was performed by dialysis in the presence of retinol, cholesterol, or progesterone as ligands. rhRBP complexation with the ligands was verified by HPLC, ELISA, and immunoprecipitation with RBP-specific antibody, A/G Sepharose, and Western blot. Presence of retinol and progesterone in the complexes was monitored through 3H-labeled ligands as a trace. Complexes were formed also with purified TTR. rhRBP-ligand and rhRBP-TTR-ligand complexes (0.1–0.4uM) were added to primary human ovarian fibroblast cell culture (FC). Ligand delivery to FC was monitored by measuring cellular 3H-ligand incorporation after 3h incubation. FF and FC were obtained at the time of vaginal oocyte retrieval from IVF patients (3 young donor patients, age <28 years) and cultured in DMEM medium. Fetal calf serum was omitted during the rhRBP-ligand and rhRBP-TTR-ligand incubation. HPLC and immunocapture were used to search for RBP-progesterone and RBP–cholesterol complexes in FF.RESULTS: We confirmed 3H-Progesterone-rhRBP and 3H-Progesterone-rhRBP-TTR complexation using purified (>99% purity) recombinant proteins. The binding affinity of progesterone-rhRBP complexation is similar to the binding affinity of the retinol-rhRBP binding. Cholesterol was found to bind rRBP and rhRBP-TTR with very low affinity (<3%). >40% of the progesterone from the progesterone-rhRBP complex was found to enter FC. The presence of TTR did not change dramatically progesterone uptake. More than 60% of the FF progesterone is present as a constituent of the RBP-TTR complex.CONCLUSIONS: rhRBP alone and in a complex with TTR is a progesterone but not cholesterol carrier. In FF, RBP-TTR is a major carrier of progesterone. In a complex with RBP or RBP-TTR progesterone is delivered into the cells. OBJECTIVE: The human retinol binding protein 4 (RBP) is a member of the lipocalin family of proteins and is the specific vitamin A carrier. It has diverse biological functions including intra- and extracellular transport of retinoids, regulation of reproduction and insulin resistance signaling. Our recent proteomic analysis of human follicular fluid (FF) demonstrate high level of RBP presence and nearly undetectable level of retinoids. This study was conducted to investigate the RBP function in human FF as a possible carrier of other than retinals hydrophobic ligands. DESIGN: Experimental study. MATERIALS AND METHODS: Recombinant human RBP (rhRBP) was isolated from bacteria as inclusion bodies and purified by HPLC. Renaturation was performed by dialysis in the presence of retinol, cholesterol, or progesterone as ligands. rhRBP complexation with the ligands was verified by HPLC, ELISA, and immunoprecipitation with RBP-specific antibody, A/G Sepharose, and Western blot. Presence of retinol and progesterone in the complexes was monitored through 3H-labeled ligands as a trace. Complexes were formed also with purified TTR. rhRBP-ligand and rhRBP-TTR-ligand complexes (0.1–0.4uM) were added to primary human ovarian fibroblast cell culture (FC). Ligand delivery to FC was monitored by measuring cellular 3H-ligand incorporation after 3h incubation. FF and FC were obtained at the time of vaginal oocyte retrieval from IVF patients (3 young donor patients, age <28 years) and cultured in DMEM medium. Fetal calf serum was omitted during the rhRBP-ligand and rhRBP-TTR-ligand incubation. HPLC and immunocapture were used to search for RBP-progesterone and RBP–cholesterol complexes in FF. RESULTS: We confirmed 3H-Progesterone-rhRBP and 3H-Progesterone-rhRBP-TTR complexation using purified (>99% purity) recombinant proteins. The binding affinity of progesterone-rhRBP complexation is similar to the binding affinity of the retinol-rhRBP binding. Cholesterol was found to bind rRBP and rhRBP-TTR with very low affinity (<3%). >40% of the progesterone from the progesterone-rhRBP complex was found to enter FC. The presence of TTR did not change dramatically progesterone uptake. More than 60% of the FF progesterone is present as a constituent of the RBP-TTR complex. CONCLUSIONS: rhRBP alone and in a complex with TTR is a progesterone but not cholesterol carrier. In FF, RBP-TTR is a major carrier of progesterone. In a complex with RBP or RBP-TTR progesterone is delivered into the cells.

Proteome analysis of human follicular fluid

We used proteomic approach to analyze the protein profile of human follicular fluid (HFF) obtained from 25 normo-ovulatory women undergoing assisted reproduction techniques due to a male infertility factor. In all HFF samples analyzed we found 695 common spots distributed in the 3 to 10 pH range and in the 10–200 kDa range. Only 625 of these spots were also present in the plasma. We used MALDI-TOF-MS analysis to unequivocally assign 183 HFF/plasma matched spots and 27 HFF/plasma unmatched spots. A large number of acute-phase proteins, including transferrin, ceruloplasmin, afamin, hemopexin, haptoglobin and plasma amyloid protein, were identified in HFF in relatively high concentration supporting the hypothesis that mammalian ovulation can be compared to an inflammatory event. We also identified several important antioxidant enzymes; i.e., catalase, superoxide dismutase, glutathione transferase, paraoxonase, heat shock protein 27 and protein disulfide isomerase. This indicates that during maturation the human follicle is well protected against toxic injury due to oxidative stress.

Quantitation of meiosis activating sterols in human follicular fluid using HPLC and photodiode array detection.

A chromatographic assay for 4,4-dimethyl-5alpha-cholesta-8,14, 24-triene-3beta-ol (FF-MAS), and its reduced species, 4, 4-dimethyl-5alpha-cholesta-8,24-triene-3beta-ol (T-MAS), has been established for analysis of human follicular fluid (huFF). The assay also quantifies lanosterol, free cholesterol and progesterone. It was established using a pool of more than 100 individual follicular fluids from women undergoing in vitro fertilization treatment. Both FF-MAS and T-MAS were found in huFF, and can be quantified with HPLC equipped with photodiode array (PDA) detection. The examination wavelength for each analyte was chosen at the absorption maximum between 200 and 300 nm. Spike-recovery experiments revealed mean recoveries of 91 +/- 7.3% for lanosterol, 103 +/- 5.1% for FF-MAS, 104 +/- 5.5% for T-MAS, 103 +/- 4.5% for free cholesterol and 85 +/- 5.1% for progesterone. The lower recovery value for progesterone was due to a sub-optimal extraction procedure for this particular analyte, as indicated by re-extraction. The minimum amounts of FF-MAS required for quantification were 4 ng/mL and 23 ng/mL for T-MAS and lanosterol. FF-MAS was assayed to approximately 1.6 microM. T-MAS and lanosterol was assayed to about half of this value. No esterification of either MAS or lanosterol could be detected in huFF. Less than 10% of cholesterol was underivatized cholesterol, as more than 10 times the amount of free cholesterol could be assayed after extended saponification. This method can be used for evaluating the accumulation of MAS in huFF and its correlation to oocyte quality and fertilization parameters in in vitro fertilization programmes.