Ovarian Follicular Fluid Research Articles

The Effects of Leptin and Irisin on Steroidogenic Enzyme Gene Expression in Human Ovarian Granulosa Cells - Initial Studies

Fertility and energy metabolism are closely associated, and the cytokines produced by the adipose and muscle tissue play a role in this association. Leptin, predominantly produced by the white adipose tissue, and irisin, produced by the brown adipose and skeletal muscle tissues, are cytokines that are important in balancing energy metabolism. This study aimed to investigate the effects of leptin and irisin on steroidogenic enzyme gene expression in human ovarian granulosa cells in vitro. Granulosa cells were retrieved and isolated from ovarian follicular fluid during in vitro fertilization (IVF) procedures. Cells were placed in primary in vitro cultures and treated with increasing concentrations of leptin (25, 50, 100, 200, and 400 ng/ml) or irisin (125, 250, 500, 1,000, and 2,000 ng/ml) for 24, 48, and 72 hours. mRNA expression levels of CYP11A1, CYP19A1, CYP21A2, HSD3B1, and HSD17B3 were measured by qRT-PCR analysis. Leptin treatment of granulosa cells resulted in significant upregulation of CYP21A2 mRNA levels, while irisin significantly downregulated mRNA levels of CYP11A1, CYP19A1, and HSD3B1. Taken together, these early experiments demonstrate that leptin and irisin may affect steroid hormone production in the ovary by targeting the gene expression of key steroidogenic enzymes. Additional experiments are in progress.

Comparison of the Effects of Stem Cell, Platelet Rich Plasma and Ovarian Follicle Fluid on Burn Stasis Zone (Experimental Study)

Objective: The basic aim in the treatment of second-degree burns is to prevent progressive cell death, and so treatments are directed at the zone of stasis. In this study, We were aimed to investigate the healing effects of using Mesenchymal Stem Cells (MSCs), Ovarian follicular fluid (OFS) and Platelet-rich plasma (PRP) on burn stasis zone in an experimental burn model. Method: Forty rats were divided randomly into four groups. Burns were created in each group according to the comb burn model. The control group received no treatment; the mesenchymal stem cell [MSC] group received MSC; the platelet-rich plasma [PRP] group received PRP; and the ovarian follicular fluid [OFF] group received OFF subcutaneously on days 1, 3 and 5. On days 1 and 21, all rats were photographed and the burn sites were calculated. At the end of day 21, all rats were sacrificed, the dorsum containing the created burns were excised, and the epithelialization, collagen amount, fibroblast density, inflammatory cell density and VEGF amounts were evaluated histopathologically. Results: The groups were assessed based on the burn site healing rate and histopathological scoring. Healing was faster in the MSC group [p<0.005], PRP group [p>0.005] and OFS group [p<0.005] than in the control group. When the treatment groups were compared with each other, the best healing was observed in the MSC, PRP and OFF groups, respectively. Conclusions: MSC, PRP and OFF were found to have a positive effect on burn healing, with MSC being the most efficient method among the three, followed by PRP and OFF, respectively, which were found to provide a faster healing than the control group.

TGFβ1 induces in-vitro and ex-vivo angiogenesis through VEGF production in human ovarian follicular fluid-derived granulosa cells during in-vitro fertilization cycle

A growing body of evidence indicates that angiogenesis in folliculogenesis contributes to oocyte developmental competence in natural and in-vitro fertilization (IVF) cycle of animals. Among the known angiogenic factors, vascular endothelial growth factor (VEGF) has an important role involved in angiogenesis. However, its expression level and regulatory mechanism in ovarian follicular fluid (FF) in patients undergoing IVF with controlled ovarian stimulation (COS) remains to be explored. In this study, the primary cultured human ovarian follicular granulosa cells (GCs) were prepared from FF and their identity was characterized by the presence of the GC specific markers. The transforming growth factor β1 (TGFβ1) was found to induce a significant increase in VEGF mRNA level and protein expression/secretion in GCs. In line with these observations, TGFβ1 could be detected in the ovarian FF, ranging from about 400 to 2000 pg/mL among three IVF patient groups with different patient’s serum Anti-Müllerian hormone level. The cellular signaling analysis revealed that TGFβ1 induced VEGF production through TGFβ receptor (TGFβR), Smad2/3, PI3 K/AKT, and JNK1/2-related signaling pathways. Finally, in a functional study, the TGFβ1-primed GC VEGF secretion promoted in-vitro angiogenesis in vascular endothelial cells and ex-vivo vessel sprouting in aortic ring. Taken together, we demonstrated here that TGFβ1 expressed in ovarian FF is an inducer for promoting VEGF production in follicular GCs through TGFβR-mediated signaling pathways and the released VEGF subsequently leads to angiogenesis. This possibly contributes to oocyte developmental competence in folliculogenesis of IVF patients with a COS protocol.

The Ratio of Mitochondrial DNA to Genomic DNA Copy Number in Cumulus Cell May Serve as a Biomarker of Embryo Quality in IVF Cycles.

Previous studies have reported that the mitochondrial DNA (mtDNA) contents of cumulus cells (CCs) in ovarian follicular fluid are correlated with embryo quality. Quantification of mtDNA CCs has been suggested as a biomarker of embryo viability. The aim of this study was to determine the relationship between mitochondrial DNA (mtDNA)/genomic DNA (gDNA) ratio in CCs and IVF outcomes such as fertilization rates and embryo quality in infertile women. This is an observational study on 144 cumulus-oocyte complexes obtained from 144 patients undergoing IVF-intracytoplasmic sperm injection (ICSI) at a single fertility center. The CCs in ovarian follicular fluid from patients undergoing IVF-ICSI were collected by ovum pick-up. A relative copy number quantification was used to determine mtDNA/gDNA ratio. Quantitative real-time PCR for various markers (β2M and mtMinArc gene) was used to determine average mtDNA/gDNA ratio of CCs. Investigation of the correlation between mtDNA/gDNA ratio in CCs and IVF outcomes showed no statistically significant correlation between the mtDNA/gDNA ratio in CCs and fertilization rates. However, mtDNA/gDNA ratio and embryo quality showed a statistically significant positive correlation. A significantly higher mtDNA/gDNA ratio was observed in the good quality embryo group compared with the poor quality embryo group (P < 0.05). In addition, the mtDNA/gDNA ratio showed negative correlation with the patient's age (correlation coefficient= -0.228, P < 0.05). Results of this study demonstrate a negative correlation of mtDNA/gDNA ratio in CCs with patient's age, and a low copy number of mtDNA in CCs may have adverse effects on embryo quality in IVF cycles. These results suggest that the ratio of mtDNA/gDNA in CCs may serve as a biomarker in predicting IVF outcomes.

Characterization of stem cells from human ovarian follicular fluid; a potential source of autologous stem cell for cell-based therapy.

Human ovarian follicular fluid (HOFF) contains proteins, extracellular matrixes necessary for growth and maturation of oocytes as well as granulosa cells. Epithelial cells and stem cells can be isolated from HOFF. However, information regarding stem cells derived from HOFF is still lacking. The objectives of the present study were to isolate, characterize, and differentiate cells derived from HOFF. HOFF was collected during the routine aspiration of oocytes in an assisted fertilization program and subjected to cell isolation, characterization, and in vitro culture. After 24h of culture, different cell morphologies including epithelial-like-, neural-like- and fibroblast-like cells were observed. Immunocytochemistry reveals the expression of pluripotent stem cell markers (OCT4, NANOG, SSEA4), epithelial marker (CK18), FSH- and LH-receptor. For in vitro culture, the isolated cells were continuously cultured in a growth medium; alpha MEM containing 10% FBS and epidermal growth factor (EGF). After 2weeks of in vitro culture, cells with fibroblast-like morphology dominantly grow in the culture vessels and resemble mesenchymal stem cells (MSCs). HOFF-derived cells exhibited MSC expression of CD44, CD73, CD90, CD105, CD146, and STRO-1, and were capable of differentiation into osteoblasts, chondrocytes, and adipocytes. After induction of neural differentiation, HOFF-derived cells formed spheroidal structures and expressed neural stem cell markers including Nestin, β-tubulin III, and O4. Besides, the oocyte-like structure was observed after prolonged culture of HOFF. In conclusion, cells derived from follicular fluidexhibited stem cell characteristics, which could be useful for regenerative medicine applications and cell-based therapies.

Suspect and non-target screening of ovarian follicular fluid and serum - identification of anthropogenic chemicals and investigation of their association to fertility.

In this work, ultra-high performance liquid chromatography-high resolution (Orbitrap) mass spectrometry-based suspect and non-target screening was applied to follicular fluid (n = 161) and serum (n = 116) from women undergoing in vitro fertilization in order to identify substances that may be associated with decreased fertility. Detected features were prioritized for identification based on (i) hazard/exposure scores in a database of chemicals on the Swedish market and an in-house database on per- and polyfluoroalkyl substances (PFAS); (ii) enrichment in follicular fluid relative to serum; and (iii) association with treatment outcomes. Non-target screening detected 20 644 features in follicular fluid and 13 740 in serum. Two hundred and sixty-two features accumulated in follicular fluid (follicular fluid: serum ratio >20) and another 252 features were associated with embryo quality. Standards were used to confirm the identities of 21 compounds, including 11 PFAS. 6-Hydroxyindole was associated with lower embryo quality and 4-aminophenol was associated with higher embryo quality. Overall, we show the complexity of follicular fluid and the applicability of suspect and non-target screening for discovering both anthropogenic and endogenous substances, which may play a role in fertility in women.

Fatty acid profiles of the plasma and follicular fluid mares fed a combination of linseed and salmon oil.

This study evaluated the presence of polyunsaturated fatty acids in circulating blood and in the ovarian follicular fluid of mares, after supplementation of the diet with linseed oil. Six Mangalarga Marchador mares, weighing 397.00±31.89 kg, were kept on native pasture, and assigned to the current study. In a switch over design, mares were randomly allocated to receive 150 ml of vegetable oil daily, containing polyunsaturated fatty acids n3 (62.23 g ALA, 20.34 g LA, 2.27 g EPA, 2.32 g DHA), (n=3) or no supplementation (n=3) in two replicates. Blood and follicular fluid samples were taken on the first day (D0) and every 30 days until the end of the supplementation period (D60). After 60 days of supplementation, mares were switched across the treatments. Plasma concentrations of linolenic acid in total fatty acids were higher (P=0.006) in the supplemented compared to the control group (1.89±0.13 vs. 1.49±0.13%). There were positive correlations between plasma linoleic acid and follicular fluid arachidonic acid (P=0.0106; r2=0.13) and between plasma alpha linolenic acid and follicular fluid EPA (P=0.0004; r2=0.2544). Data indicated a low to moderate relationship between the dietary linseed-based oil supplementation studied and circulating and follicular fluid polyunsaturated fatty acids contents in mares.

Variability of essential and non-essential trace elements in the follicular fluid of women undergoing in vitro fertilization (IVF)

Both essential and non-essential elements have been associated with female reproductive function in epidemiologic investigations, including among IVF populations. To date, most investigators have used blood or urine to assess biomarkers of exposure, with few employing ovarian follicular fluid (FF). FF may offer a more direct “snapshot” of the oocyte microenvironment than blood or urine, however previous studies report follicle-to-follicle variability in FF constituents that may contribute to exposure misclassification. Our objectives were to investigate sources of trace element variability, to estimate FF biomarker reliability among women undergoing IVF (n = 34), and to determine the minimum number of follicles required to estimate subject-specific mean concentrations. We measured As, Hg, Cd, Pb, Cu, Mn, Se, and Zn in FF samples using inductively coupled plasma tandem mass spectrometry. Inter-subject (between-women) variability contributed most of the variability in FF element concentrations, with ovarian, follicular, and analytical as smaller sources of variability. The proportion of variability attributable to sources between-follicles differed by age, body mass index (BMI), race, and cigarette smoking for Cu, Se, and Zn, by BMI and cigarette smoking for As, by primary infertility diagnosis for Hg, Cu, Se, and Zn, and by ovarian stimulation protocol for Mn and Se. Four to five individual follicles were sufficient to estimate subject-specific mean Cu, Se, and Zn concentrations, while >14 were necessary for As, Hg, Cd, Pb, and Mn. Overall, our results suggest that FF is a suitable source of biomarkers of As and Hg exposure in ovarian follicles. Although limited in size, our study offers the most comprehensive exploration of biological variation in FF trace elements to date and may provide guidance for future studies of ovarian trace element exposures.

Proteome of fluid from human ovarian small antral follicles reveals insights in folliculogenesis and oocyte maturation.

STUDY QUESTIONIs it possible to identify by mass spectrometry a wider range of proteins and key proteins involved in folliculogenesis and oocyte growth and development by studying follicular fluid (FF) from human small antral follicles (hSAF)?SUMMARY ANSWERThe largest number of proteins currently reported in human FF was identified in this study analysing hSAF where several proteins showed a strong relationship with follicular developmental processes.WHAT IS KNOWN ALREADYProtein composition of human ovarian FF constitutes the microenvironment for oocyte development. Previous proteomics studies have analysed fluids from pre-ovulatory follicles, where large numbers of plasma constituents are transferred through the follicular basal membrane. This attenuates the detection of low abundant proteins, however, the basal membrane of small antral follicles is less permeable, making it possible to detect a large number of proteins, and thereby offering further insights in folliculogenesis.STUDY DESIGN, SIZE, DURATIONProteins in FF from unstimulated hSAF (size 6.1 ± 0.4 mm) were characterised by mass spectrometry, supported by high-throughput and targeted proteomics and bioinformatics. The FF protein profiles from hSAF containing oocytes, capable or not of maturing to metaphase II of the second meiotic division during an IVM (n = 13, from 6 women), were also analysed.PARTICIPANTS/MATERIALS, SETTING, METHODSWe collected FF from hSAF of ovaries that had been surgically removed from 31 women (∼28.5 years old) undergoing unilateral ovariectomy for fertility preservation.MAIN RESULTS AND THE ROLE OF CHANCEIn total, 2461 proteins were identified, of which 1108 identified for the first time in FF. Of the identified proteins, 24 were related to follicular regulatory processes. A total of 35 and 65 proteins were down- and up-regulated, respectively, in fluid from hSAF surrounding oocytes capable of maturing (to MII). We found that changes at the protein level occur already in FF from small antral follicles related to subsequent oocyte maturation.LIMITATIONS, REASONS FOR CAUTIONA possible limitation of our study is the uncertainty of the proportion of the sampled follicles that are undergoing atresia. Although the FF samples were carefully aspirated and processed to remove possible contaminants, we cannot ensure the absence of some proteins derived from cellular lysis provoked by technical reasons.WIDER IMPLICATIONS OF THE FINDINGSThis study is, to our knowledge, the first proteomics characterisation of FF from hSAF obtained from women in their natural menstrual cycle. We demonstrated that the analysis by mass spectrometry of FF from hSAF allows the identification of a greater number of proteins compared to the results obtained from previous analyses of larger follicles. Significant differences found at the protein level in hSAF fluid could predict the ability of the enclosed oocyte to sustain meiotic resumption. If this can be confirmed in further studies, it demonstrates that the viability of the oocyte is determined early on in follicular development and this may open up new pathways for augmenting or attenuating subsequent oocyte viability in the pre-ovulatory follicle ready to undergo ovulation.STUDY FUNDING/COMPETING INTEREST(S)The authors thank the financial support from ReproUnion, which is funded by the Interreg V EU programme. No conflict of interest was reported by the authors.TRIAL REGISTRATION NUMBERN/A

Association of exosomal microRNAs in human ovarian follicular fluid with oocyte quality

The postponement of childbearing by women has led to an increase in infertility. The reproductive aging process leads to a decrease in both the quantity and quality of oocytes. The aim of this study was to investigate exosomal microRNAs in human ovarian follicular fluid and explore their potential association with oocyte quality. We collected ovarian follicle fluid from 68 patients and assigned the patients to A (superior oocyte quality) or B (poor oocyte quality) group according to their oocyte quality. Exosomal miRNAs were extracted, library constructed and sequenced using the Illumina HiSeq platform. Subsequently, we analyzed exosomal miRNA expression, predicted the miRNA target genes, and enriched Gene Ontology terms using GOSeq. Kyoto Encyclopedia of Genes and Genomes pathway analysis was performed using miRanda. A total of 47 miRNAs were found to be significantly differentially expressed between group A and group B (p < 0.05). Among nine differentially expressed miRNAs that were previously known, seven were upregulated in group B. In silico analysis indicated that several of these exosomal miRNAs were involved in pathways implicated in oocyte quality. Analysis of the expression of exosomal miRNAs in human ovarian follicular fluid showed that they were critical for maintaining oocyte quality. Our findings provide the basis for further investigations of the functions of exosomal miRNAs in the ovarian microenvironment and suggest that these exosomal miRNAs may be potential biomarkers for evaluating oocyte quality. The findings are potentially important to maintain oocyte quality in clinical settings.

Comparative Analysis of Biochemical Constituents between Ovarian Follicular Fluid and Serum in Ganjam Goat (Capra hircus)

Background: Follicular fluid is in part exudates of serum and is also partially composed of locally produced substances, which are related to the metabolic activity of the follicular cells. The knowledge of the biochemical composition of follicular fluid can provide useful information about the requirements, growth and maturation of oocyte and which may be used as a provisional guide for formulating suitable culture media for in vitro cell culture and maturation in a particular species. Methods: Follicular fluid were aspirated from small follicles ( less than 3 mm; SF), medium follicles (3-5 mm; MF) and large follicles ( greater than 5 mm; LF). Blood samples were also collected for extraction of serum. Follicular fluid and serum samples were analyzed for biochemical constituents. Conclusion: It can be concluded that some biochemical constituents of follicular fluid such as glucose, cholesterol, total protein and globulin were higher in large follicles compared to small follicles. Moreover, serum levels of most of biochemical constituents were higher than their levels in different sizes of follicular fluid. This present investigation of comparative analysis of biochemical constituents between serum and ovarian follicles in Ganjam goat of Odisha will provide clues to researchers who work on reproductive physiology in different breeds of goat.

Sphingosine-1 phosphate induces cAMP/PKA-independent phosphorylation of the cAMP response element-binding protein (CREB) in granulosa cells

Background and aimsSphingosine-1 phosphate (S1P) is a lysosphingolipid present in the ovarian follicular fluid. The role of the lysosphingolipid in gonads of the female is widely unclear. At nanomolar concentrations, S1P binds and activates five specific G protein-coupled receptors (GPCRs), known as S1P1-5, modulating different signaling pathways. S1P1 and S1P3 are highly expressed in human primary granulosa lutein cells (hGLC), as well as in the immortalized human primary granulosa cell line hGL5. In this study, we evaluated the signaling cascade activated by S1P and its synthetic analogues in hGLC and hGL5 cells, exploring the biological relevance of S1PR-stimulation in this context. METHODS AND RESULTS. hGLC and hGL5 cells were treated with a fixed dose (0.1 μM) of S1P, or by S1P1- and S1P3-specific agonists SEW2871 and CYM5541. In granulosa cells, S1P and, at a lesser extent, SEW2871 and CYM5541, potently induced CREB phosphorylation. No cAMP production was detected and pCREB activation occurred even in the presence of the PKA inhibitor H-89. Moreover, S1P-dependent CREB phosphorylation was dampened by the mitogen-activate protein kinase (MEK) inhibitor U0126 and by the L-type Ca2+ channel blocker verapamil. The complete inhibition of CREB phosphorylation occurred by blocking either S1P2 or S1P3 with the specific receptor antagonists JTE-013 and TY52156, or under PLC/PI3K depletion. S1P-dependent CREB phosphorylation induced FOXO1 and the EGF-like epiregulin-encoding gene (EREG), confirming the exclusive role of gonadotropins and interleukins in this process, but did not affect steroidogenesis. However, S1P or agonists did not modulate granulosa cell viability and proliferation in our conditions. ConclusionsThis study demonstrates for the first time that S1P may induce a cAMP-independent activation of pCREB in granulosa cells, although this is not sufficient to induce intracellular steroidogenic signals and progesterone synthesis. S1P-induced FOXO1 and EREG gene expression suggests that the activation of S1P–S1PR axis may cooperate with gonadotropins in modulating follicle development.

Ovarian follicular fluid: an innovative approach to assess internal dose for reproductive epidemiology

Ovarian follicular fluid serves as a biological window revealing metabolic processes in the microenvironment of a maturing oocyte, and so may offer a more accurate biomarker of biologically effective dose than more traditional compartments, such as blood and urine. Investigators have measured myriad environmental pollutants in ovarian follicular fluid (FF), including toxic trace elements. As a plasma ultrafiltrate, FF content reflects both blood concentrations and in situ physiology. Evidence suggests substantial follicle-to-follicle variability in the concentrations of some analytes, necessitating a “one follicle-one oocyte” approach for investigating associations between exposure and reproductive endpoints. We collected individual FF specimens from up to four follicles, two from each ovary, among 56 women undergoing in vitro fertilization (IVF) with fresh embryo transfer. We determined toxic trace element concentrations using inductively coupled plasma with tandem mass spectrometry (ICP-MS/MS) and linked individual oocytes and embryos to the corresponding FF specimen. We used linear mixed regression models to evaluate associations between natural log transformed FF As, Cd, Pb, and Hg as predictors of IVF outcomes, adjusted for other toxic trace elements, age, race, smoking, and seafood consumption. Median FF As (0.34 μg/L), Cd (27.3 ng/L), Pb (0.06 μg/L), and Hg (0.34 μg/L) concentrations were similar to or lower than those reported previously. Greater FF Cd was associated with a lower probability for a symmetric embryo per 1 ng/L (RR=0.36, 95%CI: 0.17-0.76). Greater FF Pb (1 μg/L) was associated with lower probabilities for a fertilized oocyte per 1 μg/L (RR=0.56, 95%CI=0.25-1.24), although the association was non-linear, and with live birth per 1 μg/L (RR=0.72, 95%CI: 0.53-0.99). Greater Hg was associated with a lower probability of implantation per 1 μg/L (RR=0.31, 95%CI: 0.09-1.06), although the association was non-linear. Our results suggest that FF toxic trace elements may affect IVF outcomes, including live birth, even at comparatively low doses.

Variability of essential and toxic trace elements in the follicular fluid of women undergoing in vitro fertilization (IVF)

Both essential and non-essential elements have been associated with female reproductive function in epidemiologic investigations, including those among IVF populations. To date, most investigators have used blood or urine to assess biomarkers of exposure, with few employing ovarian follicular fluid (FF). FF may offer a more direct 'snapshot' of the oocyte microenvironment, however previous studies report follicle-to-follicle variability in FF constituents which may contribute to exposure misclassification. Our objectives were to: investigate sources of trace element variability in FF among women undergoing IVF (n=34, ≤4 FF collections each; estimate biomarker reliability; and determine the minimum number of follicles to estimate mean concentrations at a 10% confidence level (m10%). We measured As, Cd, Hg, Pb, Cu, Mn, Se, and Zn in FF samples using ICP-MS/MS and employed linear mixed models using restricted maximum likelihood to generate variability components. Sources of variability between women contributed the greatest proportion of total variance for FF As (97.2%), Hg (97.8%), Cd (52.1%), Cu (60.4%), Se (48.1%), and Zn (56.8%), compared to sources between ovaries, between follicles, and analytical variation. The proportion of variability attributable to sources between follicles differed significantly (p&lt;0.05) by age, BMI, race, and cigarette smoking for Cu, Se, and Zn, by BMI and cigarette smoking for As, by primary infertility diagnosis for Hg, Cu, Se, and Zn, and by ovarian stimulation protocol for Mn and Se. To estimate m10% for Cu, Se, and Zn 4 to 5 individual follicles were sufficient, while &gt;14 were necessary for As, Cd, Hg, Pb, and Mn. Our results suggest that while FF As and Hg are suitable biomarkers, these elements and Cd, Pb, and Mn have comparatively higher between-follicle variability than the other analytes. Our findings may help elucidate the impact of trace element exposures on IVF outcomes and inform the design of future studies.

Assessment of human ovarian follicular fluid derived mesenchymal stem cells in chitosan/PCL/Zn scaffold for bone tissue regeneration

Bone tissue engineering compasses the use of mesenchymal stem cells (MSCs) along with engineered biomaterial construct to augment bone regeneration. Till now, MSCs were isolated from various sources and used in cellular constructs. For the first time, in this study, MSCs were isolated from human Ovarian Follicular Fluid (OFF) and characterized by CD 44+ and CD 105+ markers via confocal microscopy and flow cytometry. Additionally, MSCs stemness, proliferation and colony-forming unit ability, multi-lineage differentiation potential were also studied. To test its suitability for bone tissue engineering applications, we grew the MSCs with the conditioned medium obtained from biocomposite scaffold by fusing a natural polymer, Chitosan (CS) and a synthetic polymer, Polycaprolactone (PCL) and the scaffold were coated with Zinc divalent ions to impart osteogenic properties. The physico-chemical characterization of scaffold, such as FTIR, XRD, and SEM studies was carried out. The biological characterization showed that the scaffolds were compatible with MSCs and promoted osteoblast differentiation which was confirmed at both cellular and molecular levels. The cellular construct increased calcium deposition, analyzed by alizarin red staining and ALP activity at cellular level. At the molecular level, the osteoblast markers expression such as Runx2 and type 1 collagen mRNAs, and osteonectin (ON) and osteocalcin (OC) secretory proteins were increased in the presence of scaffold. Overall, the current study recommends that MSCs can be easily obtained from human waste OFF, and grown in standard in vitro conditions. Successful growth of such MSCs with CS/PCL/Zn scaffold opens new avenues in utilizing the cell source for bone tissue engineering.

Characterization of the Age-Dependent Changes in Antioxidant Defenses and Protein's Sulfhydryl/Carbonyl Stress in Human Follicular Fluid.

The oxidative stress, characterized by the imbalance between pro-oxidants and antioxidants molecules, seems to be involved in the pathogenesis of female subfertility. In particular, the presence of different markers of oxidative stress has been reported in human follicular fluid (FF) surrounding oocytes. Based on its distinctive composition and on the close proximity to the oocyte, FF creates a unique microenvironment having a direct impact on oocyte quality, implantation, and early embryo development. An imbalance in reactive oxygen species (ROS) production in ovarian follicular fluid may have a negative effect on these processes and, as a consequence, on female fertility. Therefore, the aim of this study was to evaluate the redox state of the FF through various methodological approaches. By means of 2D-electrophoresis we demonstrated that the main structural changes occurring in the proteins of the follicular fluid of normovulatory women were correlated to the age of the patients and to the antioxidant defenses present in the FF. Measurement of these parameters could have clinical relevance, since the assessment of the oxidative stress rate may be helpful in evaluating in vitro fertilization potential.

Can extracellular vesicles from bovine ovarian follicular fluid modulate the in-vitro oocyte meiosis progression similarly to the CNP-NPR2 system?

C-type natriuretic peptide (CNP) and its natriuretic peptide receptors subtype 2 (NPR2) are essential for the maintenance of oocyte meiotic arrest in different species. Extracellular vesicles (EVs) in bovine follicular fluid (FF) are important for cell communication within the ovarian follicle. This study investigated the involvement of EVs from FF of bovine ovarian follicles in the CNP-NPR2 system, first by analyzing the presence of CNP in the EV contents, followed by addition of EVs to in-vitro maturation (IVM) medium, to evaluate the effect on maintenance of oocyte meiosis arrest and improvements in in-vitro embryo production. As expected, CNP was observed in FF and granulosa cells from the ovarian follicles. To the best of our knowledge, this is the first time that CNP has been found in the EV contents. To evaluate the possible effect of EVs on the progression of oocyte meiosis, the IVM was performed under three conditions: CNP and EV supplementation and control condition. Both the CNP and EV treatments inhibited meiosis resumption in the oocyte within 9 h of IVM. CNP treatment increased cGMP levels in cumulus cells within 6 h of IVM compared to the control group, but the EV treatment did not. In contrast, the relative mRNA abundance of adenylate cyclase 3 and 9 (ADCY3 and ADCY9) was upregulated in oocytes after 6 h of IVM under EV treatment compared to the control group, but not under CNP treatment. Last, these treatments in the IVM medium had no significant effect on the in-vitro embryo production. In conclusion, we demonstrated the presence of endogenous CNP in bovine reproductive structures, especially in the EVs from the FF of antral follicles. The presence of CNP in the EVs suggests an important involvement of this cell-communication system in the CNP-NPR2 system. Therefore, we indeed observed that the EVs from FF can modulate the arrest of oocyte meiosis, acting similarly to the CNP-NPR2 system to block the oocyte in the GV state. However, the mechanism of each system might be different; the CNP-NPR2 system seems to be involved in modulating the cGMP levels, while the contents of EVs might be involved in modulating the cAMP levels.

Supplementation of follicular fluid into Garut sheep (Ovis aries) oocytes in vitro maturation media

Kaiin EM, Prasetyo AT, Gunawan M, Setiatin ET, Ondho YS. 2020. Supplementation of follicular fluid into Garut sheep (Ovis aries) oocytes in vitro maturation media. Pros Sem Nas Masy Biodiv Indon 6: 532-536. The study was conducted to determine the effect of supplementation of sheep ovarian follicular fluid added to the oocyte maturation media on the rate of expansion of cumulus cells and the appearance of polar bodies I. Follicular fluid collected from the ovaries of Garut sheep and stored at -20°C before use. The treatment of adding follicular fluid with concentrations of 0 (control), 10, 20 and 30% in the M-199 maturation medium was used for in vitro maturation of sheep oocyte, then cultured in a 5% CO2 incubator with temperature 38.5oC for 22-24 hours. Sheep oocytes used were graded A and B with 7 replications. Data were analyzed by non-parametric statistical tests for Complete Random Design (CRD) and Duncan's follow-up test. The observation of the rate of expansion of cumulus cells showed no significant difference between treatments. Addition of Garut sheep ovarian follicular fluid in oocyte maturation media had a significant effect on the appearance of polar I bodies with the highest value (56%) in the treatment of 30% follicular fluid addition. Based on the results, it can be concluded that the addition of follicular fluid into the maturation medium influences the appearance of the polar I body which is the main characteristic of oocyte maturation.

Association of taurine with ovarian follicular steroids and postpartum anestrus condition in Murrah buffaloes

Taurine is an abundant intracellular beta-amino acid majorly synthesized in the liver and transported through plasma. In mammals, taurine was reported to be involved in various physiological functions, including the enhancement of testosterone levels, the major estradiol precursor. Therefore, we hypothesize that taurine levels are associated with ovarian follicular steroids as well as with a reproductive problem called postpartum anestrus (PPA) in dairy buffaloes. To understand the taurine levels and its possible role in buffalo ovarian follicles, a correlation was established among taurine, estradiol, and testosterone levels in the ovarian follicular fluid. For this purpose, buffalo ovaries were obtained from the slaughterhouse, and follicular fluid samples were collected from small (<4 mm), medium (4–8 mm) and large (>8 mm) follicles. Taurine and steroid levels in the follicular fluid were analyzed by TLC and ELISA, respectively. Taurine and testosterone levels were significantly (P < 0.05) higher in the follicular fluid of small and medium follicles than large follicles, whereas the estradiol levels were significantly (P < 0.001) higher in the large follicles. Thus, taurine showed a positive correlation (r = 0.75) with testosterone and a negative correlation (r = −0.77) with estradiol in buffalo follicular fluid, indicating its possible role in testosterone function during follicular development. Interestingly, significantly (P < 0.001) lower plasma taurine levels in PPA (n = 50) than normal cyclic (n = 50) buffaloes represented its association with PPA. Therefore, our present study recommends the need for future nutrition studies on taurine supplementation to PPA buffaloes.

Seafood consumption is associated with higher follicular fluid arsenic (As) and mercury (Hg) concentrations in women undergoing in vitro fertilization (IVF)

Human exposure to non-essential toxic metals such as cadmium (Cd), mercury (Hg), and lead (Pb), and metalloids such as arsenic (As) commonly occurs through diet. Toxic trace element exposures have been reported in association with fertility and fecundity in epidemiologic studies even at low to moderate levels. While most previous studies employed blood and urine biomarkers of exposure, few have assessed toxic trace elements in ovarian follicular fluid (FF), which surrounds the developing oocyte and hence may better reflect concentrations potentially affecting reproductive outcomes. Our objective was to identify dietary predictors of FF toxic trace elements in n = 56 women (mean age: 38.3 years) undergoing in vitro fertilization (IVF) at the University of California at San Francisco. We determined As, Hg, Cd, and Pb in 197 FF specimens, collected on the day of oocyte retrieval, using inductively coupled plasma tandem mass spectrometry. A comprehensive food frequency questionnaire was used to assess the weekly and annual dietary “patterns” of participants. Consumption of specific seafood items and turkey were correlated with individual FF toxic trace elements. We also found that each unit higher seafood consumption in the past week dominated by mollusks, shrimp, and bass was associated with 60% higher FF As (95% confidence interval (CI): 25%, 105%) and FF Hg (95%CI: 7%, 136%) concentrations. Higher annual seafood consumption dominated by urchin, crab, and trout was associated with 16% higher FF As (95%CI: −2%, 38%) and 31% higher FF Hg (95%CI: 7%, 60%) concentrations per unit intake. No associations were noted between diet and Cd and Pb levels in FF. Overall, our results suggest that higher seafood consumption contributes to elevated levels of As and Hg in FF. These findings are consistent with previous IVF studies that assessed toxic trace element exposures in blood and urine. To our knowledge, this is the first study to report that diet might be a source of As, Hg, Cd, and Pb in FF.