Components Of Follicular Fluid Research Articles

High coverage of targeted lipidomics revealed lipid changes in the follicular fluid of patients with insulin-resistant polycystic ovary syndrome and a positive correlation between plasmalogens and oocyte quality.

Polycystic ovary syndrome with insulin resistance (PCOS-IR) is the most common endocrine and metabolic disease in women of reproductive age, and low fertility in PCOS patients may be associated with oocyte quality; however, the molecular mechanism through which PCOS-IR affects oocyte quality remains unknown. A total of 22 women with PCOS-IR and 23 women without polycystic ovary syndrome (control) who underwent in vitro fertilization and embryo transfer were recruited, and clinical information pertaining to oocyte quality was analyzed. Lipid components of follicular fluid (FF) were detected using high-coverage targeted lipidomics, which identified 344 lipid species belonging to 19 lipid classes. The exact lipid species associated with oocyte quality were identified. The number (rate) of two pronuclear (2PN) zygotes, the number (rate) of 2PN cleaved embryos, and the number of high-quality embryos were significantly lower in the PCOS-IR group. A total of 19 individual lipid classes and 344 lipid species were identified and quantified. The concentrations of the 19 lipid species in the normal follicular fluid (control) ranged between 10-3 mol/L and 10-9 mol/L. In addition, 39 lipid species were significantly reduced in the PCOS-IR group, among which plasmalogens were positively correlated with oocyte quality. This study measured the levels of various lipids in follicular fluid, identified a significantly altered lipid profile in the FF of PCOS-IR patients, and established a correlation between poor oocyte quality and plasmalogens in PCOS-IR patients. These findings have contributed to the development of plasmalogen replacement therapy to enhance oocyte quality and have improved culture medium formulations for oocyte in vitro maturation (IVM).

Correlation of Metabolic and Ionic Components of Follicular Fluid with Estrogen Concentration

This study was aimed to assess the correlation between the metabolic and ionic composition of ovarian follicular fluid and its relationship to the concentration of estrogen and prolactin hormones as the follicle size changes in the local female cows. Ovaries were collected (80 ovaries) from non- pregnant 40 adult females cow at Hilla slaughterhouse at Babil province. The gathered ovaries were transported to the laboratory within two hours. Follicular fluid was pulled from small (2-5 mm) follicles, medium (6-10 mm), and large (11-20 mm) follicles, then it stored at 5 ° C until assayed. Follicular fluid samples were analyzed for metabolites composition of (cholesterol, total protein, and glucose), and ionic composition (calcium, phosphorus, copper, magnesium, zinc) ,and hormone concentration ( estrogen and prolactin). The results of the study showed that the total protein and glucose concentrations were increased significantly(p> 05.0) with increased of follicle size. The study also showed the calcium, copper, and phosphorus concentration were increased significantly(p> 05.0) with changes of follicular size, as well as the estrogen concentration was significantly increased (p > 05.0) With increased follicular size. A significantly (p> 05.0) correlation was found between both magnesium, zinc ions, and estrogen hormone.

Follicular fluid C3a-peptide promotes oocyte maturation through F-actin aggregation

BackgroundImmature cumulus-oocyte complexes are retrieved to obtain mature oocytes by in vitro maturation (IVM), a laboratory tool in reproductive medicine to obtain mature oocytes. Unfortunately, the efficiency of IVM is not satisfactory. To circumvent this problem, we therefore intended to commence with the composition of ovarian follicular fluid (FF), an important microenvironment influencing oocyte growth. It is well known that FF has a critical role in oocyte development and maturation. However, the components in human FF remain largely unknown, particularly with regard to small molecular peptides.ResultsIn current study, the follicular fluid derived from human mature and immature follicles were harvested. The peptide profiles of FF were further investigated by using combined ultrafiltration and LC–MS/MS. The differential peptides were preliminary determined by performing differentially expressed analysis. Human and mouse oocyte culture were used to verify the influence of differential peptides on oocyte development. Constructing plasmids, cell transfecting, Co-IP, PLA etc. were used to reveal the detail molecular mechanism. The results from differentially expressed peptide as well as cultured human and mouse oocytes analyses showed that highly conserved C3a-peptide, a cleavage product of complement C3a, definitely affected oocytes development. Intriguingly, C3a-peptide possessed a novel function that promoted F-actin aggregation and spindle migration, raised the percentage of oocytes at the MII stage, without increasing the chromosome aneuploidy ratio, especially in poor-quality oocytes. These effects of C3a-peptide were attenuated by C3aR morpholino inhibition, suggesting that C3a-peptide affected oocytes development by collaborating with its classical receptor, C3aR. Specially, we found that C3aR co-localized to the spindle with β-tubulin to recruit F-actin toward the spindle and subcortical region of the oocytes through specific binding to MYO10, a key regulator for actin organization, spindle morphogenesis and positioning in oocytes.ConclusionsOur results provide a new perspective for improving IVM culture systems by applying FF components and also provide molecular insights into the physiological function of C3a-peptide, its interaction with C3aR, and their roles in enabling meiotic division of oocytes.

Biological profile of the follicular fluid. A pilot study

BACKGROUND: One of the most important research areas in the field of reproductive medicine is the search for biochemical and immunological parameters of oocyte quality and predicting the effectiveness of assisted reproductive technology protocols.
 AIM: The aim of this study was to evaluate the expression of follicular fluid soluble human leukocyte antigen-G, human leukocyte antigen-E, human leukocyte antigen-C, progesterone-inducing blocking factor, and relaxin levels in women with reproductive disorders.
 MATERIALS AND METHODS: This prospective cohort study included 22 patients undergoing infertility treatment in a superovulation stimulation protocol using gonadotropin-releasing hormone antagonists. The inclusion criteria were age from 25 to 39 years, tubal-peritoneal factor infertility, and voluntary participation informed consent. The levels of soluble human leukocyte antigen-G, human leukocyte antigen-E, human leukocyte antigen-C, progesterone-inducing blocking factor, and relaxin in follicular fluid samples were determined on the day of transvaginal follicle puncture by enzyme immunoassay.
 RESULTS: We established an inverse correlation between the expression levels of progesterone-inducing blocking factor and relaxin (r = 0.450) in the follicular fluid, antibodies to thyroperoxidase (r = 0.649), and thyroid-stimulating hormone (r = 0.519). We also found a direct correlation between human leukocyte antigen-E parameters in the follicular fluid, age (r = 0.813) and Body Mass Index (r = 0.866), as well as between human leukocyte antigen-C expression levels and total testosterone (r = 0.960). No data were obtained on any significant correlations between the studied biomarkers and the number of received oocytes.
 CONCLUSIONS: In this comprehensive study, we were the first who found the expression levels of five different follicular fluid components, namely, soluble human leukocyte antigen-G, human leukocyte antigen-E, human leukocyte antigen-C, progesterone-inducing blocking factor, and relaxin. Such a complex assessment of the follicular fluid can allow for establishing the quality of the oocyte to predict the onset of pregnancy in an in vitro fertilization protocol.

Evaluation of cortisol, thyroxine (T3) and triiodothyronine (T4) concentrations in preovulatory follicles in jennies.

Reference values of follicular fluid components at different stages of follicular development in jennies are unknown. It is important to estimate such values because the biochemical composition of follicular fluid provides a suitable microenvironment for oocyte development, growth and maturation. The experimental protocol was carried out in the Equine Reproduction Clinic of the FMVZ-UNAM and was approved by an Institutional Sub-committee for the Care and Use of Experimental Animals (SICUAE.MMVZ-2020/4-2). Seven healthy female donkeys “Mexican burro” with average body condition score of 3 on a scale of 1 to 5 (Svendsen et al. The professional Handbook of the Donkey. 2008), were monitored by transrectal ultrasonography through the Veterinary Digital Diagnostic Ultrasound System 5 MHz (Model, DUS 60 VET) with a multi-frequency linear transducer. The ovum pick up (OPU) by transvaginal aspiration (TVA) was performed in a total of 70 preovulatory follicles, 10 for each jenny using a real- time B- mode ultrasound vaginal 6.5 MHz (Sonovet 600) with curvilinear transducer and 12 gauge (12 G) needle. Follicular fluid components were measured and analyzed by Luminex Multiplex Assays at the Universidad Nacional Autónoma de México. The relationship between cortisol, thyroxine (T3) and triiodothyronine (T4) concentrations in preovulatory follicles and follicular size was assessed. The preovulatory follicles are classified in three groups accordingto the diameter: small (27-30 mm), medium (31-35 mm) and large (36-38 mm). Concentrations of cortisol were lower in the large follicles group (18.8 ng/ml) compared with small (721.44 ng/ml) and medium follicles (361 ng/ml). The concentrations of thyroxine were higher in the medium follicle group (49 ng/ml) than in the small (21ng/ml) and large follicle groups (15.9 ng/ml). Finally, the concentration of triiodothyronine was higher in the small follicles group (1043 ng/ml) followed by the medium (545 ng/ml) and the lowest concentration was in the large follicles group (22 ng/ml). It is concluded that thyroxine concentrations are lower than T4 concentrations in preovulatory follicles irrespective of size as reported for human follicular fluid (Wakim. et. al. Fertil Steril 1993; 59(6) 1187–1190). Furthermore, no relationship was found between preovulatory follicular size and thyroxine hormone concentrations. It has been suggested that cortisol and triiodothyronine concentrations are related to preovulatory follicle size. Cortisol concentrations are higher in preovulatory follicles that in subordinate follicles and cortisol is a mediator of apoptosis in granulosa cells, differentiation, development and maturation of tissues, specifically in the oocyte (Acosta & Miyamoto. Anim Reprod Sci 2004; 82:127-140, Scarlett. et al. Dom Anim Endocrinol 2016; 59:11-22). The role of cortisol within the different sizes of preovulatory follicles is unknown.

Nuclear magnetic resonance-based metabolomics in goat ovarian follicular fluid

During folliculogenesis, follicular fluid (FF) supplies many of the nutrients, metabolic compounds, and signaling molecules that are essential for the accumulation of metabolic substrate stores within the oocyte. The use of metabolomics can identify metabolites produced in FF that are related to oocyte quality. Thus, this work aimed to carry out a qualitative metabolomic study using Hydrogen Nuclear Magnetic Resonance Spectroscopy (1H NMR) to characterize FF components of goats after hormonal estrus synchronization (HES) and no estrus synchronization (NES). Fluids of small (< 3 mm) and large (≥ 3 mm) follicles were collected and evaluated. 1H NMR analyses permitted to characterize seventeen metabolites, comprising sugars, amino acids, short chain organic acids, fatty acids residues, besides acetylated glycoprotein, choline phosphate and choline glycerophosphate. Qualitative differences were observed between follicles at different stages of development (small vs large). Finally, further quantitative studies will be needed to clarify the FF synthesis in goats and the effects on the quality of oocytes.

Effect of PFOA exposure on diminished ovarian reserve and its metabolism

Owing to its difficulty in degrading and ease of accumulation in the body, perfluorooctanoic acid (PFOA) has a detrimental effect on reproduction. This study aimed to examine the effect of PFOA concentration in follicular fluid during ovulation stimulation on embryo quality and the impact of PFOA exposure on the metabolic components of follicular fluid. This was a single-center prospective study that included 25 patients with diminished ovarian reserve (DOR), 25 with normal ovarian reserve (NOR), and 25 with polycystic ovary syndrome (PCOS). Follicular fluid samples were analyzed using ultra-high performance liquid chromatography-tandem mass spectrometry. We demonstrated that the PFOA levels of follicular fluid in the DOR group were higher than those in the NOR group and PCOS group (P < 0.05). PFOA concentration in the PCOS group was negatively correlated with high-quality embryos (P < 0.05). To gain more insight into the impact of PFOA on the metabolic composition of follicular fluid, we classified the DOR group based on the PFOA concentration, for which metabolomic analysis was performed. In the high-concentration PFOA group, there was an increase and a decrease in three and nine metabolites, respectively, compared to that in the low-concentration group. These results suggest that PFOA may alter the metabolic composition of follicular fluid, thus, affecting ovarian reserve function.

Follicular fluid progesterone downregulated HPGD and COX2 in granulosa cells via suppressing NF-ĸB in endometriosis†.

Increasing evidences showed that ovulatory dysfunction, possibly caused by luteinized unruptured follicular follicle syndrome (LUFS), is one of the reasons for endometriosis-related infertility. The present study was conducted to explore the potential effect of elevated progesterone in follicular fluid (FF) on ovulation in endometriosis. A prospective study including 50 ovarian endometriosis patients and 50 control patients with matched pairs design was conducted with alterations in FF and peritoneal fluid (PF) components identified by metabolomics analyses and differentially expressed genes in granulosa cells (GCs) identified by transcriptome analysis. Patients with endometriosis exhibited a significantly higher progesterone level in serum, FF, and PF. Granulosa cells from endometriosis patients revealed decreased expression of HPGD, COX-2, and suppressed NF-ĸB signaling. Similarly, progesterone treatment in vitro downregulated HPGD and COX2 expression and suppressed NF-ĸB signaling in granulosa tumor-like cell line KGN (Bena Culture Collection, China) and primarily cultured GCs, as manifested by decreased expressions of IL1R1, IRAK3, reduced pIĸBα/IĸBα ratio, and nucleus translocation of p65. On the contrary, TNF-α treatment increased expression of IL1R1, IRAK3, pIĸBα, p65, and HPGD in GCs. One potential p65 binding site was identified in the promoter region of HPGD by chromatin immunoprecipitation. In conclusion, we found that intrafollicular progesterone might downregulate HPGD and COX-2 in GCs via suppressing the NF-ĸB signaling pathway, shedding light on the mechanism underlying the endometriosis-related ovulatory dysfunction.

Follicular Fluid Components in Reduced Ovarian Reserve, Endometriosis, and Idiopathic Infertility.

Follicular fluid (FF) molecules, and their increase or decrease, can contribute to appropriate follicular growth and oocyte maturation, thus being related to female infertility conditions. In this paper, we studied the changes and the relationships of some biochemical components, hormones, antioxidant enzymes, F2-Isoprostanes (F2-IsoPs), and resolvin (Rv) D1 in the FF of infertile women with different reproductive conditions such as endometriosis, reduced ovarian reserve, and idiopathic infertility during assisted reproductive techniques (ART). In the whole population, positive correlations between albumin (ALB)/iron (Fe), ALB/beta-2-microglobulin (B2MG), and F2-IsoPs/RvD1 were detected in the FF. In FF from aged women, increased levels of follicle stimulating hormone (FSH) and reduced anti-Müllerian hormone (AMH) levels were associated with a worse oocyte quality. The negative ART outcome was influenced by patient age and AMH, B2MG, and FSH levels. Moreover, the reduced ovarian reserve condition was characterised by a significant decrease in oocyte number and quality, AMH amount, and lactate dehydrogenase (LDH) activity, as well as by an increase in age and FSH levels. In the presence of endometriosis, high levels of MDA and RvD1 were detected in FF, with a decrease in luteinising hormone (LH). Finally, among the molecules examined, none characterised the condition of idiopathic infertility. These data could support the identification of new FF markers in different reproductive disorders, suggesting the need for personalised therapeutic approaches and optimised ART outcomes. In particular, the evaluation of resolvins and lipid mediators in FF could be a promising field of investigation with which to understand the entity of oxidative stress and inflammation in some female infertility conditions.

Follicular Fluid Growth Factors and Interleukin Profiling as Potential Predictors of IVF Outcomes.

Growth hormone (GH) has gained attention as an anti-aging compound enhancing oocyte quality. In fact, GH is known to activate intrafollicular metabolic events for oocyte maturation. Insulin growth factor I (IGF1) is another ovarian growth factor that mediates the FSH and GH actions. Cytokines could also increase IVF outcomes. Indeed, IL-6 is a pleiotropic cytokine with multiple cellular effects that can vary based on the physiological environment. IL-6 may also play an important role in follicular development (Yang et al., J Assist Reprod Genet, 2020, 37 (5), 1171–1176). Clinical studies have been performed to explore the potential role of IL-6 in human oocyte maturation and subsequent embryonic development. To date, the answers are not conclusive. During peri-implantation, many cytokines balances are regulated like pro-inflammatory and anti-inflammatory interleukins. The pro-inflammatory properties of IL-17 and its impact on the tumor microenvironment or autoimmune diseases are characterized, but new dimensions of IL-17 activity that promotes embryo implantation are not well explored. In the search for answers, our study compared concentrations of growth factors IGF1, GH, and interleukins IL-6 and IL-17 in the follicular fluid (FF) from 140 women divided into two groups depending on bad (G1) or good prognosis (G2) and investigated the relationships between these FF components’ levels and the main parameters of IVF. GH, IGF1, and IL-6 were significantly higher for G2. For GH, it was negatively correlated to patient age and positively correlated to maturity rate and IGF1. Moreover, GH and IGF1 were correlated to the top embryo rate and cumulative pregnancy rate. Regarding IL-6, it was correlated to IGF1 level, endometrium thickness, and implantation rate. As for IL-17, it was only correlated to IL-6. Consequently, all these FF components were predictive of oocyte quality except IL-17. GH seemed to be the best biomarker of this quality.

Cocultured porcine granulosa cells respond to excess non-esterified fatty acids during in vitro maturation

BackgroundNon-esterified fatty acids (NEFAs) are one of the main lipid components of follicular fluid at concentrations that depend on circulating levels. Elevated levels of NEFAs impair oocyte quality, development potential, and may subsequently influence the metabolism and reproductive fitness of offspring. Granulosa cells (GCs) are the follicular cells that are closely communicating with the oocyte. However, the responses of GCs exposed to high levels of NEFAs when cocultured with cumulus-oocyte complexes (COCs), and how they attenuate the negative effects of NEFAs on oocytes, are unclear.ResultsTo better understand this protective effect, monolayers of porcine GCs were cocultured with COCs during in vitro maturation (IVM) in the presence of elevated levels of NEFAs. Genomic expression analysis was conducted to explore the responses of the GCs to the elevated levels of NEFAs. After limma algorithm analysis, 1,013 genes were differentially expressed between GCs cultured with and without elevated NEFAs. Among them, 438 genes were upregulated and 575 were downregulated. The differentially expressed genes were enriched in pathways related to metabolism, inflammation, and epithelial-mesenchymal transition.ConclusionsThe pathways and upstream regulators suggested that the cocultured GCs responded to the elevated NEFAs with (1) inhibition of the transition from granulosa to luteal cell, (2) interactions of metabolism change, anti-inflammation, mitochondrial function, and cell transition, (3) intercommunication with cocultured COCs of anti-inflammatory factors.

UHPLC-MS-MS analysis of oxylipins metabolomics components of follicular fluid in infertile individuals with diminished ovarian reserve

BackgroundDiminished ovarian reserve (DOR) refers to a decrease in the number and quality of oocytes in the ovary, which results in a lack of sex hormones and a decline of fertility in women. DOR can potentially progress to premature ovarian failure (POF), which has a negative impact on women's quality of life and is a major cause of female infertility. Oxidative stress is a major contributor to fertility decrease in DOR patients, affecting the follicular microenvironment, oocyte maturation, fertilization, and embryo development. Understanding intracellular signal transduction can be achieved by defining specific oxidized lipid components in follicular fluid (FF) of DOR infertile patients.MethodsThe oxylipins metabolic signatures in the FF of DOR patients and females with normal ovarian reserve (NOR) enrolled for the in vitro fertilization (IVF) cycle were analyzed using UHPLC-MS-MS technology. Principal component analysis (PCA) and orthogonal projections to latent structure discriminant analysis (OPLS-DA) were used to analyze the derived metabolomic profiles. Pathway enrichment analysis was carried out using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and MetaboAnalyst databases. Furthermore, the Spearman rank correlation coefficient was used to determine the correlation between age, FSH, AMH, AFC, oocytes retrieved, MII oocytes, fertilization, high-quality embryos, and the concentration of differential oxidized lipid metabolites in FF.ResultsFifteen oxylipins metabolites were found to be lower in the FF of DOR patients than those in the NOR group, including ±20-HDoHE, ±5-iso PGF2α-VI, 12S-HHTrE, 15-deoxy-Δ12,14-PGJ2, 1a,1b-dihomo PGE2, 1a,1b-dihomo PGF2α, 20-COOH-AA, 20-HETE, 8S,15S-DiHETE, PGA2, PGD2, PGE1, PGF1α, PGF2α, and PGJ2. The pathway enrichment analysis revealed that the 15 differentially oxidized lipid metabolites were closely related to the arachidonic acid metabolic pathway. Correlation analysis revealed that the concentration of 8 different oxidized lipid metabolites in FF was negatively correlated to FSH and positively correlated with AFC. AMH, the number of oocytes retrieved, MII oocytes and fertilization, were all positively correlated with 9 different oxidized lipid metabolites, but only one metabolite was positively correlated with the number of high-quality embryos.ConclusionsMetabolomic analysis of FF revealed that oxylipins metabolism disorders were closely related to ovarian reserve function. Among these oxylipins metabolites, arachidonic acid metabolism undergoes significant changes that may be related to oocyte development, resulting in decreased fertility in DOR patients.Trial registrationChiCTR, ChiCTR2000038182, Registered 12 September 2020-Retrospectively registered

Concentrations of homocysteine in follicular fluid and embryo quality and oocyte maturity in infertile women: a prospective cohort

Homocysteine is one of the components of follicular fluid (FF), so that any disruptions in its concentration may affect oocyte development. The aim of this study was to determine the relationship between FF homocysteine concentration and embryo quality, oocyte maturity, and pregnancy rate. Oocytes and embryos of 44 infertile women were categorised into different groups based on their maturity and quality, respectively. FF homocysteine levels, oocyte maturity, embryo quality, and pregnancy status were measured. A significant association was observed between the levels of FF homocysteine ​and oocyte maturation rate (p = .00). The concentration of FF homocysteine was higher than 9.8 µm/L in women with oocyte maturation < 80%. Most of the good quality embryos belonged to homocysteine levels < 9.8 µm/L. Decreased FF homocysteine concentrations can significantly improve the oocyte maturation rate and embryo quality. Aging may be an indirect factor contributing to decreased embryo quality and oocyte maturation through increasing FF homocysteine levels. IMPACT STATEMENT What is already known on this subject? It has been demonstrated that homocysteine is one of the components of follicular fluid (FF), but no information is available about the link between its concentration in FF and oocyte development. What do the results of this study add? The data indicated that decreased FF homocysteine concentrations at a younger age may remarkably improve the oocyte maturity and embryo quality of infertile patients undergoing assisted reproductive treatment (ART). What are the implications of these findings for clinical practice and/or further research? Based on the findings and considering the ease of measuring serum homocysteine and its direct correlation with FF homocysteine, homocysteine level measurement is recommended in patients who are candidates for infertility treatment in order to estimate oocyte maturation rate, embryo quality, and ART outcomes. Future studies are suggested to investigate patients with PCOS, endometriosis, and male factor infertility.

Advanced High-Coverage Targeted Metabolomics Method (SWATHtoMRM) for Exploring the Relationship of Follicular Fluid Components with Age

Background: The complexity of follicular fluid metabolome presents a huge challenge for qualitative and quantitative metabolite profiling and discovery of the comprehensive biomarkers. Objective: In order to address this challenge, novel SWATHtoMRM metabolomics method was used for providing broad coverage and excellent quantitative capability to discover the human follicular fluid metabolites related to age and evaluate their relationship with pregnancy outcome and oocyte senescence. Methods: The patients were divided into four groups according to age, including group A (28 cases, 21- 27 years old), group B (42 cases, 28-34 years old), group C (31 cases, 35-41 years old), and group D (24 cases, 42-48 years old). Follicular fluid samples from 125 IVF patients were analyzed. The differential ions among the four groups were identified by principal components analysis according to accurate mass, isotope ratio, and tandem mass spectroscopic spectra. Then, the differential metabolic pathways were further identified by a KEGG cluster analysis. Results: A total of 18 metabolites in the follicular fluid differed among the four groups, including amino acids, lipids, hormones, and vitamins. A total of 15 metabolites, including 6-oxohexanoate, phenylalanine, proline, hexadecanoic acid, linoleate, arachidonate, oleic acid, docosahexaenoic acid, LysoPC(16:1), LysoPC(20:5), LysoPC (20:3), 25-hydroxyvitamin D3, 5-dehydroepisterol, 27- hydroxycholesterol, and 5beta-cholestane-3alpha,7alpha,12alpha,23,25-pentol, were down-regulated with age and 3 metabolites, including LysoPC(18:3), LysoPC(18:1), and 13,14-dihydroretinol, were upregulated with age. Conclusion: Our study provides useful information for revealing the relationship between age and female reproductive capability.

Human and mouse activin genes: Divergent expression of activin A protein variants and identification of a novel heparan sulfate-binding domain in activin B

Activins are members of the transforming growth factor-β (TGF-β) superfamily of signaling proteins and were originally identified as components of follicular fluid. The proteins are now known to play critical roles in numerous normal and pathological processes and conditions, but less is clear about the relationships between their gene organization and protein variant expression and structure. The four human and mouse activin (Act) genes, termed INHβA, INHβB, INHβC and INHβE, differ in exon numbers. Human INHβA is the most complex with 7 exons and elicits production of three Act A variants (Act A X1, X2 and X3) differing in their pro-region, as we showed previously. Here we further analyzed the mouse INHβA gene and found that its 4 exons encode for a single open reading frame (mouse Act A), corresponding to the shortest human Act A X3 variant. Activins are synthesized and secreted as large complexes made of a long pro-region and a short mature C- terminal ligand and are known to interact with the heparan sulfate (HS) chains of cell surface and matrix proteoglycans. Human Act A X1 and X2 variants do have a HS-binding domain (HBD) with Cardin/Weintraub traits in their pro-region, while the X3 variant does not as shown previously. We found that the mouse Act A lacks a HBD as well. However, we identified a typical HBD in the pro-region of both mouse and human Act B, and synthetic peptides containing that domain interacted with immobilized HS and cell surface with nanomolar affinity. In sum, human and mouse Act A genes elicit expression of different variant sets, while there is concordance in Act B protein expression, reflecting possible evolutionary diversity in function of, and responses to, these signaling proteins in the two species.

Changes of follicular fluid composition during estrous cycle: The effects on oocyte maturation and embryo development in vitro

Oocytes are bathed in extracellular fluid of the antral follicles, which is termed follicular fluid (FF). Follicular fluid is synthesized from secretions of theca, granulosa, and cumulus cells and from a transudate of blood plasma. Oocytes persist in meiotic arrest in antral follicles until luteinizing hormone (LH) surge or removal the oocytes from the ovarian follicles. This suggests that FF before LH surge might contain meiosis inhibiting factor(s). The microvasculatory bed of the follicular wall and the composition of FF undergo changes during follicular growth and development, which is important for oocyte maturation and subsequent embryo development. Therefore, it is expected that FF composition and components might change according to timing of FF aspiration from follicles. Hence, negative or positive effects could be expected when FF supplemented during oocyte maturation in vitro. Nutrition effects on microvasculatory bed of follicles and their sizes. Thus, the nutritional status of animals is a factor affected on oocyte maturation and embryo development. The present article reviews and discusses these effects.

Effects of Follicular Fluid Components on Oocyte Maturation and Embryo Development In vivo and In vitro

Effects of Follicular Fluid Components on Oocyte Maturation and Embryo Development In vivo and In vitro

Periovulatory follicular fluid levels of estradiol trigger inflammatory and DNA damage responses in oviduct epithelial cells.

ObjectiveOvarian steroid hormones (mainly E2 and P4) regulate oviduct physiology. Serum-E2 acts on the oviduct epithelium from the basolateral cell compartment. Upon ovulation, the apical compartment of the oviduct epithelium is temporarily exposed to follicular fluid, which contains much higher levels of E2 than serum. The aim of this study was to evaluate the effects of human periovulatory follicular fluid levels of E2 on oviduct epithelial cells using two porcine in vitro models.MethodsA cell line derived from the porcine oviductal epithelium (CCLV-RIE270) was characterized (lineage markers, proliferation characteristics and transformation status). Primary porcine oviduct epithelial cells (POEC) were cultured in air-liquid interface and differentiation was assessed histologically. Both cultures were exposed to E2 (10 ng/ml and 200 ng/ml). Proliferation of CCLV-RIE270 and POEC was determined by real-time impedance monitoring and immunohistochemical detection of Ki67. Furthermore, marker gene expression for DNA damage response (DDR) and inflammation was quantified.ResultsCCLV-RIE270 was not transformed and exhibited properties of secretory oviduct epithelial cells. Periovulatory follicular fluid levels of E2 (200 ng/ml) upregulated the expression of inflammatory genes in CCLV-RIE270 but not in POEC (except for IL8). Expression of DDR genes was elevated in both models. A significant increase in cell proliferation could not be detected in response to E2.ConclusionsCCLV-RIE270 and POEC are complementary models to evaluate the consequences of oviduct exposure to follicular fluid components. Single administration of periovulatory follicular fluid E2 levels trigger inflammatory and DNA damage responses, but not proliferation in oviduct epithelial cells.

Current knowledge on the transport and fate of spermatozoa in the reproductive tract of the bitch.

Canine sperm transport, distribution, storage and detachment is a complex, dynamic and highly regulated process. Transport of sperm within the bitch's reproductive tract is rapid and is influenced by the method of semen deposition (natural mating or artificial insemination) and by the timing of breeding in relation to the day of ovulation. The fertile lifespan of spermatozoa in the reproductive tract of the bitch is considerably longer than in most other domestic species, and the main sperm reservoirs appear to be the uterine crypts and the distal part of the uterotubal junction, where spermatozoa attach by their heads to uterine epithelium. While several in vitro studies demonstrated prolonged motility and viability of canine spermatozoa after coincubation with uterine tube explants, spermatozoal storage has not been documented in the canine uterine tube isthmus or ampulla in vivo. Several factors, including exposure to progesterone, solubilized zona pellucida proteins and post-ovulation uterine tube fluid, appear to trigger membrane events resulting in capacitation-like changes with subsequent motility pattern changes (transitional and hyperactivated) that are associated with sperm detachment. After mating or insemination, a normal low-magnitude post-mating uterine inflammatory response occurs, evidenced by an influx of polymorphonuclear neutrophils (PMNs), increased uterine contractions and an increased uterine artery blood flow. Recently, it was also shown that normal dogs with cystic endometrial hyperplasia develop a more significant endometritis, show fewer mating-induced uterine contractions, a decreased ability of spermatozoa to bind to uterine explants in vitro and a slower uterine clearance after mating.

Personalized treatment of infertility – way to increase treatment efficiency

Objectives To overcome the still not sufficient implantation rate following in vitro fertilization (IVF) and intracytoplasmatic sperm injection (ICSI) in humans, more than two embryos are commonly replaced, potentially leading to high number of multiple pregnancies with associated significantly elevated risks like low birth weight. One of the solutions for avoiding multiple pregnancies could be optimalization of embryo selection and single embryo transfer [1]. The traditional systems for assessing gamete and embryo viability have limited ability to accurately select those with best developmental potential. Morphological evaluation remains the primary method of the oocyte and embryo assessment during IVF cycles, but its modest predictive power and inherent inter- and intra-observer variability limits its value. Prolonged embryo culture to blastocyst stage was introduced as a possible strategy for selecting the best embryos, though not even this method brought the solution. The extended period of in-vitro culture may have its side effects – e.g. predispose the embryo to phenomena such as ‘large offspring syndrome’, which is probably linked to altered gene expression, particularly of imprinted genes. The unexplained repeated implantation failure (RIF) is still another unsolved and one of the principal problems to affect the outcome of assisted reproductive technology (ART). The successful embryo implantation requires accurate temporal regulation of maternal immune function (to accommodate a semi-allogeneic embryo) and endocrine system [2]. The past few years have witnessed a virtual explosion in the identification of gene mutations or polymorphisms that cause or are linked to human infertility and that are inherited in a polygenic/ multifactorial fashion. Suggested technologic approaches The “best viable” embryo selection and also the optimal implantation regulation, both are going to be solved by new diagnostics and treatment methods based on personalized omics profiling [3]. Molecules of embryo-endometrial cross talk that are engaged in regulation of the embryo implantation and selected components of follicular fluid as well as embryo media will play a role on a developmental potential of the embryo and treatment efficiency. Expected impact if the project will succeed The goal of our effort is to point out new and potentially useful biomarkers that can be used for the improvement of pregnancy rates after the assisted reproduction techniques (ART) in near future and also to emphasize some of the novel molecular targets and biomarkers that have been reported recently and might contribute to new strategies of infertility management that will return a hope of natural conception for selected couples. Evaluation of such markers may help clinicians to predict pregnancy outcome and detect occult implantation deficiency. Moreover, these novel molecular targets are expected to apply to the clinical practice from bench to bedside and improve the implantation efficiency in ART.