Follicular Liquid Research Articles

Does Endometriosis Impact the Composition of Follicular Fluid in IL6 and AMH? A Case-Control Study

The aim of this study was to compare follicular liquid levels of IL6 and AMH in women with and without endometriosis and to evaluate their potential impact on ICSI outcomes. It is a prospective case-control study conducted on 25 women with proven endometriosis and 50 patients diagnosed with other causes of infertility. All these patients were candidates for ICSI cycles. Their follicular fluid was collected at the time of oocyte retrieval and used to evaluate IL-6 and AMH titers by electro-chemiluminescent immunoassay (Cobas e411-Roche). The IL-6 levels in follicular fluid were higher in the endometriosis group than in the control group (152.3 vs. 19.9 pg/mL; p = 0.02). The median level for AMH was 2.2 ± 1.88 ng/mL with no statistical difference between the two groups (2.2 vs. 2.7 ng/mL, p = 0.41). No significant correlation between the follicular IL6 and AMH levels was observed. The oocyte quality seems to be preserved in patients with endometriosis with the adequate response to ovarian stimulation. High levels of follicular IL6 are in accordance with the inflammatory phenomenon of the disease; however, this increase has no impact on ICSI outcomes.

Higher IL-1beta level in the follicular liquid of endometriosis compared with non-endometriosis patients

Objective: To ascertain the IL-1b level in the follicular liquid of endometriosis sufferers compared with that of non-endometriosis sufferers.Materials and Methods: The ELISA method, for detecting the IL-1b, is taken from follicular fluid of endometriosis as well as non-endometriosis patients that diagnosed according to the ASRM criteria.Results: The 54 subjects were dominantly endometriosis <34 year old (88.9%). The IL-1b levels of endometriosis patients and non-endometriosis patients were 82.86 ± 30.15 and 56.05 ± 23.27 respectively (p=0.01). The ROC value of IL-1b was found at a cut-off point of 26 (with the IL-1b cut-off point of 65.16). The discriminating value of AUC (area under curve) was 59.3%.Conclusion: It was concluded that the IL-1b level in endometriosis is higher than that in non- endometriosis.

Некоторые острофазовые белки и цитокины сыворотки крови и фолликулярной жидкости инфертильных пациенток с наружным генитальным эндометриозом 1–2 степени

Некоторые острофазовые белки и цитокины сыворотки крови и фолликулярной жидкости инфертильных пациенток с наружным генитальным эндометриозом 1–2 степени

Stem cells from follicular fluid present multilineage differentiation capacity and form oocyte-like structures under the influence of FSH hormone

Background & Aim Recent studies highlight the existence of putative stem cells derived from human ovarian follicular liquid collected after routine procedures for in vitrofertilization techniques (Riva F et al, 2009; Polimeni M et al, 2012).The aim of this study was to isolate, characterize and evaluate the pluripotency of this heterogeneous cell population of stem cells isolated from follicular fluid. For this purpose human ovarian follicular fluids were collected from women during the IVF procedure at the Embryoclinic Assisted Reproduction Unit (Thessaloniki, Greece). Methods, Results & Conclusion The collection was performed during the retrieval process of oocytes by transvaginal ultrasound-guided aspiration needle. After removal of the cumulus oophorus-oocyte complex the remained follicular fluid was centrifuged and the pellet was analyzed by flow cytometry for total immunophenotypic characterization. Cells were seeded on plastic surfaces in DMEM medium supplemented with fetal bovine serum, penicillin/streptomycin and gentamycin for incubation at 37οC/5% CO2. After 10-day cultivation the cells were analyzed for the expression of mesenchymal and pluripotent stem cell markers with flow cytometry. The induced differentiation capacity of isolated cells towards bone, adipose and endothelium in specific culture mediums was estimated via alizarin red, oil red staining and flow cytometry respectively. Cultures of follicular stem cells were checked for their ability to form oocyte like structures with the addition of FSH hormone in different concentrations. Real time PCR was performed for primordial germ stem cells markers Sox-2 Nanog and OCT4. A population of CD34-/CD45+/CD90+/CD105+/CD133-/SSEA4-/OCT4-/CD44+ stem cells were isolated. Osteogenic, adipogenic and endothelial multilineage differentiation capacity of cells derived from follicular liquid after culture was successfully confirmed. Oocyte like structure were observed in different FSH concentrations in the culture medium. After the presence of FSH hormone, cells were positive for the primordial stem cells markers Sox-2, Oct4 and Nanog while expression was gradually decreased in larger FSH concentrations. Conclusively, stem cells from follicular fluid show multilineage potential capacity and express primordial stem cell markers in specific conditions. Follicular fluid could be used as an alternative source of pluripotent stem cells and it might be a hopeful cure to women suffer from premature ovarian failure.

Complement C3 and anti-testosterone antibody in the 150 kDa Bali cattle (Bos sondaicus/javanicus) subordinate follicular fluid peptide observed using MALDI-TOF/TOF-MS

Objective : This study aimed to elucidate the proteome profile of the 150 kDa protein isolated from the subordinate follicle of Bali cattle ( Bos sondaicus / javanicus ). Some researchers have revealed several factors in the follicular subordinate with a 150 kDa protein substance, which play important roles in the bovine ovulation. Materials and methods : In the present study, subordinate follicles (~ 5 mm in diameter) were collected from 10 female Bali cattle from slaughterhouses in Taliwang, Sumbawa of West Nusa Tenggara Province, Indonesia. The follicular liquid was pooled; fractionated using SDSPAGE 10%, the 150 kDa band was sliced and then analyzed using MALDI-TOF/TOF-MS. Results : Mascot search results significantly revealed the presence of four species of proteins/peptides. Of the four peptides, two were predominant i.e. complement C3 and anti-testosterone antibody, which both were 100% identical to complement C3 and anti-testosterone antibody of Bos Taurus cattle. Conclusion : Complement C3 and anti-testosterone antibody are present in the follicle fluid of Bos sondaicus/javanicus cows. These findings are novel in Bali cattle follicles. http://doi.org/10.5455/javar.2018.e240

Natural infection in ovarian structures by bovine herpesvirus 1: molecular and serological detection

In this study, the polymerase chain reaction (PCR) was used to evaluate the presence of viral DNA in ovarian tissue, in the cumulus- oocyte complex (COC), follicular liquid, and blood of animals naturally infected with bovine herpesvirus-1 (BoHV-1). The serum profile of the sampled animals was also evaluated. Samples of serum, blood, ovarian tissue, follicular liquid, and COC were collected from 147 slaughterhouse animals that were not vaccinated against BoHV-1. Contaminated or insufficient samples were disregarded. Serological tests allowed the identification of serum-positive animals with neutralizing antibodies against BoHV-1. Analysis of samples by PCR revealed the presence of viral DNA in 0.9% (1/115) of the COC samples, in 4.3% (5/117) of the ovarian tissue samples, and in 2.8% (3/108) of the blood samples. Viral DNA was not detected in any of the follicular liquid samples. In serological samples, a positivity of 83.6% (117/140) was observed for BoHV-1. All PCR-positive animals, regardless of the samples analyzed, showed positivity in the serum neutralization test for the detection of BoHV-1-specific antibodies. According to these results, a high prevalence of antibodies against BoHV-1 was detected in naturally infected animals from different herds, and the molecular tests revealed the presence of viral DNA in bovine ovarian tissue, providing evidence that this might be a site of BoHV-1 infection in naturally infected animals.

3D culture of multipotent cells derived from waste human ovarian follicular liquid and seeded onto gelatin cryogel

Current tissue engineering uses 3D biomaterials in combination with stem cells, since mature cells are often not available in sufficient amounts or quality. Biomaterial scaffolds have been widely used in reconstructive bone surgery not only as cell carriers providing mechanical support, but also as promoters of cell attachment and proliferation (1). In particular, gelatine cryogel scaffolds are promising new biomaterials owing to their biocompatibility and to substain the differentiation of mesenchymal stromal stem cells (MSCs) (2). Human MSC proliferate onto the surfaces with fibroblastic morphology and can differentiate into osteoblasts, chondrocytes and adipocytes (3). These cells can be isolated from several sources, including bone marrow and adipose tissue (4). Our previously studies showed the possibility to obtain MSCs also from the human ovarian follicular liquid (FL) that is usually wasted during in vitro fertilization (5). In this study, we tested the ability of these FL cells to grow and differentiate on gelatine cryogel in comparison with MSCs derived from human bone marrow. Samples and controls were analyzed with confocal and scanning electron microscopes. Results demonstrated that FL cells could grow on the biomaterial not only on the top but also in the layers below till 60mm of deepness. Data suggested that the observed cells are mesenchymal since positive for vimentin and CD44 (a typical MSC marker). Preliminary results showed also the capability of induced FL cells to osteogenic differentiation to produce bone extracellular matrix, expressing some specific proteins (i.e.osteopontin). In conclusion, MSCs derived from waste human ovarian follicular liquid showed promising affinity with 3D gelatine cryogel, opening new potential developments in biotech and medical applications.

Heterogeneous cell population derived from human ovarian follicular liquid: morphological studies and molecular screening

The origin of oocytes and primary follicles in ovaries of adult mammalian females is still a matter of dispute [1]. The components of new primary follicles, primitive granulosa and germ cells, differentiate sequentially and de novo from mesenchymal progenitor cells residing in the ovarian tunica albuginea (TA). It appears that mesenchymal progenitor cells contribute to the generation of epithelial cells similar to granulosa cells (GCs). The multipotency of a subset of granulosa cells was also established by in vitro differentiation into other cell types [2]. Up to now, luteinizing GCs were considered to be terminally differentiated, unavoidably becoming apoptotic a few days after ovulation. Previously, we have provided evidence for the existence of putative stem cells derived from human ovarian follicular liquid collected after routine procedures for in vitro fertilization techniques [3]. These cells grow in minimal medium condition, without any growth factor (i.e. LIF), that is considered essential according to other procedures [4]. Using immunocytochemistry and flow cytometry we showed that these cells are positive for several mesenchymal stemness markers, including CD90, CD73, CD44, CD105. However, morphological analysis revealed a heterogeneous cell population, with cells displaying a fibroblast-like, epithelial- like and neural-like shapes. These observations are also supported by the identification of cells expressing specific neural markers, such as neurofilaments and PGP9.5, in addition to vimentin and cytocheratin positive cells. All these data are suggestive of the presence of different cell populations in follicular fluids. To verify this hypothesis we select a panel of markers specific for the different cell populations previously identified and we plan a molecular screening to follow their expression in the follicular fluid derived cells at different times of minimal culture conditions in vitro. Bone marrow derived MSCs were used as a control. For each sample we performed semiquantitative RT-PCR experiments normalizing the cDNAs used as templates on the basis of the number of pseudo-mesenchymal cells morphologically identified in the sample. For this purpose OCT-4 was selected as a stem marker to follow the mesenchymal stem cell population, while FSH-R was used to identify granulosa derived cells; CNTF and beta-3-tubuline were used to discriminate between neural and neuronal cells populations; epithelial and hematopoietic cells were followed using cytokeratin (CK8 and CK10) and CD45 markers, respectively. GAPDH and β-actin specific primers were used on all samples for normalization. Here we compare the results of this molecular screening with the previously obtained immunocytochemical and morphological data to confirm the presence of these different cytotypes in the samples purified from the follicular liquid and their persistence, loss or amplification at different times of in vitro minimal culture conditions.

Fatty Acid Composition in Blood Plasma and Follicular Liquid in Cows Supplemented with Linseed or Canola Grains

This study was carried out to evaluate the fatty acid composition in Nellore cows supplemented with either linseed (n- 3) or canola grains (n-6 and n-9). Fifteen Nellore cows, aged five years and bodyweight 550 kg±48 kg, were randomly distributed to the following treatments: CON (control), LIN (linseed) and CAN (canola grains). The cows were fed for 80 days. The concentrations of C18:0, C18:2 n-6 and C20:3 n-6 fatty acid were higher (p 0.10) among the treatments. C14:0, C16:1 n-7, C18:2 n-6 and n-6 contents were higher (p<0.10) for CON and CAN than LIN. C17:1 n-7, C20:4 n-6 and C 22:0 contents were higher (p<0.10) for CAN than CON and LIN. C18:1 n-9, C18:3 n-3, MUFA and n-3 contents were higher (p<0.10) for LIN and CAN than CON. C20:3 n-6 content and n-6:n-3 ratio were higher (p<0.10) for CON than LIN and CAN. C22:5 n-3 content were higher (p<0.10) for CON and LIN than CAN. The concentrations of fatty acids in blood plasma and follicular liquid were not correlated for any fatty acid, independent of the treatment studied. Canola grain added to the diet of Nellore cows resulted in increased concentrations of fatty

Changes in the glucose-6-phosphate dehydrogenase activity in granulosa cells during follicular atresia in ewes

Apoptosis of granulosa cells during follicular atresia is preceded by oxidative stress, partly due to a drop in the antioxidant glutathione (GSH). Under oxidative stress, GSH regeneration is dependent on the adequate supply of NADPH by glucose-6-phosphate dehydrogenase (G6PD). In this study, we analyzed the changes of G6PD, GSH, and oxidative stress of granulosa cells and follicular liquid and its association with apoptosis during atresia of small (4-6 mm) and large (>6 mm) sheep antral follicles. G6PD activity was found to be higher in granulosa cells of healthy small rather than large follicles, with similar GSH concentration in both cases. During atresia, increased apoptosis and protein oxidation, as well as a drop in GSH levels, were observed in follicles of both sizes. Furthermore, the activity of G6PD decreased in atretic small follicles, but not in large ones. GSH decreased and protein oxidation increased in follicular fluid. This was dependent on the degree of atresia, whereas the changes in G6PD activity were based on the type of follicle. The higher G6PD activity in the small follicles could be related to granulosa cell proliferation, follicular growth, and a lower sensitivity to oxidative stress when compared with large follicles. The results also indicate that GSH concentration in atretic follicles depends on other factors in addition to G6PD, such as de novo synthesis or activity of other NADPH-producing enzymes. Finally, lower G6PD activity in large follicles indicating a higher susceptibility to oxidative stress associated to apoptosis progression in follicle atresia.

Effect of the dry matter intake level on the sanguine profile of glucose, insulin, urea, estrogen and progesterone and concentration of IGF-I in the follicular liquid of crossbred heifers

The aim of this work was to study effect of the dry matter intake level on the concentrations of glucose, insulin, urea, estrogen and progesterone in the blood and the level of IGF-I in the follicular liquid of heifers. Twenty seven crossbred heifers were used, in three treatments: 1.2, 1.6 and 2.6% of the live weight of dry matter intake (DMI) daily. The sanguine concentrations of glucose and estrogen were higher in the animals with 1.6% of DMI and the insulin levels were higher in the animals with 2.6% of DMI. There was an increase in the sanguine levels of urea with the increase of the DMI. There was no effect of the DMI on the progesterone levels in the sanguine plasma collected from the jugular. The progesterone levels in caudal vena were higher in the animals with 2.6% of DMI. There was no effect of the DMI on the follicular levels of IGF-I.

The role of pH values in porcine reproductive tracts of male and female individuals

For a long time, in practice, and science, has been known that pH values of sperm and vagina are important for successful fertilization. In this investigation this fact was confirmed, and the goal was to investigate the role of pH values through whole reproductive tract of male and female individuals: testis, rete testis, epididymis, ductus deferens, Cowper's gland, vesicula seminalis, prostata, corpus cavernosus, corpus spongiosus, epitel tissue of penis tube, sperm, vagina, uterus, horn of uterus, oviduct, fimbrie ovarica, ovarium, follicular fluid. Measurement was performed in reproductive active males as well as before and after ovulation in females. Porcine reproductive tracts (per 15 female and male individuals) were collected from institute's slaughterhouse, immediately post mortem dissected, homogenised and pH values were measured (according to method Rede&amp;Rahelic (1969)). Ovarium and follicular liquid have the highest pH values (7,4) in females, but a small peak in preovulatory oviduct is also present and corresponded to pH of sperm of reproductive fully active male individuals (pH=app.7,2). After fertilization pH in surrounding of zygot (through depolarisation of its membrane) in oviduct, and zygot which then has external decreased pH value moves to less pH values regions by the same principle, that means to uterus, (pH between 7,2 (horn) and 7,07(cervix)) in postovulatory female reproductive tract, where nidation of blastocyst occurs. This investigation could help to elucidate knowledge about reproductive physiology in vivo, giving importance to role of pH values along reproductive tract of male and female individuals.

Fine structure of micropylar cell in the ovarian follicle of the teleost, Plecoglossus altivelis.

The micropylar cell in the ovarian follicle of the teleost, Plecoglossus altivelis has been studied with light and electron microscopy in order to clarify the mechanism of maintenance of its highly modified structure, and to determine the role of the cell during egg maturation. As oogenesis proceeds a monolayers of squamous follicle cells at the animal pole transform into stratified cuboidal cells in a depression in the attaching layer of the chorion. The micropylar cell assumes the contours of a mushroom and differentiates to form the cell body and the micropylar process. In the cytoplasm of cell body 10 nm filament form bundles about 1μm in diameter which encircle the upper part of nucleus. Branched bundles extend to the periphery and into the micropylar process. Well developed endoplasmic reticulum with cisternae which seem to be empty make the cell appear like a sponge. The micropylar process, which inserts into the cortex of oocyte to a depth of 1μm, contains a diplosome oriented nearly longitudinally with respect to the axis of the process. Many amorphous dotted satellites are associated with the diplosome. Microtubules extended to the periphery of the process and to the cell body. Functionally, the micropylar cell has three kinds of secretory systems. Two of them, which appear also in the follicular epithelium, are forthe production of the follicular liquid and the attaching layer. The third kind is particular to the micropylar cell and involves secretion from the tip of the micropylar process directly into the vitellus.