Proliferative potential and phenotypic analysis of long-term cultivated human granulosa cells initiated by addition of follicular fluid.

PURPOSE: The aim of this study was to develop and optimize a strategy for long-term cultivation of luteinizing human granulosa cells (GCs). METHODS: GCs were cultivated in DMEM/F12 medium supplemented with 2% fetal calf serum. In vitro proliferation of GCs was supported by follicular fluid as well as FSH and growth factors. RESULTS: The cultured GCs were maintained for 45 days with a doubling time of 159 ± 24 h. GCs initiated by the addition of follicular fluid and cultivated under low serum conditions reached 10 ± 0.7 population doublings. GCs maintain the typical phenotypic expression and the telomere length according to specific culture conditions. CONCLUSION: Our present study has demonstrated that GCs can be maintained in vitro for at least 45 days and this cell model can be beneficial when studying hormonal regulation associated with follicular maturation and preparation of oocytes for fertilization.

Development of ovarian hyperstimulation syndrome: interrogation of key proteins and biological processes in human follicular fluid of women undergoing in vitro fertilization.

Ovarian hyperstimulation syndrome (OHSS) is an iatrogenic complication and potentially life-threatening condition resulting from excessive ovarian stimulation during assisted reproductive technologies. Our aim was to identify candidate proteins in follicular fluid (FF) using various proteomic approaches which may help to identify patients at risk of OHSS. We analysed the proteome alterations in FF from patients suffering from severe forms of OHSS (OHSS+) compared with a control group of women without or with only mild signs of OHSS (OHSS-). The 12 abundant proteins of FF were removed using an immunoaffinity system. Pools of remaining depleted proteins were applied to the two-dimensional (2D) electrophoresis and 2D liquid chromatography and proteins in differentially expressed protein spots/fractions were identified by mass spectrometry. Among a total of 19 candidate proteins differentially expressed (P< 0.05) between OHSS+ and OHSS- FF samples, three proteins, namely ceruloplasmin, complement C3 and kininogen-1, were found using both 2D techniques. Computer modelling highlighted the important role of kininogen-1 as an anchor for mediated interactions with other identified proteins including ferritin light chain and ceruloplasmin, hepatocyte growth factor-like protein, as well as complement C3 and gelsolin, thus linking various biological processes including inflammation and angiogenesis, iron transport and storage, blood coagulation, innate immunity, cell adhesion and actin filament polymerization. The delineation of such processes may allow the development of informed corrective therapeutic intervention in patients at risk of OHSS and a set of key proteins of the FF may be helpful as potential biomarkers for monitoring IVF therapy.

Proteome mining of human follicular fluid reveals a crucial role of complement cascade and key biological pathways in women undergoing in vitro fertilization.

In vitro fertilization (IVF) is fraught with problems and currently proteomics approaches are being tried out to examine the microenvironment of the follicle in order to assess biological and immunological parameters that may affect its development. Additionally, better understanding of reproductive process may help increase IVF birth rate per embryo transfer and at the same time avoid spontaneous miscarriages or life threatening conditions such as ovarian hyperstimulation syndrome. The primary aim of this study was to search for specific differences in protein composition of human follicular fluid (HFF) and plasma in order to identify proteins that accumulate or are absent in HFF. Depletion of abundant proteins combined with multidimensional protein fractionation allowed the study of middle- and lower-abundance proteins. Paired comparison study examining HFF with plasma/serum from women undergoing successful IVF revealed important differences in the protein composition which may improve our knowledge of the follicular microenvironment and its biological role. This study showed involvement of innate immune function of complement cascade in HFF. Complement inhibition and the presence of C-terminal fragment of perlecan suggested possible links to angiogenesis which is a vital process in folliculogenesis and placental development. Differences in proteins associated with blood coagulation were also found in the follicular milieu. Several specific proteins were observed, many of which have not yet been associated with follicle/oocyte maturation. These proteins together with their regulatory pathways may play a vital role in the reproductive process.

Comparison of follicular fluid and serum levels of Inhibin A and Inhibin B with calculated indices used as predictive markers of Ovarian Hyperstimulation Syndrome in IVF patients.

BACKGROUND: Ovarian Hyperstimulation Syndrome (OHSS) is a severe health complication observed in some patients undergoing hormonal stimulation during IVF. Presence of OHSS is often associated with a high count of growing follicles responding to FSH hyperstimulation. However, the number of responding follicles may not be sufficient enough to predict the onset and severity of OHSS. The aim of this study was to find whether follicular fluid (FF) and serum concentrations of Inhibin A and Inhibin B in patients undergoing IVF treatment may serve as a predictor of OHSS status independent of the growing follicles count. METHODS: Serum and follicular fluid of fifty-three women undertaking the IVF program were separated into four groups according to their OHSS status and growing follicles count and analyzed for serum and FF concentrations of Inhibin A and Inhibin B. The resulting data were combined with clinical and demographic data to calculate indices independent of the growing follicles count. RESULTS: Serum Inhibin A and Inhibin B concentrations showed no significant difference between the severe OHSS group and the control group without OHSS. Moreover, the serum concentrations of Inhibin A and Inhibin B were strongly correlated with the growing follicles count. Their concentrations in the high responders group (>18 follicles) were significantly higher (p < 0.00001, p < 0.0001) when compared with normal and low responders (<18 follicles). To suppress the dependence on the growing follicle count, three indices were constructed and calculated. The best association with OHSS status and independence of the growing follicle count was achieved by using the Inhibin B TFF/SBM index calculated as follows: [concentration in FF] x [growing follicle count]/[concentration in serum] x [body mass]. The Inhibin B TFF/SBM index showed a clear difference (p = 0,00433) between the group with severe OHSS and the control group, while showing no apparent correlation with the growing follicle count. CONCLUSION: These observations demonstrated that while neither serum nor FF concentrations of Inhibin A nor Inhibin B can be used as an OHSS predictor independent of the growing follicle count, calculated indices may meet the criteria.

Follicular fluid and serum levels of inhibin A and pregnancy-associated plasma protein A in patients undergoing IVF.

OBJECTIVE: To elucidate transport of intrafollicular proteins Inhibin A and pregnancy-associated plasma protein A (PAPP-A) across the follicular fluid (FF)/blood barrier. DESIGN: A retrospective study. SETTING: IVF lab at a university hospital, academic, and industrial research labs. PATIENT(S): Fifty-five women undertook the IVF program. INTERVENTION(S): Follicular fluid aspirations and analysis, blood sample drawing, and serum analysis. MAIN OUTCOME MEASURE(S): Concentrations of Inhibin A, PAPP-A, and major serum proteins in FF and serum, total amount of PAPP-A, and Inhibin A in FF. RESULT(S): The FF/blood barrier permeability was calibrated using major serum proteins. The FF/serum ratio decreased with the molecular mass of proteins, and their FF and serum concentrations were well correlated. In contrast, concentrations of Inhibin A in paired serum and FF samples showed a weak correlation (r = 0.563), whereas serum and FF concentrations of PAPP-A were independent of each other. The total amount of Inhibin A in FF correlated well with concentrations of Inhibin A in paired serum samples (r = 0.858), whereas the correlation between the total amount of FF PAPP-A and PAPP-A serum concentrations remains poor (r = 0.215). CONCLUSION(S): These observations suggest that at the day of oocyte retrieval, FF is a major source of serum Inhibin A but not of serum PAPP-A.

The cultivation of human granulosa cells.

The major functions of granulosa cells (GCs) include the production of steroids, as well as a myriad of growth factors to interact with the oocyte during its development within the ovarian follicle. Also FSH stimulates GCs to convert androgens (coming from the thecal cells) to estradiol by aromatase. However, after ovulation the GCs produce progesterone that may maintain a potential pregnancy. Experiments with human GCs are mainly focused on the purification of GCs from ovarian follicular fluid followed by FACS analysis or short-term cultivation. The aim of our study was to cultivate GCs for a long period, to characterize their morphology and phenotype. Moreover, we have cultivated GCs under gonadotropin stimulation in order to simulate different pathological mechanisms during folliculogenesis (e.g. ovarian hyperstimulation syndrome). GCs were harvested from women undergoing in vitro fertilization. Complex oocyte-cumulus oophorus was dissociated by hyaluronidase. The best condition for transport of GCs was optimized as short transport in follicular fluid at 37 degrees C. GCs expansion medium consisted of DMEM/F12, 2% FCS, ascorbic acid, dexamethasone, L-glutamine, gentamycine, penicillin, streptomycin and growth factors (EGF, bFGF). GCs transported in follicular fluid and cultivated in 2% FCS containing DMEM/F12 medium supplemented with follicular fluid presented increased adhesion, proliferation, viability and decreased doubling time. Cell viability was 92% and mean cell doubling time was 52 hrs. We have optimized transport and cultivation protocols for long-term cultivation of GCs.