Individual Preovulatory Follicles Research Articles

Ovarian adipocytokines are associated with early in vitro human embryo development independent of the action of ovarian insulin

We aimed to characterize the association between levels of serum and follicular fluid (FF) adipocytokines, reflected by the leptin to adiponectin ratio (L:A ratio), and oocyte quality and in vitro embryo development in women undergoing assisted reproduction. We also aimed to assess whether follicular hormonal pathways mediate this interaction. We prospectively collected FF from up to four individual preovulatory follicles (n = 76) and fasting sera from women (n = 31) without endocrinopathies undergoing in vitro fertilization (IVF) at a university-based center for assisted reproduction. Leptin, total adiponectin, insulin, insulin-like growth factor 1 (IGF-1), and ovarian steriods were measured using enzyme immunoassay. Oocyte maturity, fertilization, and embryo development were assessed. FF leptin was similar to serum levels while FF adiponectin was lower. FF leptin (27.10 ± 4.05ng/mL) and the L:A ratio (11.48E-3 ± 2.57E-3) were related to FF insulin (R (2) = 0.370 and 0.419, p < 0.001) but not to ovarian steroids or IGF-1, whereas FF adiponectin ( 4.22 ± 0.52ug/mL) correlated only with leptin (R (2) = -0.138, p = 0.001). Oocytes from a high FF L:A ratio environment were 81% (RR 1.81 [95%CI 0.97-3.37]) more likely to undergo successful cleavage and 117% (RR 2.17 [95% CI 1.06-4.44]) more likely to obtain viable cleavage morphology compared to a low FF L:A ratio environment, even when adjusted for FF insulin, an independent predictor of cleavage. Certain adipocytokines, particularly the L:A ratio in the FF of the preovulatory follicle, are related to successful in vitro embryo development. This action may be independent of FF insulin.

Specific genes are selectively expressed between cumulus and granulosa cells from individual human pre-ovulatory follicles

During folliculogenesis, the granulosa cells differentiate into two cell types: cumulus cells (CCs) and mural granulosa cells (MGCs). The objective of the study was to generate and compare the transcriptomes of MGCs and CCs from the pre-ovulatory follicle to characterize the detailed profile of the two cell populations shortly before ovulation. Twenty-one IVF/ICSI patients undergoing controlled ovarian stimulation (COS) donated CCs and MGCs from individual follicles containing metaphase II oocytes. Cells were prepared immediately after recovery and mRNA was isolated for whole-genome gene expression analysis and reverse transcriptase-polymerase chain reactions. Paired (within the individual follicle) comparisons between the CC and MGC expression profiles were performed and corrected for multiple comparisons. A total of 1562 genes were differentially expressed by >2-fold (P < 0.01) in the two cell types. Of these, 156 genes were >8-fold changed and represented specialized cellular functional categories such as inflammatory response, extracellular matrix and cell-cell communication, whereas the 1406 genes were 2-8-fold changed and represented functional categories such as proliferation and lipid metabolism. Transcripts not previously linked to the follicle were found to be differentially expressed between CCs and MGCs, suggesting specialized function in these compartments, e.g. pepsinogen A was selectively expressed in MGCs, whereas ryanodine receptor-2 (RYR2) was selectively expressed in CCs. Positive correlations were present between expression levels of RYR2 and the amphiregulin and gap-junction proteins. In conclusion, the transcriptomes of corresponding CCs and MGCs from individual pre-ovulatory follicles clearly revealed two distinct cell types. New as well as known genes representing specific cell functions close to ovulation were highlighted.

Impact of the Reproductive Cycle on the Timing of the Circadian System.

The circadian clock, an autoregulatory transcriptional oscillator, has been implicated in the timing of endocrine physiology and rhythms of clock gene expression have been described in tissues of the hypothalamo-pituitary-gonadal (HPG) axis. It is well known that reproductive physiology depends on the timing of hierarchical control by the central clock in the suprachiasmatic nucleus (SCN), though recent evidence suggests that timing in this system may also depend on peripheral clock function. Limited evidence reveals that gonadal steroid hormones may alter circadian rhythms of locomotor activity and clock gene expression in tissues of the female reproductive tract. It has been suggested that these effects are mediated by the direct action of steroid hormone receptors on clock- and clock-controlled gene expression in target tissues. However, a systematic analysis of the effects of the reproductive cycle on the coordinated timing of peripheral clocks has not yet been reported. We have examined circadian timing in peripheral clocks across the reproductive cycle. Cycling adult (>60 day old) rats expressing the Period1-luciferase (Per1-luc) clock gene reporter were sacrificed across the four-day cycle. The liver, kidney, lung, pituitary, cornea, oviduct, adipose tissue and individual pre-ovulatory follicles were cultured and Per1-luc expression was analyzed for at least 5 days in vitro. We examined phase synchrony among tissues and phase relationship within tissue across animals as a function of cycle day. Though we saw only modest, organ-specific effects in those tissues associated with metabolism (liver, lung, WAT), we did observe a significant effect of the cycle on synchrony and phase relationships in tissues of the HPG axis (follicles, pituitary) as a function of cycle day. These data suggest that fluctuations in ovarian steroid hormones across the cycle can alter the timing of the circadian system, presumably as a means to coordinate the timing of clock-dependent gene expression in target tissues. It remains to be seen if diseases that have substantial effects on steroid hormone secretion, such as polycystic ovarian syndrome, compound their effects by disrupting phase coordination between central and peripheral oscillators of the metabolic system and HPG axis.

Serum and follicular fluid ghrelin levels negatively reflect human oocyte quality and in vitro embryo development

To characterize serum and follicular fluid (FF) ghrelin in women undergoing assisted reproduction and to evaluate ghrelin's impact on ovarian steroidogenesis, oocyte quality, and embryo development. Prospective cohort study. University-based center for assisted reproduction. Normal weight women (n = 31) without endocrinopathies undergoing IVF. Fasting serum and FF from individual preovulatory follicles (n = 81) were collected. Ghrelin, insulin, ovarian steroids, and IVF outcomes including oocyte maturity and embryo development were assessed. The FF ghrelin correlated highly with serum levels and was 13% (3%-24%) lower. Levels were not associated with body mass or exogenous gonadotropins. Serum ghrelin correlated negatively with the cleavage rate and number of viable day 3 embryos. Embryos with successful cleavage and viable morphology came from follicles with lower ghrelin-odds ratios were 3.67 (1.02-13.14) and 3.46 (1.01-11.81), respectively. The FF ghrelin correlated negatively with FF insulin and progesterone, and positively with estradiol. The FF insulin was more than 50% higher (Δ = 1.88 [0.31-3.45] uIU/mL) in follicles with successfully cleaved embryos. The FF ghrelin does not reflect ovarian production and likely represents transudation. Ghrelin in FF correlates with local steroidogenesis and FF insulin expression. Serum and FF ghrelin are reflective of embryo development, possibly through interactions with FF insulin.

Perifollicular blood flow doppler indices, but not follicular pO 2, pCO 2, or pH, predict oocyte developmental competence in in vitro fertilization

Objective: To assess the relationships among perifollicular blood flow; follicular fluid pO 2, pCO 2, and pH; oocyte developmental capacity; preimplantation embryo quality. Design: Prospective study. Setting: Academic, tertiary care institution. Patient(s): Unselected, gonadotropin-stimulated IVF cycles. Intervention(s): Color, pulsed Doppler analysis of perifollicular blood flow, and follicular pO 2, pCO 2, and pH determinations of randomly designated, mapped ovarian follicles. Main Outcome Measure(s): Fertilization and day 3 embryo cleavage and morphology. Result(s): Perifollicular vascularity indices were significantly and negatively correlated with day 3 embryo cleavage. Pulsatility index and S-D ratio also were significantly and negatively correlated with follicular pO 2. The same correlation was found between resistance index and the fertilization rate of preovulatory oocytes. No relationship existed between follicular metabolic analysis and fertilization or embryo quality. The resistance index had a sensitivity of 0.57 and a specificity of 0.71 for the prediction of advanced embryo cleavage status. Conclusion(s): Results confirm and extend previous reports demonstrating that color, pulsed Doppler ultrasound analysis of individual preovulatory follicles during IVF therapy may provide an indirect index of the developmental competence of the corresponding oocyte. Although these methods may provide means to select embryos for transfer with the highest implantation potential, the moderate predictive power showed so far may limit their clinical applicability.

Application of image analysis cytometry in follicular fluid cells obtained from in-vitro fertilization cycles: relationships to patient's age, oocyte maturity, fertilizability and in-vitro fertilization outcome.

In an in-vitro fertilization (IVF)/embryo transfer programme granulosa cells obtained from 59 individual preovulatory follicles were analysed using multiparameter image analysis cytometry, in an attempt to determine whether their morphometric and DNA-cytometric parameters could prove useful in assessing follicle and oocyte maturity and in predicting fertilizability and outcome of these IVF cycles. Almost all morphometric and DNA-cytometric parameters were not correlated with either the patient's age or oocyte maturity, and did not predict oocyte fertilization or occurrence of a clinical pregnancy. The only possible relevant parameter which, despite its inverse correlation to total luteinizing hormone administration, also proved to be inversely correlated to pregnancy outcome (in the seven cases in which a pregnancy occurred), was the percentage of granulosa cell nuclei with increased DNA content (> 5c). Finally, if granulosa cells do not reveal euploid polyploidization in spontaneous or induced ovulatory cycles, the detected cells with increased DNA content should be interpreted as aneuploid, i.e. with chromosomal aberrations, and so their presence could also be discussed in connection with the hypothetical risk of prospective neoplastic transformation of the tissue.

ACTIVIN-A INHIBITS OXYTOCIN AND PROGESTERONE PRODUCTION BY PREOVULATORY BOVINE GRANULOSA CELLSIN VITRO

The aim was to examine the effect of activin on luteinization of preovulatory bovine granulosa cells in vitro. Bovine activin-A was found to inhibit the production of oxytocin (OT) and progesterone by bovine granulosa cells from individual preovulatory follicles cultured in serum-free medium. The minimal response on OT production (25% inhibition) occurred with 0.1-1 ng/ml activin-A, and the maximal inhibition (83%) occurred with 10 ng/ml activin-A after 2-3 days in culture. Progesterone showed a similar response (30% inhibition for 0.1-1 ng/ml and 74% for 10 ng/ml). Inhibin production was not consistently effected by activin-A. Inhibin (75 U/ml) had no detectable effect upon OT or progesterone production. When activin-A was withdrawn from the cell culture after 72 h and the incubation continued for a further 72 h, a recovery in OT was seen on day 4 and 5 after activin-A doses of 0.1-1 ng/ml, but not after higher doses (3 and 10 ng/ml). Progesterone did not show a recovery, but the levels remained constant for 3 days (0.1 and 0.3 ng/ml activin-A) or for 1 day (1-10 ng/ml activin-A) and then fell to control levels by day 6 of culture. We conclude that bovine activin-A has an autocrine action on bovine granulosa cells in vitro, to inhibit basal production of OT and progesterone, consistent with the role of activin-A in delaying the process of luteinization.

Oxytocin and progesterone secretion by bovine granulosa cells of individual preovulatory follicles cultured in serum-free medium

To examine the secretion of oxytocin (OT) and progesterone (P) from a homogeneous population of cells during luteinization, we developed a serum-free culture technique for granulosa cells, obtained from individual preovulatory bovine follicles. Granulosa cells from earlier stages of the follicle development did not have the capability to secrete OT under the in vitro conditions used. For optimal stimulation of the cells the medium (a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12) was supplemented with bovine serum albumin (BSA) and insulin. OT was detectable from day 1 of culture reaching a maximum level between days 2 and 4 and then declined towards day 5. In the absence of insulin OT declined from day 1 onwards and was undetectable from day 4. When cells were cocultured with theca tissue or theca-conditioned medium (TCM), there was an enhancement in OT secretion, but not in P secretion. Other tissues including liver, kidney, aorta, muscle and adrenal incubated with the cells induced a similar increase in OT production. In the presence of insulin progesterone secretion was increased and was correlated with OT production, but did not show a consistent pattern among follicles. We conclude that (a) culture of granulosa cells from an individual follicle in a serum-free medium can be used to study the secretion of OT and P from bovine granulosa cells, (b) insulin is essential for the optimal production of OT and P by these cells, and (c) the addition of theca or other tissues enhanced OT secretion by a mechanism not understood.

Effects of transforming growth factors and inhibin-related proteins on rat preovulatory graafian follicles in vitro.

In view of recent reports on ovarian production and action of transforming growth factors (TGFs) and inhibin-related proteins (inhibin, activin, and follistatin), we have examined the effects of these hormones on the function of preovulatory follicles in vitro. Individual preovulatory follicles were obtained from PMSG-treated rats and incubated with these hormones in the absence or presence of LH. Oocyte maturation and progesterone production were monitored. Treatment with TGF alpha alone, but not with TGF beta or inhibin-related proteins, mimicked the action of LH on oocyte maturation by inducing the resumption of meiosis in follicle-enclosed oocytes (56.6% and 80.6% oocytes resumed meiosis in the presence of 0.5 and 1.0 microgram/ml TGF alpha, respectively). In follicle cultures treated with LH to induce oocyte maturation, cotreatment with inhibin and TGF beta (30-50 ng/ml), but not other related hormones, partially inhibited LH-induced meiosis in follicle-enclosed oocytes (from 82% mature ova in the presence of LH to 51% and 55% mature ova with TGF beta and inhibin, respectively). In contrast to follicle cultures, none of the hormones tested significantly affected the spontaneous maturation of rat oocytes explanted from their follicles and cultured within their cumulus mass for 4 h. Treatment with TGF alpha, but not with TGF beta, inhibin, activin, or follistatin, stimulated progesterone production. The present study demonstrated that TGF alpha, like LH, induces oocyte maturation and progesterone production in preovulatory rat follicles. Furthermore, inhibin and TGF beta suppressed LH-induced resumption of meiosis in follicle-enclosed oocytes. Because these growth factors and inhibin-related proteins are synthesized by follicle cells, they may play important roles in regulating follicular development and activity.

Plasminogen Activator and Plasminogen Activator Inhibitor in Human Preovulatory Follicular Fluid*

To evaluate the roles of plasminogen activator (PA) and PA inhibitor (PAI) in human ovulation, we obtained follicular fluid and granulosa cells from individual preovulatory follicles of patients undergoing gamete intrafallopian tube transfer. The follicular fluid samples (n = 25) were analyzed for total tissue-type PA antigen, PA enzyme activity by fibrin autography, PAI activity, PAI type 1 (PAI-1) antigen, and PAI-1 mRNA. The follicular fluid of preovulatory follicles contained low levels of total tissue-type PA antigen (less than 1 ng/mL). Fibrin autography experiments indicated little or no detectable PA activity associated with free or unbound PA. The results of the PAI activity assay and PAI-1 antigen determination support the concept of a relative abundance of PAI compared with PA. Hybridization analysis was used to measure the relative amounts of granulosa cell PAI-1 mRNA. The levels of PAI-1 mRNA correlate with follicular fluid PAI concentrations in individual follicles. Taken together, these results support the idea that there is very little free, or active, PA in follicular fluid of human preovulatory follicles, but there is an abundance of PAI. Furthermore, PAI-1 produced by the granulosa cells may represent a major form of PAI in follicular fluid.

A preliminary assessment of the source of oestrogen within the ovary of the domestic fowl Gallus domesticus.

Oestradiol was measured by radioimmunoassay in blood draining individual preovulatory follicles and in different ovarian tissues of the hen. The concentration of oestradiol in blood from follicles 20 to 50 hr before ovulation ranged from 66 to 264 pg/ml and was less than that in peripheral blood collected concurrently, suggesting a net uptake rather than secretion of oestradiol by the follicle at these times. In one bird approximately 6 hr before ovulation, the highest content of oestradiol in tissue (35 ng) was in the small (less than 5 mm) follicles and ovarian stroma; this represented 87.5% of the total ovarian content. Only 0.86 to 1.02 ng oestradiol (2.0 to 2.5) was found in the large preovulatory follicles and 2.22 ng (5.5%) in the postovulatory follicles. High concentrations of oestradiol (2.4 ng/g) were also found in the liver. These results suggest that the small follicles and/or ovarian stroma are the main site of oestradiol production in the fowl; they do not exclude the possibility that oestradiol is secreted at a high rate by the mature follicle for a short period immediately before ovulation, thus influencing the release of LH.