Localisation and Function of the Endocannabinoid System in the Human Ovary

Although anandamide (AEA) had been measured in human follicular fluid and is suggested to play a role in ovarian follicle and oocyte maturity, its exact source and role in the human ovary remains unclear.Immunohistochemical examination of normal human ovaries indicated that the endocannabinoid system was present and widely expressed in the ovarian medulla and cortex with more intense cannabinoid receptor 2 (CB2) than CB1 immunoreactivity in the granulosa cells of primordial, primary, secondary, tertiary follicles, corpus luteum and corpus albicans. The enzymes, fatty acid amide hydrolase (FAAH) and N-acyclphosphatidylethanolamine-phospholipase D (NAPE-PLD), were only found in growing secondary and tertiary follicles and corpora lutea and albicantes. The follicular fluid (FF) AEA concentrations of 260 FF samples, taken from 37 infertile women undergoing controlled ovarian hyperstimulation for in vitro fertilisation and intracytoplasmic sperm injection with embryo transfer, were correlated with ovarian follicle size (P = 0.03). Significantly higher FF AEA concentrations were also observed in mature follicles (1.43+/-0.04 nM; mean+/-SEM) compared to immature follicles (1.26+/-0.06 nM), P = 0.0142 and from follicles containing morphologically assessed mature oocytes (1.56+/-0.11 nM) compared to that containing immature oocytes (0.99+/-0.09 nM), P = 0.0011. ROC analysis indicated that a FF AEA level of 1.09 nM could discriminate between mature and immature oocytes with 72.2% sensitivity and 77.14% specificity, whilst plasma AEA levels and FF AEA levels on oocyte retrieval day were not significantly different (P = 0.23).These data suggest that AEA is produced in the ovary, is under hormonal control and plays a role in folliculogenesis, preovulatory follicle maturation, oocyte maturity and ovulation

The role of anandamide in human folliculogenesis, implantation and early pregnacny

Plasma AEA levels were measured by ultra-performance liquid chromatography and mass-spectrometry throughout the menstrual cycle in healthy women both cross-sectionally and longitudinally. Plasma and follicular fluid (FF) AEA levels were also determined in women undergoing in-vitro fertilisation and embryo transfer (IVF-ET) and correlations between FF AEA and follicle size and oocyte maturity were undertaken. Plasma AEA levels were then measured in these women on the day of oocyte retrieval (OR), embryo transfer (ET) and at pregnancy test (PT) and comparisons made between pregnant and non-pregnant women and between those with viable pregnancies and those who had miscarried at 6 weeks. The relationship between plasma AEA and sex steroids in the stimulated cycles was also examined. AEA was found to be hormonally regulated, mainly by gonadotrophins and estradiol, and its highest levels were at ovulation and low at the implantation window in natural and stimulated cycles. The endocannabinoid system was localised in human ovary, FF AEA was associated with folliculogenesis, ovulation and oocyte maturity. ROC analysis indicated that a FF AEA concentration of 1.09 nM discriminated between mature and immature oocytes. In successful pregnancies a significant decline in plasma AEA levels from ovulation to the day of ET than a significant rise from the day of ET to the day of PT appeared to be important Plasma AEA levels were high at 4 and 5 weeks gestation and then significantly declined at 6 weeks gestation. These data may show the potential for using AEA as a biomarker test for oocyte assessment and to improve pregnancy outcomes in stimulated IVF/ICSI cycles

Spearman correlation between follicular fluid AEA concentration and follicle size.

<p>A positive correlation between the size of preovulatory follicle (in ml) and the concentrations of follicular fluids AEA from IVF/ICSI patients is shown. Follicular fluids from 193 follicles were analysed, R = 0.1304; P = 0.03.</p

Immunohistochemical staining for CB1, CB2, FAAH and NAPE-PLD on control tissues.

<p>Images on the left side of the panel represent the negative control (IgG/serum) and images on the right side of the panel are sections incubated with specific antibodies/antisera. The tissues used in a–f are human fetal membranes whereas g and h are human endometrium. CB1, CB2 and FAAH Immunoreactivity was observed in the amnion (Am), chorion (Ch) and decidua (Dec) of term fetal membranes, whilst NAPE-PLD immunoreactivity was observed mainly in the endometrial glands (Gl) with sparse staining in endometrial stroma (S). The images are representatives from at least six samples and were taken at 200× magnification. Bar = 50 µm.</p

Details of the ovulation stimulation regimen, number of oocytes collected per cycle and number of embryos on the day of embryo transfer.

<p>hMG = human menopausal gonadotrophins; rFSH = recombinant follicle stimulating hormone.</p

Immunohistochemical staining for FAAH.

<p>Images on the left side of the panel represent the negative control (non-immune rabbit serum and images on the right are for FAAH. Images a and b are primordial follicle; c and d are primary follicles; e and f are secondary follicles; g and h are low power images of tertiary follicles; i and j are high power images of tertiary follicles, k and l are corpus luteum, and m and n are images of the corpus albicans. Granulosa cells (G), theca cell layers (Th), the oocyte (O) and follicular fluid (ff) demonstrated FAAH immunoreactivity as did lutein-granulosa cells of the corpus luteum (CL) and corpus albicans (CL), and septa (S). There was a gradation of staining in the granulosa cells of the tertiary follicle with greatest intensity in the mural cells (panel j). The images are representatives from at least two structures and taken at 50×, 200× or 400× magnification. Bar = 50 µm.</p

Immunohistochemistry localisation of the various components of the endocannabinoid system in the human ovary.

<p>Scoring system (−) = staining absent; (+) = staining visible; (++) = strong staining.</p

Immunohistochemical staining for CB1 and CB2 receptors.

<p>Images on the left side of the panel represent the negative control (IgG) and images in the middle are for CB1 on the right are for CB2. Images a, b and c are primordial follicle; d, e and f are primary follicles; g, h and i are secondary follicles; j, k and l are low power images of tertiary follicles; m, n an o are high power images of tertiary follicles, p, q and r are corpus luteum, r, s and t are images of the corpus albicans. Granulosa cells (G), theca cell layers (Th), the oocyte (O) and follicular fluid (ff) demonstrated CB1 and CB2 immunoreactivity as did lutein-granulosa cells of the corpus luteum (CL) and corpus albicans (CL), and septa (S) between lobes of these postovulatory bodies. The images are representatives from at least two structures and taken at 50×, 200× or 400× magnification. Bar = 50 µm. Ovarian follicles were classified as primordial, primary, secondary, tertiary, corpus luteum, and corpus albicans <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004579#pone.0004579-Ross1" target="_blank">[43]</a>.</p

Prediction of oocyte maturity from follicular fluid AEA measurements.

<p>Panel a shows the follicular fluid AEA concentrations from follicles that gave rise to mature and immature oocytes during ICSI cycles. The long horizontal bar represents the mean and the shorter bars the sem. Panel b shows a receiver-operating characteristic curve (ROC) analysis for the prediction of the production of mature oocytes from follicular AEA concentration. The sensitivity and specificity relationship for measurements of anandamide in follicular fluid is plotted. The optimum cut off point for the identification of mature oocytes at 1.09 nM, provided a sensitivity of 72.2%, (95CI = 46.52% to 90.31%) and a specificity of 77.14% (95CI = 59.86 to 89.58%) and likelihood ratio of 3.16. The area under the curve was 0.768±0.067, (Mean±sem; 95%CI = 0.63–0.89, P = 0.001).</p

Immunohistochemical staining for NAPE-PLD.

<p>Images in the left side of the panel represent the negative control (rabbit IgG) and images on the right are for NAPE-PLD. Images a, and b are primordial follicles, c and d are primary follicles, e and f are secondary follicles; g and h are low power images of tertiary follicles, i and j are high power images of tertiary follicles, k and i are images of corpus luteum, and m and n are images of corpus albicans. Granulosa cells (G), theca cell layers (Th), the oocyte (O) and follicular fluid (ff) demonstrated NAPE-PLD immunoreactivity as did lutein-granulosa cells of the corpus luteum (CL) and corpus albicans (CL) and septa (S). The images are representative from at least two structures and taken at 50×, 200×, or 400× magnification. Bar = 50 µm.</p