Anthony H. Taylor

Oestrogen receptor beta is the predominant oestrogen receptor in human scalp skin.

Abstract: Oestrogens play a major role in non‐classic target tissues in both sexes, yet there have been few studies on estrogens and skin. Recently a second oestrogen receptor (ERβ) has been discovered. Therefore, we have compared the expression of oestrogen receptor alpha (ERα), beta (ERβ), the androgen receptor (AR) and a cell proliferation marker in male and female non‐balding scalp skin. ERβ was the major steroid receptor expressed in human skin. It was highly expressed in epidermis, blood vessels and dermal fibroblasts, in contrast to ERα and AR. In the hair follicle, ERβ expression was localized to nuclei of outer root sheath, epithelial matrix and dermal papilla cells, in contrast to ERα, and the AR, which was only expressed in dermal papilla cells. Serial sections also showed strong nuclear expression of ERβ in the cells of the bulge, while neither ERα nor AR was expressed. In the sebaceous gland, ERβ was expressed in both basal and partially differentiated sebocytes. ERα exhibited a similar pattern of expression, while the AR was expressed in the basal and very early differentiated sebocytes. There was no obvious difference in the expression of either oestrogen receptor in male or female skin. The wide distribution of ERβ in human skin suggests that oestrogens may play an important role in the maintenance of skin and in the regulation of the pilosebaceous unit, and provides further evidence for oestrogen action in non‐classic target tissues. The differential expression of ERα, ERβ and AR in human skin suggests that the mechanisms by which steroid hormones mediate their effects may be more complex than previously thought.

Localisation and function of the endocannabinoid system in the human ovary.

Background 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. Methods and Findings 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). Conclusions 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.

Ultra performance liquid chromatography tandem mass spectrometry method for the measurement of anandamide in human plasma.

Anandamide (N-arachidonoylethanolamine, AEA) is an endocannabinoid present in human plasma that is associated with several physiological functions and disease states. Significant variability in AEA plasma concentrations has been reported between studies, because quantification of AEA is fraught with methodological difficulties. A rapid, highly sensitive, robust, specific, and highly reproducible ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method is described here for the analysis of AEA in human plasma. This fully validated method using octa-deuterated AEA (AEA-d8) as an internal standard represents an improvement over previous analyses in terms of run time (4 min), limit of detection (0.055 fmol on column, 18.75 fmol/ml plasma), precision (relative standard deviations of 3.7, 3.9, and 4.8% for 1.66, 6.65, and 133 fmol on column), and accuracy (97.5-104.5%). AEA analysis was linear over the range 0.23 to 19 nM (1.66 to 133 fmol on column). To demonstrate the usefulness of this method for the measurement of AEA levels in clinical samples, plasma samples obtained from female volunteers at different stages of the menstrual cycle and pregnant women were assayed. Plasma AEA concentrations were significantly (P=0.0078) lower in the luteal phase of the menstrual cycle compared to the follicular phase. In pregnancy, the concentrations were lowest in the first and second trimesters with levels comparable to those observed in the luteal phase of the menstrual cycle and modestly increased in the third trimester. The highest plasma AEA levels were observed in women in active labour, and these were significantly (P=0.0147) higher than those observed in women at term but not in active labour. Postmenopausal women had AEA concentrations comparable to levels observed during the luteal phase of premenopausal women and were significantly (P=0.0389) lower than AEA plasma concentrations obtained during the follicular phase. The sensitivity and precision of the validated method described here suggests that this method is suitable for the analysis of AEA in clinical samples and may be utilised for the investigation of biomatrices containing limited amounts of AEA.

Plasma anandamide concentration and pregnancy outcome in women with threatened miscarriage.

motherapy for patients who have received treatment in the past. Our recommendation to treat patients with impending immunosuppression (from human immunodeficiency virus infection, induction for organ transplant, or other etiologies) who have not previously received antitrypanosomal therapy should be interpreted in the context of our overall BII recommendation for T cruzi–infected adults up to age 50 years without advanced heart disease. The major objective of etiologic treatment in this setting is to prevent development or progression of cardiomyopathy. Our intention in explicitly listing anticipated immunosuppressionasapatientcategoryfortreatmentwastolendadditional weight to the recommendation since we assume that antitrypanosomal therapywouldbebetter tolerated,andtheoretically more effective, before immunosuppression develops or is induced.Althoughdataarelackingonthispoint,asecondarytheoreticalbenefit is apossible reduction in the riskof reactivation. Advancedrenal,hepatic,orcardiacdysfunctionwouldcertainly complicate therapy and constitutes a contraindication. We agree with Altclas et al that posttransplantation monitoring is indicatedwhetherornotacourseofantitrypanosomal treatment was completed prior to transplant. As stated in the Clinical Review, we recommend that treatment decisions be individualized for all adult patients with Chagas disease, balancing the potential benefit vs the risk of drug toxicity.

Expression of the Endocannabinoid System in Human First Trimester Placenta and Its Role in Trophoblast Proliferation

The endocannabinoid, anandamide, which binds to two major receptor proteins, the cannabinoid receptors (CBs) 1 and 2 (CB1 and CB2), has been shown to play a role in first trimester miscarriage possibly through impairment of the developing trophoblast. Although the precise molecular mechanisms underlying this are unknown, plasma anandamide levels are known to be regulated by the progesterone-induced enzyme, fatty acid amide hydrolase (FAAH). Here, we tested the hypothesis that temporal-spatial expression of FAAH, CB1, and CB2 is regulated during early pregnancy and that anandamide detrimentally alters trophoblast proliferation. Transcripts for CB1, CB2, and FAAH were demonstrated in first trimester trophoblast extracts with only the CB1 transcript being significantly regulated. The significant 4.7-fold increase in expression at wk 10 gestation was reduced to 8.9% of the peak value by wk 12. Transcripts for CB2 showed a similar pattern of expression but were not significantly induced. By contrast, FAAH transcript levels appeared to increase toward the end of the first trimester, but again did not reach significance. These observations were supported by immunohistochemical studies that demonstrated a similar pattern of expression at the protein level, with cellular localization for all three proteins concentrated within the syncytiotrophoblast layer. Anandamide also prevented BeWo trophoblast cell proliferation in a dose-dependent manner, with a 50-60% significant inhibition of cell proliferation with concentrations in excess of 3 mum. This effect was mediated through CB2. Together, these data provide insights into how elevated plasma anandamide levels increase the risk of first trimester miscarriage.

Estrogen and progesterone receptor isoform distribution through the menstrual cycle in uteri with and without adenomyosis.

OBJECTIVE To test the hypothesis that the expression of the different isoforms of the estrogen receptor alpha (ER-α) and beta (ER-β) and the progesterone receptor A (PR-A) and B (PR-B) would be differentially modulated in uteri with adenomyosis compared with controls and that modulation would be related to the menstrual cycle. DESIGN Case control, blinded comparison. SETTING University department. PATIENT(S) 54 premenopausal women with and 35 without uterine adenomyosis as the sole pathology. INTERVENTION(S) Multiple samples studied using immunohistochemistry for estrogen and progesterone receptors. MAIN OUTCOME MEASURE(S) Histomorphometric analysis of receptor expression. RESULT(S) The ER-α expression in the adenomyotic endometrium was different from that of the normal endometrium and the foci in the midsecretory phase of the cycle, but expression of ER-α in the inner and outer myometrium was not statistically significantly different. The ER-β expression was statistically significantly elevated in the adenomyotic functionalis gland during the proliferative phase and throughout the myometrium across the entire menstrual cycle. Expression of PR-A was similar to that of PR-B, with reduced expression in the basalis stroma, and inner and outer myometrium in the adenomyotic samples. The pattern of ER-β, PR-A, and PR-B expression was similar in the endometrial basalis and adenomyotic foci. CONCLUSION(S) These data suggest ER-β expression and the lack of PR expression are related to the development and/or progression of adenomyosis and might explain the poor response of adenomyosis-associated menstrual symptoms to progestational agents.

The relationship between plasma levels of the endocannabinoid, anandamide, sex steroids, and gonadotrophins during the menstrual cycle

OBJECTIVE To further investigate the relationship between plasma anandamide (AEA), sex steroids, and gonadotrophins to improve our understanding of how AEA may be involved in human fertility. DESIGN Cross-sectional and longitudinal study. SETTING University Hospital of Leicester NHS Trust, Leicester Royal Infirmary. PATIENT(S) Healthy premenopausal and postmenopausal volunteers. INTERVENTION(S) UPLC-MS/MS-measured plasma AEA and ELISA-measured serum FSH, LH, estradiol, and progesterone levels at five different phases of the menstrual cycle and postmenopause. MAIN OUTCOME MEASURE(S) Plasma AEA, serum steroids and gonadotrophins. RESULT(S) Changes in AEA levels were similar in the two cohorts. The mean +/- SEM levels in the early follicular phase (0.89 +/- 0.06) for the cross-sectional cohort and the longitudinal cohort (0.73 +/- 0.03) were higher than those in the late follicular phase (0.77 +/- 0.09 cross-sectional; 0.63 +/- 0.08 longitudinal). The highest AEA levels were measured at ovulation (1.38 +/- 0.14 and 1.33 +/- 0.16) and the lowest level was measured in the late luteal phase (0.66 +/- 0.07 and 0.56 +/- 0.06). There was a statistically significant positive correlation between AEA, estradiol (P=0.0015), LH (P<0.0001) and FSH levels but not progesterone (P=0.022). CONCLUSION(S) Peak plasma AEA occurred at ovulation and positively correlated with estradiol and gonadotrophin levels suggesting that these may be involved in the regulation of AEA levels.

Fluctuation in anandamide levels from ovulation to early pregnancy in in-vitro fertilization-embryo transfer women, and its hormonal regulation

BACKGROUND Low levels of plasma arachidonoylethanolamide (anandamide) (AEA) (<2 nM) are associated with a successful early pregnancy in the mouse, and are thought to be regulated by sex steroid hormones. A similar association in the human may exist, although it has never been studied. The objective of this study was to investigate plasma AEA concentrations from the time of ovulation to implantation in pregnant and non-pregnant women, and whether AEA is hormonally regulated. METHODS Women who had undergone IVF/ICSI-embryo transfer were divided into pregnant (n = 12) and non-pregnant (n = 12) groups, based on serum beta-hCG >5 IU at 4 weeks and a viable intrauterine singleton pregnancy confirmed by ultrasound at 6 weeks gestation. Blood samples for plasma AEA and sex steroid hormonal measurements were taken at the time of oocyte collection, embryo transfer and pregnancy test, and an extra sample was also taken from the pregnant group at the viability ultrasound scan. RESULTS In pregnant women, there was a significant initial decrease in plasma AEA levels from the day of oocyte retrieval to that of embryo transfer. In addition, in the viable pregnancy group, plasma AEA was high at 4 and 5 weeks gestation, and a decline was observed at 6 weeks gestation (P = 0.003). No correlations were seen between plasma AEA and serum estradiol (E2), progesterone (P4) or beta-hCG in pregnant women; however, there was a significant correlation between plasma AEA and E2 (P = 0.022), but not between plasma AEA and serum P4, in non-pregnant women. CONCLUSION Our observations suggest that in successful pregnancy, a higher plasma AEA level at ovulation and a significantly lower level during implantation are required. The drop in AEA levels could be used as a biomarker for the appropriate timing of embryo transfer.

Histomorphometric evaluation of cannabinoid receptor and anandamide modulating enzyme expression in the human endometrium through the menstrual cycle.

Plasma anandamide (AEA) levels fluctuate throughout the menstrual cycle and in early pregnancy in a pattern suggesting its involvement in implantation and early pregnancy maintenance through mechanisms that might involve its binding to cannabinoid receptors CB1 and CB2. Plasma AEA levels are maintained by the actions of the enzymes fatty acid amide hydrolase (FAAH) and N-acylphosphatidylethanolamine-phospholipase D (NAPE-PLD). All of these component parts of the ‘endocannabinoid system’ have been demonstrated in rodent but not in human uteri. This study aimed to demonstrate the presence of the endocannabinoid system in the human uterus and catalogue its modulation. Immunohistochemical techniques were employed to localise and determine the distribution of immunoreactive CB1, CB2, FAAH, and NAPE-PLD in well-characterised menstrual cycle biopsy samples. Immunoreactive CB1 and CB2 were widely distributed throughout the uterine tissue. In the myometrium and endometrium, smooth muscle cells were immunoreactive, although the vascular smooth muscle cells in both tissues were more so. In the endometrium, CB1 and CB2 immunoreactivity was primarily restricted to the glandular epithelium and expression was unrelated to the phase of the cycle. FAAH immunoreactivity in the endometrium was highest in the mid-proliferative gland and mid-secretory stroma, whilst NAPE-PLD immunoreactivity was down-regulated in the secretory epithelial gland compared to the proliferative epithelial gland and unaffected in the stroma. These data indicate that elements of the ‘endocannabinoid system’ coexist in many cell types within the uterus and may provide insight into the sites of action of endogenous and exogenous cannabinoids during endometrial transformation.

A solid-phase method for the extraction and measurement of anandamide from multiple human biomatrices

N-Arachidonoylethanolamine (AEA, anandamide) was the first endocannabinoid to be identified and has since become associated with the mediation of several physiological functions and disease states. AEA has been isolated from numerous tissues and biofluids, in the low nanomolar range, using lipid extraction techniques with organic solvents. These techniques require the drying down of relatively large volumes of solvents, making them unsuitable for high-throughput analysis. Here we describe a solid-phase extraction (SPE) method for the investigation of AEA concentrations in human plasma, serum, milk, urine, amniotic fluid, peritoneal fluid, saliva, follicular fluid, and fluid from an ovarian cyst. AEA was detected in serum and plasma from blood isolated from 20 adult women (means+/-standard deviations: 0.68+/-0.29 and 0.64+/-0.28 nM, respectively), from pregnant women at term (1.37+/-0.42 nM), and from umbilical vein (1.26+/-0.33 nM) and umbilical artery (1.14+/-0.35nM), in milk (0.12+/-0.05 nM) and from amniotic (0.03+/-0.02 nM), peritoneal (0.93+/-0.27 nM), follicular (1.17+/-0.51 nM), and ovarian cyst (0.32+/-0.01 nM) fluids. AEA was undetectable in saliva and urine. The 60% AEA extraction efficiency achieved with SPE from plasma was superior to the 19% efficiency achieved using the existing organic solvent extraction method. Limits of quantification and detection for AEA were also improved dramatically using SPE (8 and 4 fmol/ml) compared with organic extraction (25 and 18.75 fmol/ml plasma). These improvements allow the use of smaller plasma samples with SPE. Intra- and interday variability were comparable, and the mean AEA concentration of pooled plasma samples (1.18 nM, n=15) was identical with the two techniques. Similarly, when 56 plasma samples from laboring and nonlaboring women were analyzed using both techniques, no extraction method-dependent differences were observed. Consequently, we provide evidence for a robust SPE technique for the extraction of AEA from biomatrices to replace the existing liquid extraction methods, with the SPE technique being superior in terms of speed, extraction efficiency, and sample size required.