Akwasi A. Amoako

Smoking induces differential miRNA expression in human spermatozoa: A potential transgenerational epigenetic concern?

Recent work has suggested that environmental chemicals, including those contained in cigarette smoke, can have adverse effects on the exposed individuals as well as their future progeny. The mechanisms underlying transmission of environmentally induced phenotypes through the germ line are not well understood. However, a predominant process appears to be the establishment of permanent heritable epigenetic alterations, and a number of studies have implicated microRNAs in such processes. Here, we show that cigarette smoke induces specific differences in the spermatozoal microRNA content of human smokers compared with non-smokers, and that these altered microRNAs appear to predominantly mediate pathways vital for healthy sperm and normal embryo development, particularly cell death and apoptosis. microRNA-mediated perturbation of such pathways may explain how harmful phenotypes can be induced in the progeny of smokers.

Anandamide levels in human female reproductive tissues: solid-phase extraction and measurement by ultraperformance liquid chromatography tandem mass spectrometry.

Anandamide (N-arachidonoylethanolamide), a bioactive lipid, is reported to play a role in pregnancy maintenance and parturition. Our aims were to (1) evaluate AEA levels at the human maternal:fetal interface and (2) validate the use of solid-phase extraction of AEA from tissues. AEA was analyzed in cord and maternal blood, amniotic fluid, placenta, and fetal membranes collected during Caesarean section (n=14). Extraction efficiencies were 42 and 36% for the placenta and the fetal membranes, respectively. Tissue AEA was quantified using an isotope-dilution method and UPLC-ESI-MS/MS giving intra- and inter-day variability for tissues spiked with 0.2, 1, and 5pmol/g AEA of less than 12%. Accuracy for these spiked samples was between 95% and 103% for fetal membranes and between 99% and 114% for placenta. Mean AEA concentrations were 2.72 + or - 1.04 pmol/g for placenta and 1.19 + or - 0.68 pmol/g for fetal membranes, and 0.93 + or - 0.28, 0.88 + or - 0.33, 0.77 + or - 0.30, and 0.06 + or - 0.04nM for maternal, umbilical vein, and umbilical artery plasma and amniotic fluid. Higher AEA concentrations were found in placenta compared to fetal membranes (P<0.0001), in umbilical vein compared with umbilical artery (P=0.0015), and in plasma from maternal circulation compared with umbilical artery (P=0.0152). The relevance of these changes in AEA concentrations at the maternal:fetal interface requires further investigation.

Anandamide modulates human sperm motility: implications for men with asthenozoospermia and oligoasthenoteratozoospermia

STUDY QUESTION What are the levels of anandamide (N-arachidonoylethanolamide, AEA) in human seminal plasma and how are these related to abnormal spermatozoa? SUMMARY ANSWER Seminal plasma AEA levels were lower in men with asthenozoospermia and oligoasthenoteratozoospermia compared with normozoospermic men. WHAT IS KNOWN ALREADY AEA, a bioactive lipid, synthesized from membrane phospholipids may signal through cannabinoid receptors (CB1 and CB2) to regulate human sperm functions and male reproduction by modulating sperm motility, capacitation and the acrosome reaction in vitro. Local AEA levels are regulated by the synthetic and degradative enzymes, NAPE-PLD and FAAH, respectively. How the deregulation of this endogenous signalling pathway affects human sperm function(s) is not clear. STUDY DESIGN, SIZE AND DURATION This was a cross-sectional study of 86 men presenting at an infertility clinic for semen analysis over a period of 2 years. PARTICIPANTS/MATERIALS, SETTING, METHODS AEA was quantified, by ultra-high performance liquid chromatography-tandem mass spectrometry, in seminal plasma from 86 volunteers. Using qRT-PCR, CB1, CB2, NAPE-PLD and FAAH transcript levels were determined in spermatozoa from men with normozoospermia, asthenozoospermia, oligoasthenoteratozoospermia and teratozoospermia. Normal spermatozoa were exposed in vitro to methanadamide (meth-AEA) to determine its effect on sperm motility, viability and mitochondrial activity. MAIN RESULTS AND THE ROLE OF CHANCE Seminal plasma AEA levels (mean ± SEM) were significantly lower in men with asthenozoospermia (0.080 ± 0.01 nM; P < 0.05) or oligoasthenoteratozoospermia (0.083 ± 0.01 nM; P < 0.05) compared with normozoospermic men (0.198 ± 0.03 nM). In addition, the levels of spermatozoal CB1 mRNA were significantly decreased in men with asthenozoospermia (P < 0.001) or oligoasthenoteratozoospermia (P < 0.001) compared with normozoospermic controls. Supra-physiological levels of meth-AEA decreased sperm motility and viability, probably through CB1-mediated inhibition of mitochondrial activity. LIMITATIONS, REASONS FOR CAUTION The inhibitory effect of meth-AEA was only shown in vitro and may not reflect what happens in vivo. WIDER IMPLICATIONS OF THE FINDINGS As the regulation of the endocannabinoid system appears to be necessary for the preservation of normal sperm function and male fertility, there may be implications for the adverse reproductive consequences of marijuana use. Exocannabinoids, such as Δ(9)-THC, are likely to compete with endocannabinoids at the cannabinoid receptors, upsetting the finely balanced endocannabinoid signalling system. The importance of the endocannabinoid system makes it an attractive target for pharmacological interventions to control male fertility. STUDY FUNDING/COMPETING INTEREST(S) This work was funded in part by miscellaneous educational funds from the University Hospitals of Leicester National Health Services Trust to support the Endocannabinoid Research Laboratory of University of Leicester. The authors declare no competing interests.

Quantitative analysis of anandamide and related acylethanolamides in human seminal plasma by ultra performance liquid chromatography tandem mass spectrometry

The endocannabinoids anandamide, palmitoylethanolamide and oleoylethanolamide have been detected in human seminal plasma and are bioactive lipids implicated in regulation of sperm motility, capacitation and acrosome reaction. Several methods exist for endocannabinoid quantification but none have been validated for measurement in human seminal plasma. We describe sensitive, robust, reproducible solid phase and isotope-dilution UHPLC-ESI-MS/MS methods for the extraction and quantification of anandamide, palmitoylethanolamide and oleoylethanolamide in human seminal plasma. Precision and accuracy were evaluated using pooled seminal plasma over a 4 day period. For all analytes, the inter- and intraday precision (CV%) was between 6.6-17.7% and 6.3-12.5%, respectively. Analyses were linear over the range 0.237-19nM for anandamide and oleoylethanolamide and 0.9-76nM for PEA. Limits of detection (signal-to-noise >3) were 50, 100 and 100fmol/mL and limits of quantification (signal-to-noise >10) were 100, 200 and 200fmol/mL, respectively for anandamide, palmitoylethanolamide and oleoylethanolamide. Anandamide and oleoylethanolamide were stable at -80°C for up to 4 weeks, but palmitoylethanolamide declined significantly. We assessed seminal plasma from 40 human donors with normozoospermia and found mean (inter-quartile range) concentrations of 0.21nM (0.09-0.27), 1.785nM (0.48-2.32) and 15.54nM (7.05-16.31) for anandamide, oleoylethanolamide and palmitoylethanolamide, respectively. Consequently, this UHPLC-ESI-MS/MS method represents a rapid, reliable and reproducible technique for the analysis of these endocannabinoids in fresh seminal plasma.

Variation in Stability of Endogenous Reference Genes in Fallopian Tubes and Endometrium from Healthy and Ectopic Pregnant Women

RT-qPCR is commonly employed in gene expression studies in ectopic pregnancy. Most use RN18S1, β-actin or GAPDH as internal controls without validation of their suitability as reference genes. A systematic study of the suitability of endogenous reference genes for gene expression studies in ectopic pregnancy is lacking. The aims of this study were therefore to evaluate the stability of 12 reference genes and suggest those that are stable for use as internal control genes in fallopian tubes and endometrium from ectopic pregnancy and healthy non-pregnant controls. Analysis of the results showed that the genes consistently ranked in the top six by geNorm and NormFinder algorithms, were UBC, GAPDH, CYC1 and EIF4A2 (fallopian tubes) and UBC and ATP5B (endometrium). mRNA expression of NAPE-PLD as a test gene of interest varied between the groups depending on which of the 12 reference genes was used as internal controls. This study demonstrates that arbitrary selection of reference genes for normalisation in RT-qPCR studies in ectopic pregnancy without validation, risk producing inaccurate data and should therefore be discouraged.

Impact of reference gene selection for type 2 cannabinoid receptor gene expression studies in human spermatozoa.

Quantitative real‐time polymerase chain reaction (qRT‐PCR) has been employed to study the gene expression profiles in human spermatozoa, but accurate analysis is dependent upon normalisation of data against an endogenous control. β‐Actin (ACTB) and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) are the most commonly used reference genes for normalisation of gene expression in human spermatozoa, but the expression of these genes in many tissues has considerable variation under different physiological, pathological and experimental conditions which limits their effectiveness in normalisation. The expression stability of a panel of 12 reference genes was studied in normal and pathological human spermatozoa using geNorm and NormFinder software. Although there were some discrepancies in the ranking of reference gene stability, each software program ranked B2M, ACTB, CYC1 and 18S RNA within the top 5 and recommended the combined use of at least two reference genes. Normalisation of qRT‐PCR data for the cannabinoid receptor type 2 in spermatozoa using the different housekeeping genes demonstrated how, without validation, conflicting results are obtained. We recommend that the arbitrary use of reference genes should be avoided and the validation of reference gene stability should be undertaken prior to every study. For normalisation of CB2 expression, we would recommend using the geometric mean of B2M and ACTB.

Laparoscopic Surgery for Ovarian Pregnancy using Diathermy Hook with Conservation of Ovary: A Case Report and Literature Review

A 31-year-old woman presented with a 7-week history of irregular vaginal bleeding without abdominal pain. She had been using the intrauterine contraceptive device (IUD) for the last 3 years. A pregnancy test was positive and subsequent serum beta human chorionic gonadotropin (β-HCG) was 4992 mIU/mL. A transvaginal ultrasound scan demonstrated an empty uterus with an associated adnexal mass but no free fluid. A right primary ovarian ectopic pregnancy was diagnosed a laparoscopy. This was managed laparoscopically using monopolar diathermy hook with conservation of the ovary and minimal blood loss. Ovarian pregnancy is rare, especially in women without the classical risk factors for tubal pregnancy, and efforts should be made to exclude ectopic pregnancy in the absence of abdominal pain in a woman of reproductive age presenting with prolonged and irregular vaginal bleeding. Methods to conserve the ovary are also encouraged in cases of ovarian pregnancy.

Female Infertility: Diagnosis and Management

Infertility is defined as failure of a couple not using any form of contraception to conceive after 12 months of regular sexual intercourse. It constitutes a major social and psychological burden amongst couples, and the prevalence is increasing. Conditions in the female partner account for the majority of cases of infertility. This chapter summarises the different etiological factors in female infertility, baseline investigative evaluation and the management options for couples presenting with female factor infertility.

Fibroids and Infertility: Principles and Techniques of Management

Uterine fibroids occur commonly in women of reproductive age group and may play an etiological role in infertility and pregnancy wastage in a significant number of women. Several studies have suggested an association between uterine fibroids and adverse pregnancy outcomes such as reduced fertility and increased miscarriage. Management of uterine fibroids to improve fertility and reduce pregnancy wastage is currently surrounded by considerable uncertainty and remains controversial. In managing uterine fibroid-associated infertility, clinicians must balance the potential benefits in the context of fertility enhancement with complications of the procedure.

Differential Expression of the Orphan G-Protein Coupled Receptor Gpr55 in Human Spermatozoa

OBJECTIVE: This study assessed whether endocrine treatment increases the rate of obtaining sperm by ejaculation or surgical retrieval in patients with NOA. DESIGN: Prospective, non-randomized, controlled study. MATERIALS AND METHODS: 612 NOA patients were included. 116 patients chose immediate microsurgical sperm extraction (micro-TESE) and served as the control group. 496 remaining patients were administered clomiphene and were classified and treated based on their response to medication. Patients with an increase in follicle stimulating hormone (FSH) and testosterone continued clomiphene treatment and were the primary study group. For patients with increased FSH and diminishing or no increase in luteinizing hormone (LH) and testosterone, human chorionic gonadotropin (hCG) was added. For patients with no increase in testosterone, LH or FSH, or with decreasing testosterone, clomiphene was replaced by hCG and hMG. After 9 monthly semen analyses, micro-TESE was performed for those who remained azoospermic. Success rates were compared to the control group using the chi-squared test. RESULTS: With medical treatment, 10.9% of patients developed sperm in their semen. Treated patients, who remained azoospermic underwent micro-TESE with a 57% success rate. In total, sperm was made available for 61.7% of medically treated patients compared to 33.6% in the control group (P<0.001), and each treatment group showed a significantly increased rate of sperm retrieval compared to the control group (maximum P1⁄40.01). CONCLUSION: For NOA patients, a course of medical treatment may result in sperm in the ejaculate and increases the likelihood of successful micro-TESE for those remaining azoospermic.