Gene expression of the endocannabinoid system in endometrium through menstrual cycle.

Endocannabinoids mediate cellular functions and their activity is controlled by a complex system of enzymes, membrane receptors and transport molecules. Endocannabinoids are present in endometrium, a cyclical regenerative tissue requiring tightly regulated cellular mechanisms for maturation. The objective of this study was to investigate the gene expression of key elements involved in the endocannabinoid system across the menstrual cycle. RNA was isolated from endometrial tissue and genome-wide gene expression datasets were generated using RNA-sequencing. An a priori set of 70 genes associated with endocannabinoid system were selected from published literature. Gene expression across the menstrual cycle was analyzed using a moderated t test, corrected for multiple testing with Bonferroni's method. A total of 40 of the 70 genes were present in > 90% of the samples, and significant differential gene expression identified for 29 genes. We identified 4 distinct regulation patterns for synthesizing enzymes, as well as a distinct regulation pattern for degradations and transporting enzymes. This study charts the expression of endometrial endocannabinoid system genes across the menstrual cycle. Altered expression of genes that control endocannabinoid may allow fine control over endocannabinoid concentrations and their influence on cellular function, maturation and differentiation as the endometrium matures through the menstrual cycle.

The association between induction of labour in nulliparous women at term and subsequent spontaneous preterm birth: a retrospective cohort study.

OBJECTIVES: To evaluate the rate of subsequent spontaneous preterm birth in patients with previous induction of labour at term compared to women with previous spontaneous labour at term. METHODS: This was a retrospective cohort study of all women with consecutive births at the Royal Brisbane and Women's Hospital between 2014 and 2018. All nulliparous women with a singleton pregnancy and induction of labour at term or in spontaneous labour at term in the index pregnancy were included. Data was extracted from electronic medical records. The outcome of spontaneous preterm birth in the subsequent pregnancy was compared between patients with previous term induction of labour and in previous term spontaneous labour. RESULTS: A total of 907 patients with consecutive births met the inclusion criteria; of which 269 (29.7%) had a term induction of labour and 638 (70.3%) had a term spontaneous labour in the index pregnancy. The overall subsequent spontaneous preterm birth rate was 2.3%. Nulliparous women who underwent term induction of labour were less likely to have a subsequent preterm birth compared to nulliparous women in term spontaneous labour (0.74 vs. 2.98%; odds ratio [OR], 0.25; 95% confidence interval, 0.06-1.07; p=0.0496) in the index pregnancy. This however was not significant once adjusted for confounders (adjusted OR, 0.29; p=0.10). Spontaneous preterm birth was associated with a previous spontaneous labour compared to induction of labour between 37 to 37+6 and 38 to 38+6 weeks (adjusted OR 0.18 and 0.21; p=0.02 and 0.004 respectively). CONCLUSIONS: Term induction of labour does not increase the risk of subsequent spontaneous preterm birth compared to spontaneous labour at term in nulliparous women. Further research is needed to validate these findings in a larger cohort of women and to evaluate the effect of elective IOL among low-risk nulliparous women.

The risk of preterm delivery and pregnancy outcomes in women with asymptomatic short cervix: a retrospective cohort study.

OBJECTIVE: Routine cervical length measurement in asymptomatic pregnant women to prevent preterm birth has not been universally adopted due to poor predictive accuracy. The purpose of our study was to evaluate the risk of preterm delivery and pregnancy outcomes in women with asymptomatic short cervix and examine the implications of gestational age at presentation on these outcomes. STUDY DESIGN: This was a retrospective cohort study of women with singleton pregnancies who presented prior to or at 32 + 0 weeks with an asymptomatic short cervix (≤25 mm) between April 2014 to March 2018 at a single tertiary maternity center. Women with cervical length ≤25 mm were grouped into four cohorts according to gestational age at presentation: Obstetric outcomes were compared between the cohorts and the general cohort of women delivering during the same period. Outcomes were compared using Mann-Whitney U, chi-square tests, and logistic regression. Survival analysis was carried out to compare the probability of delivery for each subgroup. RESULTS: The rate of spontaneous preterm birth <37 weeks was highest in the cohort presenting at 25 + 0-27 + 6 weeks, and lowest in the first cohort presenting at <22 + 0 (60.0 versus 22.2%, p < .05). When compared with the general cohort, the rate of spontaneous preterm birth at <37-week gestation was significantly higher in the asymptomatic short cervix cohort (40.4 versus 8.7%, p < .001), with a 7.1-fold increase in the relative risk of spontaneous PTB. CONCLUSIONS: In asymptomatic women, cervical shortening showed significant increase in the risk of preterm birth. Our study findings suggest that routine cervical screening may be helpful in predicting risk of preterm birth even in women who are considered low-risk for preterm birth.

Does the length of second stage of labour or second stage caesarean section in nulliparous women increase the risk of preterm birth in subsequent pregnancies?

OBJECTIVES: This study aimed to investigate the role of prolonged second stage of labour and second stage caesarean section on the risk of spontaneous preterm birth (sPTB) in a subsequent pregnancy. METHODS: This was a retrospective cohort study of nulliparous women with two consecutive singleton deliveries between 2014 and 2017 at a tertiary centre. In the vaginal delivery cohort, subsequent pregnancy outcomes for women with a prolonged second stage (>2 h) were compared with those with a normal second stage (≤2 h). In the caesarean delivery cohort, women with a first stage or a second stage were compared with the vaginal delivery cohort. The primary outcome was subsequent sPTB. RESULTS: A total of 821 women met inclusion criteria, of which 74.8% (614/821) delivered vaginally and 25.2% (207/821) delivered by caesarean section. There was no association between a prolonged second stage in the index pregnancy and subsequent sPTB (aOR 0.70, 95% CI 0.13-3.83, p=0.7). The risk of subsequent sPTB was threefold for those with a second stage caesarean section; however this did not reach statistical significance. CONCLUSIONS: A prolonged second stage of labour in the index pregnancy is not associated with an increased risk of subsequent sPTB. A second stage caesarean section in the index pregnancy may be associated with an increased risk of subsequent sPTB, however there was no statistically significant difference. These findings are important for counseling and suggest that the effects of these factors are not clinically significant to justify additional interventions in the subsequent pregnancy.

The role of the endocannabinoid system in aetiopathogenesis of endometriosis: A potential therapeutic target.

Endometriosis affects a large proportion of women during their reproductive years and is associated with pain and infertility, also affecting psychological wellbeing and quality of life. The pathogenesis of the disease remains unclear, although it is believed to be multifactorial. The endocannabinoid system (ECS) consists of a number of ligands, receptors and enzymes, and has gained interests in endometriosis research. This review aims to summarise all available evidence reporting the roles of the ECS in endometriosis. A literature search of the PubMed, EMBASE, and Web of Science electronic medical databases was performed. Original and review articles published in peer-reviewed journals were included. No publication date or publication status restrictions were imposed. Significant differences in the concentrations and expressions of the components of the ECS were reported in the eutopic and ectopic endometrium, and the systemic circulation of women with endometriosis compared to controls. Endometriosis appears to be associated with downregulation of CB1 receptors and upregulation of TRPV1 receptors. The role of CB1 and progesterone in anti-inflammatory action and the role of TRPV1 in inflammation and pain are of particular interests. Furthermore, the ECS has been reported to be involved in processes relevant to endometriosis, including cell migration, cell proliferation, apoptosis, inflammation, and interacts with sex steroid hormones. The ECS may play a role in disease establishment, progression, and pain in endometriosis. However, reports are based on studies of limited size and there are inconsistencies among the definition of their control groups. There are also conflicting reports regarding precise involvement of the ECS in endometriosis. Future research with larger numbers, strict inclusion and exclusion criteria and detailed clinical information is imperative.

Unilateral Ureteric Endometriosis at the Pelvic Brim Resulting in Complete Loss of Renal Function.

Deep infiltrating endometriosis of the urinary tract is rare but can result in ureteric obstruction, hydroureteronephrosis and renal failure. Ureteric endometriosis usually affects the distal third of the left ureter among women of reproductive age. Greater awareness of ureteric endometriosis and a multidisciplinary approach in the management is essential to achieve optimal outcomes. We present an atypical case of right ureteric obstruction due to endometriosis at the pelvic brim resulting in complete loss of renal function and necessitating nephroureterectomy.

Epigenetic Influences During the Periconception Period and Assisted Reproduction.

The periconception period starts 6 months before conception and lasts until the tenth week of gestation. In this chapter, we will focus on epigenetic modifications to DNA and gene expression within this period and during assisted reproduction. There are two critical times during the periconception window when significant epigenetic 'reprogramming' occur: one during gametogenesis and another during the pre-implantation embryonic stage. Furthermore, assisted conception treatments, laboratory protocols and culture media can affect the embryo development and birth weights in laboratory animals. There is, however, an ongoing debate as to whether epigenetic changes in humans, causing embryo mal-development, placenta dysfunction and birth defects, result from assisted reproductive technologies or are consequences of pre-existing medical and/or genetic conditions in the parents. The periconception period starts from ovarian folliculogenesis, through resumption of oogenesis, fertilisation, peri-implantation embryo development, embryogenesis until the end of organogenesis. In men, it is the period from spermatogenesis to epididymal sperm storage and fertilisation. Gametes and developing embryos are sensitive to environmental factors during this period, and epigenetic modifications can occur in response to adverse lifestyles and environmental factors. We now know that lifestyle factors such as advanced parentage age, obesity or undernutrition, smoking, excessive alcohol and caffeine intake and recreational drugs used during gamete production and embryogenesis could induce epigenetic alterations, which could impact adversely on pregnancy outcomes and health of the offspring. Furthermore, these can also result in a permanent and irreversible effect in a dose-dependent manner, which can be passed on to the future generations.

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 (ß-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.

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, ß-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.

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.

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.

Endocannabinoids and pregnancy.

Acylethanolamides such as anandamide (AEA), and monoacylglycerols like 2-arachidonoylglycerol are endocannabinoids that bind to cannabinoid, vanilloid and peroxisome proliferator-activated receptors. These compounds, their various receptors, the purported membrane transporter(s), and related enzymes that synthesize and degrade them are collectively referred to as the "endocannabinoid system (ECS)". Poorly defined cellular and molecular mechanisms control the biological actions of the ECS. Over the last decade evidence has been emerging to suggest that the ECS plays a significant role in various aspects of human reproduction. In this review, we summarize our current understanding of this role especially the involvement of AEA and related ECS elements in regulating oogenesis, embryo oviductal transport, blastocyst implantation, placental development and pregnancy outcomes, and sperm survival, motility, capacitation and acrosome reaction. Additionally, the possibility that plasma and tissue AEA and other cannabinoids may represent reliable diagnostic markers of natural and assisted reproduction and pregnancy outcomes in women will be discussed.

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.