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BACKGROUND: Tubal ectopic pregnancies can cause substantial morbidity or even death. Current treatment is with methotrexate or surgery. Methotrexate treatment fails in approximately 30% of women who subsequently require rescue surgery. Gefitinib, an epidermal growth factor receptor inhibitor, might improve the effects of methotrexate. We assessed the efficacy of oral gefitinib with methotrexate, versus methotrexate alone, to treat tubal ectopic pregnancy. METHODS: We performed a multicentre, randomised, double-blind, placebo-controlled trial across 50 UK hospitals. Participants diagnosed with tubal ectopic pregnancy were administered a single dose of intramuscular methotrexate (50 mg/m2) and randomised (1:1 ratio) to 7 days of additional oral gefitinib (250 mg daily) or placebo. The primary outcome, analysed by intention to treat, was surgical intervention to resolve the ectopic pregnancy. Secondary outcomes included time to resolution of ectopic pregnancy and serious adverse events. This trial is registered at the ISRCTN registry, ISCRTN 67795930. FINDINGS: Between Nov 2, 2016, and Oct 6, 2021, 328 participants were allocated to methotrexate and gefitinib (n=165) or methotrexate and placebo (n=163). Three participants in the placebo group withdrew. Surgical intervention occurred in 50 (30%) of 165 participants in the gefitinib group and in 47 (29%) of 160 participants in the placebo group (adjusted risk ratio 1·15, 95% CI 0·85 to 1·58; adjusted risk difference -0·01, 95% CI -0·10 to 0·09; p=0·37). Without surgical intervention, median time to resolution was 28·0 days in the gefitinib group and 28·0 days in the placebo group (subdistribution hazard ratio 1·03, 95% CI 0·75 to 1·40). Serious adverse events occurred in five (3%) of 165 participants in the gefitinib group and in six (4%) of 162 participants in the placebo group. Diarrhoea and rash were more common in the gefitinib group. INTERPRETATION: In women with a tubal ectopic pregnancy, adding oral gefitinib to parenteral methotrexate does not offer clinical benefit over methotrexate and increases minor adverse reactions. FUNDING: National Institute of Health Research.

STUDY QUESTION What is the capacity of the change between Day 1 and Day 4 post-treatment serum human chorionic gonadotropin (hCG) levels for predicting single-dose methotrexate treatment success in tubal ectopic pregnancy? SUMMARY ANSWER Any fall in Days 1–4 serum hCG signified an 85% (95% CI 76.8–90.6) likelihood of treatment success for women with tubal ectopic pregnancy (initial hCG of ≥1000 and ≤5000 IU/l) managed with single-dose methotrexate. WHAT IS KNOWN ALREADY For those with tubal ectopic pregnancy managed by single-dose methotrexate, current guidelines advocate intervention if Days 4–7 hCG fails to fall by >15%. The trajectory of hCG over Days 1–4 has been proposed as an early indicator that predicts treatment success, allowing early reassurance for women. However, almost all prior studies of Days 1–4 hCG changes have been retrospective. STUDY DESIGN, SIZE, DURATION This was a prospective cohort study of women with tubal ectopic pregnancy (pre-treatment hCG of ≥1000 and ≤5000 IU/l) managed with single-dose methotrexate. The data were derived from a UK multicentre randomized controlled trial of methotrexate and gefitinib versus methotrexate and placebo for treatment of tubal ectopic pregnancy (GEM3). For this analysis, we include data from both treatment arms. PARTICIPANTS/MATERIALS, SETTING, METHODS Participants were categorized according to single-dose methotrexate treatment success or failure. Treatment success for this analysis was defined as complete and uneventful resolution of tubal ectopic pregnancy to serum hCG MAIN RESULTS AND THE ROLE OF CHANCE A total of 322 women with tubal ectopic pregnancy were treated with single-dose methotrexate. The overall single-dose methotrexate treatment success rate was 59% (n = 189/322). For any fall in serum hCG on Days 1–4, likelihood ratios were >3, while for any fall of serum hCG >20% on Days 1–7, likelihood ratios reached 5. Any rise of serum hCG on Days 1–7 and 4–7 strongly reduced the chance of success. Any fall in Days 1–4 hCG predicted single-dose methotrexate treatment success with a sensitivity of 58% and specificity 84%, resulting in positive and negative predictive values of 85% and 57%, respectively. Any rise in Days 1–4 serum hCG LIMITATIONS, REASONS FOR CAUTION Our findings may be limited by intervention bias resulting from existing guidelines which influences evaluation of hCG changes reliant on Day 7 serum hCG levels. WIDER IMPLICATIONS OF THE FINDINGS Examining a large prospective cohort, we show the value of Days 1–4 serum hCG changes in predicting single-dose methotrexate treatment success in tubal ectopic pregnancy. We recommend that clinicians provide early reassurance to women who have a fall or only a modest ( STUDY FUNDING/COMPETING INTEREST(S) This project was supported by funding from the Efficacy and Mechanism Evaluation programme, a Medical Research Council and National Institute for Health Research partnership (grant reference number 14/150/03). A.W.H. has received honoraria for consultancy for Ferring, Roche, Nordic Pharma and AbbVie. W.C.D. has received honoraria from Merck and Guerbet and research funding from Galvani Biosciences. L.H.R.W. has received research funding from Roche Diagnostics. B.W.M. is supported by a NHMRC Investigator grant (GNT1176437). B.W.M. also reports consultancy for ObsEva and Merck and travel support from Merck. The other authors declare no competing interests. TRIAL REGISTRATION NUMBER This study is a secondary analysis of the GEM3 trial (ISRCTN Registry ISRCTN67795930).

Lay summary Q fever is a bacterial disease that passes between animals and humans and causes disease in both. The disease has been associated with pregnancy complications including miscarriage. This study was undertaken to identify if Q fever exposure was correlated with miscarriage in 369 women attending a pregnancy support unit in Edinburgh. The women in the study were in two groups, the miscarriage group with 251 women who had experienced a miscarriage and a control group of 118 women who had not experienced miscarriage. Three women were found to be positive for Q fever antibodies, suggesting that they had previously been exposed to the infection and all of them were from the group who had experienced miscarriage. The study indicates that Q fever is relatively rare in women attending an urban Scottish hospital suggesting that the infection is not a major cause of miscarriage in this population. However, as Q fever antibodies could only be found in women within the miscarriage group, it suggests that the infection cannot be ruled out as a potential cause of miscarriage in individual cases.

This case-control study assesses the association between past Chlamydia trachomatis infection with first-trimester miscarriage by using an enzyme-linked immunosorbent assay to detect antibodies to the protein Pgp3.

Polycystic ovary syndrome (PCOS) affects over 10% of women. Insulin resistance, elevated free fatty acids (FFAs) and increased adiposity are key factors contributing to metabolic dysfunction in PCOS. We hypothesised that aberrant adipogenesis during adolescence, and downstream metabolic perturbations, contributes to the metabolic phenotype of adult PCOS. We used prenatally androgenized (PA) sheep as a clinically realistic model of PCOS. During adolescence, but not during fetal or early life of PA sheep, adipogenesis was decreased in subcutaneous adipose tissue (SAT) accompanied by decreased leptin, adiponectin, and increased FFAs. In adulthood, PA sheep developed adipocyte hypertrophy in SAT paralleled by increased expression of inflammatory markers, elevated FFAs and increased expression of genes linked to fat accumulation in visceral adipose tissue. This study provides better understanding into the pathophysiology of PCOS from puberty to adulthood and identifies opportunity for early clinical intervention to normalise adipogenesis and ameliorate the metabolic phenotype.

1–2% of pregnancies are ectopic, the majority implanting in the Fallopian tube. A single, systemic dose of methotrexate, a DNA-synthesis (S phase) inhibitor, has been used since 1991 for outpatient treatment of women with stable EP. However, methotrexate has limited clinical and cost effectiveness, restricting its use to 25–30% of these women. There is an unmet need for better medical treatment for EP. Colony stimulating factor-1 (CSF-1) promotes placentation and creates a pro-inflammatory environment that is fundamental for the maintenance of a normal pregnancy. We hypothesised that CSF-1 is also involved in the placentation and maintenance of an EP. Herein, we demonstrate the immunolocalisation of the CSF-1 receptor (CSF-1R) as well as its ligand (CSF-1) in immortalised first trimester trophoblast cells. We show that a specific CSF-1R kinase inhibitor, GW2580, abolishes CSF-1 induced trophoblast cell proliferation and migration and can be cytotoxic. We then demonstrate the expression of CSF-1R and CSF-1 in the cytotrophoblast and syncytiotrophoblast within ectopic implantation sites from women with EP. Our data suggests that CSF-1 is involved in the survival and proliferation of trophoblast cells in EP. This suggests that pharmacological disruption of CSF-1/CSF-1R signaling axis could be the basis of a new therapeutic for EP.

Contrast Echocardiography (CE) ultrasound with microbubble contrast agents (UCA) has significantly advanced our capability for assessment of cardiac function, including myocardium perfusion quantification. However in standard CE techniques obtained with line by line scanning, the frame rate and image quality are limited. Recent research has shown significant frame rate improvement in non-contrast cardiac imaging. In this work we present and initially evaluate, both in-vitro and in-vivo, a high frame rate (HFR) CE imaging system using diverging waves and pulse inversion sequence. An imaging frame rate of 5500 frames per second before and 250 frames per second after compounding is achieved. A destruction-replenishment sequence has also been developed. The developed HFR CE is compared with standard CE in-vitro on a phantom and then in-vivo on a sheep heart. The image signal to noise ratio, contrast between the myocardium and the chamber are evaluated. Results show up to 13.4 dB improvement in contrast for HFR CE over standard CE when compared at the same display frame-rate even when the average spatial acoustic pressure in HFR CE is 36% lower than the standard CE. It is also found that when coherent compounding is used the HFR CE image intensity can be significantly modulated by the flow motion in the chamber.

Background Ectopic pregnancies are a leading cause of maternal mortality. Most are treated surgically. We evaluated the efficacy and safety of combining oral gefitinib (epidermal growth factor receptor inhibitor) with methotrexate to treat larger ectopic pregnancies. Methods We performed a phase II, single arm, open label study across four hospitals in Edinburgh and Melbourne. We recruited women with a stable tubal ectopic pregnancy and a pre-treatment serum hCG between 1000 and 10,000 IU/L. We administered intramuscular methotrexate (50 mg/m2) once, and oral gefitinib (250 mg) for seven days. The primary outcome was the percentage successfully treated without needing surgery. To show the treatment is at least 70% effective, 28 participants were required, and 24 or more successfully treated without surgery. Secondary outcomes were safety, tolerability, and time to resolution. This study is registered (ACTRN12611001056987). Findings 30 participants with stable tubal ectopic pregnancies were recruited but two withdrew, leaving 28 participants. The median (± range) pre-treatment serum hCG was 2039 (1031–8575) IU/L and nine had pre-treatment hCGs levels >3000 IU/L. The treatment successfully resolved 86% (24/28) cases with a median (±range) time to resolution of 32 (18–67) days. The treatment caused transient rash and diarrhoea, but no serious adverse events. Interpretation Combination gefitinib and methotrexate is at least 70% effective in resolving ectopic pregnancies with a pre-treatment serum hCG 1000–10,000 IU/L. This may be a new way to treat most stable ectopic pregnancies, but needs to be validated via a randomised clinical trial., Highlights • Methotrexate and seven tablets of gefitinib resolved 86% of ectopic pregnancies with a starting hCG 1000-10,000 IU/L. • The treatment commonly caused a transient rash and diarrhea, but no serious adverse events • This could be more effective than methotrexate alone to treat ectopic pregnancies, allowing more to avoid surgery. Evidence before this study: We searched PubMed for articles before May 31st without restriction on the start date. We included the search terms “ectopic pregnancy”, AND “treatments” AND “gefitinib”. There is already an extensive literature evaluating the use of methotrexate to treat ectopic pregnancies, and reviewing each of these trials was beyond the remit of our current study. Hence for methotrexate treatment we examined meta-analyses', reviews and international clinical guidelines on the medical management of ectopic pregnancies. We also searched for general reviews on the topic of ectopic pregnancy treatment (search terms “ectopic pregnancy” AND “review”). Most ectopic pregnancies are treated surgically. An injection of methotrexate is now widely used clinically to treat ectopic pregnancy. However, the efficacy of methotrexate declines with increasing pre-treatment serum human chorionic gonadotrophin (hCG) levels, and declines when other clinical parameters are present suggesting the ectopic pregnancy is large (demonstrable fetal cardiac activity or a large size seen on ultrasound). One systematic meta-analysis published 10 years ago concluded that methotrexate treatment is only more cost economical than surgery if the pre-treatment serum hCG is

Background: Ectopic pregnancies are a leading cause of maternal mortality. Most are treated surgically. We evaluated the efficacy and safety of combining oral gefitinib (epidermal growth factor receptor inhibitor) with methotrexate to treat larger ectopic pregnancies. Methods: We performed a phase II, single arm, open label study across four hospitals in Edinburgh and Melbourne. We recruited women with a stable tubal ectopic pregnancy and a pre-treatment serum hCG between 1000 and 10,000 IU/L. We administered intramuscular methotrexate (50 mg/m2) once, and oral gefitinib (250 mg) for seven days. The primary outcome was the percentage successfully treated without needing surgery. To show the treatment is at least 70% effective, 28 participants were required, and 24 or more successfully treated without surgery. Secondary outcomes were safety, tolerability, and time to resolution. This study is registered (ACTRN12611001056987). Findings: 30 participants with stable tubal ectopic pregnancies were recruited but two withdrew, leaving 28 participants. The median (± range) pre-treatment serum hCG was 2039 (1031–8575) IU/L and nine had pre-treatment hCGs levels >3000 IU/L. The treatment successfully resolved 86% (24/28) cases with a median (±range) time to resolution of 32 (18–67) days. The treatment caused transient rash and diarrhoea, but no serious adverse events. Interpretation: Combination gefitinib and methotrexate is at least 70% effective in resolving ectopic pregnancies with a pre-treatment serum hCG 1000–10,000 IU/L. This may be a new way to treat most stable ectopic pregnancies, but needs to be validated via a randomised clinical trial. Keywords: Gefitinib, Methotrexate, Ectopic pregnancy, Medical treatment, Phase II

A diagnosis of recurrent pregnancy loss can be considered after the loss of two or more pregnancies before 24 weeks' gestation. In most couples no pathological cause is identified. The outcome can be improved for women with recurrent pregnancy loss and antiphospholipid syndrome with treatment with aspirin and/or heparin. Couples with unexplained recurrent pregnancy loss have an excellent prognosis with supportive care alone and to date no pharmacological intervention has been proven to be beneficial. This review describes the causes of recurrent pregnancy loss, the clinical work-up and an evidence-based approach to management.

Androgen signalling is a critical driver of male development. Fetal steroid signalling can be dysregulated by a range of environmental insults and clinical conditions. We hypothesised that poor adult male health was partially attributable to aberrant androgen exposure during development. Testosterone was directly administered to developing male ovine fetuses to model excess prenatal androgenic overexposure associated with conditions such as polycystic ovary syndrome (PCOS). Such in utero androgen excess recreated the dyslipidaemia and hormonal profile observed in sons of PCOS patients. 1,084 of 15,134 and 408 of 2,766 quantifiable genes and proteins respectively, were altered in the liver during adolescence, attributable to fetal androgen excess. Furthermore, prenatal androgen excess predisposed to adolescent development of an intrahepatic cholestasis-like condition with attendant hypercholesterolaemia and an emergent pro-fibrotic, pro-oxidative stress gene and protein expression profile evident in both liver and circulation. We conclude that prenatal androgen excess is a previously unrecognised determinant of lifelong male metabolic health.

Sialylation creates a negative charge on the cell surface that can interfere with blastocyst implantation. For example, α2,6-sialylation on terminal galactose, catalyzed by the sialyltransferase ST6GAL1, inhibits the binding of galectin-1, a β-galactoside-binding lectin. We recently reported the potential involvement of galectin-1 and -3 in the pathogenesis of tubal ectopic pregnancy; however, the precise role of galectins and their ligand glycoconjugates remain unclear. Here, we investigated the expression of the genes encoding α2,3- and α2,6-galactoside sialyltransferases (ST3GAL1 − 6 and ST6GAL1 − 2) and the localization of sialic acids in the Fallopian tube of women with or without ectopic implantation. ST6GAL1 expression was higher in the mid-secretory phase than the proliferative phase of non-pregnant women (P

STUDY QUESTION: Is inhibitor of DNA binding protein 2 (ID2) a mediator of the transforming growth factor (TGF)-β1-induced Warburg-like effect seen in the peritoneum of women with endometriosis?STUDY FINDING: The TGF-β1-induced changes in the metabolic phenotype of peritoneal mesothelial cells from women with endometriosis are mediated through the ID2 pathway.WHAT IS KNOWN ALREADY: TGF-β1 induces the metabolic conversion of glucose to lactate via aerobic glycolysis (the 'Warburg effect') in the peritoneum of women with endometriosis, through increased expression of the transcription factor hypoxia inducible factor α (HIF-1α). ID proteins are transcriptional targets of TGF-β1.STUDY DESIGN, SAMPLES/MATERIALS, AND METHODS: Expression of ID2 was investigated in luteal phase peritoneal biopsies from women with regular menstrual cycles, with and without endometriosis (n = 8-10 each group) by quantitative RT-PCR (qRT-PCR) and immunohistochemistry. ID2 mRNA expression in primary human peritoneal mesothelial cells (HPMC) and immortalized mesothelial cells (MeT-5A) was assessed by qRT-PCR (n = 6). The effects of TGF-β1 and ID2 siRNA on HIF-1α mRNA expression and lactate secretion was assessed using qRT-PCR and a colorimetric lactate assay.MAIN RESULTS AND THE ROLE OF CHANCE: ID2 is localised to peritoneal mesothelial and stromal cells of women with and without endometriosis. ID2 mRNA expression is lower in peritoneum adjacent to the endometriosis lesions compared to distal sites (p < 0.01). Exposure of HPMC and MeT-5A cells to physiological concentrations of TGF-β1 decreases ID2 mRNA expression (P < 0.01, P < 0.001, respectively, versus control). ID2 knockdown increases HIF-1α mRNA expression (p < 0.01) and lactate secretion (p < 0.05 versus scrambled control) to the same degree as with exposure to TGF-β1.LIMITATIONS, REASONS FOR CAUTION: Primary human cell cultures and a cell line were used in this study, and thus the results may not fully represent the situation in vivo. The results should also be replicated using a larger number of samples.WIDER IMPLICATIONS OF THE FINDINGS: Novel therapeutics that target the TGFβ/ID pathway offer a potential role in the treatment of endometriosis.LARGE SCALE DATA: N/A STUDY FUNDING AND COMPETING INTERESTS: This work was funded by a Wellbeing of Women research grant (R42533) awarded to AWH, JKB and WCD; and an MRC Centre Grant G1002033. VJY received grant support from Federation of Women Graduates (134225) and a PhD studentship from the College of Medicine and Veterinary Medicine at the University of Edinburgh. There are no competing interests to declare.

Recurrent miscarriage is traditionally defined as three or more consecutive miscarriages and this occurs in approximately 1% of couples trying to conceive. In most couples no pathological cause is identified. The outcome can be improved for women with recurrent miscarriage and antiphospholipid syndrome with treatment with aspirin and/or heparin. Couples with unexplained recurrent miscarriage have an excellent prognosis with supportive care alone and to date no pharmacological intervention has been proven to be beneficial. This review describes the causes of recurrent miscarriage, the clinical work-up and an evidence-based approach to management.

Doppler ultrasound was performed under moderate sedation (ketamine and azaperone) for 30min to monitor umbilical arterial (UA) blood flow in one uterine horn of Large White×Landrace gilts (n=23) at Gestational Days (GD) 30, 45, 60 and 90. Gilts were scanned before they were killed to examine relationships between litter size, sex ratio and five UA parameters (peak systolic velocity (PSV), end diastolic velocity (EDV), A/B (PSV to EDV) ratio, fetal heart rate (FHR) and resistance index (RI)). In gilts in which scans were obtained from all fetuses in the scanned horn, relationships between UA parameters, and fetal weight and sex were examined. A subset of gilts were sedated, scanned and recovered (SSR) earlier in gestation (GD30 or GD45) to assess the effects of sedation on later fetal development by comparison with control litters (no previous sedation). Temporal changes were observed in all UA parameters (P≤0.001). At GD60 and GD90, FHR decreased with increasing duration of sedation (P≤0.001). Sex ratio and fetal weight affected UA blood flow, whereas litter size and fetal sex did not. SSR at GD30 and GD45 was associated with decreased fetal weight at GD60 (P≤0.001) and GD90 (P=0.06) respectively, compared with controls. These results suggest maternal sedation during gestation affects fetal development, which should be investigated further. Measuring UA blood flow in growth-restricted porcine fetuses throughout gestation may be feasible.

Polycystic ovary syndrome (PCOS) is the commonest endocrine disorder affecting women of reproductive age. Some 20% of women will have polycystic ovaries on an ultrasound scan and around 7% of women have the additional clinical or biochemical features of PCOS. As a complex multisystem disorder its background can be confusing to understand. They key feature, however, is an increased production of androgen by the ovaries. This review uses ovarian biology to describe a strategy to aid understanding and explanation of PCOS. This framework can be also be used to teach about PCOS and to inform different approaches to its management.

Prenatal androgenization induces a polycystic ovary syndrome-like phenotype in adult female offspring, which is associated with alterations that can be detected in the fetal ovary, suggesting gestational origins of this condition. We therefore investigated whether increased prenatal androgen exposure also altered testicular development using ovine animal models. Biweekly maternal testosterone propionate (TP; 100 mg) from day 62 to day 70/day 90 of gestation altered male developmental trajectory. In male fetuses serum LH was decreased (P < .01), and testicular STAR, CYP11, and CYP17 abundance were reduced. Coincident with this, basal testicular T synthesis was decreased in vitro (P < .001). Leydig cell distribution was severely perturbed in all testes prenatally exposed to TP (P < .001). To examine the contribution of estrogens, fetuses were injected with TP (20 mg), the potent estrogen agonist, diethylstilbestrol (DES; 20 mg), or vehicle control at day 62 and day 82 and assessed at day 90. The effects of fetal (direct) TP treatment, but not DES, paralleled maternal (indirect) TP exposure, supporting a direct androgen effect. Cessation of maternal androgenization at day 102 returned Leydig cell distribution to normal but increased basal T output, at day 112, demonstrating Leydig cell developmental plasticity. Earlier maternal androgen exposure from day 30 similarly influenced Leydig cell development at day 90 but additionally affected the expression of Sertoli and germ cell markers. We show in this study that increased prenatal androgen exposure alters development and function of Leydig cells at a time when androgen production is paramount for male development. This supports the concept that gestational antecedents associated with polycystic ovary syndrome may have effects on the male fetus.

STUDY QUESTION Do intraluteal prostaglandins (PG) contribute to luteal regulation in women? SUMMARY ANSWER Prostaglandin E (PGE), which is produced in human granulosa-lutein cells stimulated with luteotropic hCG, exerts similar luteotropic effects to hCG, and the expression of PG synthetic and metabolic enzymes in the human CL is driven toward less PGE but more prostaglandin F (PGF) during luteolysis. WHAT IS KNOWN ALREADY Uterine PGF is a major luteolysin in many non-primate species but not in women. Increases in the PGF synthase, aldo-ketoreductase family one member C3 (AKR1C3), have been observed in the CL of marmoset monkeys during luteolysis. PGE prevents spontaneous or induced luteolysis in domestic animals. STUDY DESIGN, SIZE, DURATION Human CL tissues staged as the early-luteal (n = 6), mid-luteal (n = 6), late-luteal (n = 5) and menstrual (n = 3) phases were obtained at the time of hysterectomy for benign gynecological conditions. Luteinized granulosa cells (LGCs) were purified from follicular fluids obtained from patients undergoing assisted conception. PARTICIPANTS/MATERIALS, SETTING, METHODS Upon collection, one half of the CL was snap-frozen and the other was fixed with formalin and processed for immunohistochemical analysis of a PGE synthase (PTGES). Quantitative RT-PCR was employed to examine changes in the mRNA abundance of PG synthetic and metabolic enzymes, steroidogenic enzymes, and luteolytic molecules in the staged human CL and in human LGCs in vitro treated with hCG, PGE and PGF. A PGE withdrawal experiment was also conducted in order to reveal the effects of the loss of PGE in LGCs. Progesterone concentrations in the culture medium were measured. MAIN RESULTS AND THE ROLE OF CHANCE The key enzyme for PGE synthesis, PTGES mRNA was abundant in the functional CL during the mid-luteal phase (P < 0.01), while mRNA abundance for genes involved in PGF synthesis (AKR1B1 and AKR1C1-3) increased in the CL during the late-luteal phase and menstruation (P < 0.05-0.001). PTGES mRNA expression positively correlated with that of 3β-hydroxysteroid dehydrogenase (HSD3B1; r = 0.7836, P < 0.001), while AKR1C3 expression inversely correlated with that of HSD3B1 (r = -0.7514, P = 0.0012) and PTGES (r = -0.6923, P = 0.0042). PGE exerted similar effects to hCG-promoting genes, such as steroidogenic acute regulatory protein (STAR) and HSD3B1, to produce progesterone and luteotropic PGE, suppress PGF synthetic enzymes and down-regulate luteolytic molecules such as βA- and βB-inhibin subunits (INHBA and INHBB) and bone morphogenetic proteins (BMP2, BMP4 and BMP6). PGE withdrawal resulted in reductions in the enzymes that produce progesterone (STAR; P < 0.001) and PGE (PTGES; P < 0.001), and the capacity to produce PGE decreased, while the capacity to produce PGF increased during the culture. The addition of PGF did not recapitulate the luteolytic effects of PGE withdrawal. LARGE SCALE DATA None. LIMITATIONS, REASONS FOR CAUTION Changes in mRNA expression of PG synthetic and metabolic enzymes may not represent actual increases in PGF during luteolysis in the CL. The effects of PGF on luteal cells currently remain unclear and the mechanisms responsible for decreases in the synthesis of PGE in vitro and at luteolysis have not been elucidated in detail. WIDER IMPLICATIONS OF THE FINDINGS The results obtained strongly support a luteotropic function of PGE in regulation of the human CL. They suggest that the main PG produced in human luteal tissue changes from PGE to PGF during the maturation and regression of the CL, and the loss of PGE is more important than the effects of PGF during luteolysis in women. This may be accompanied by reduced effects of LH/hCG in luteal cells, particularly decreased activation of cAMP/protein kinase A; however, the underlying mechanisms remain unknown. STUDY FUNDING AND COMPETING INTEREST(S) This study was supported by the Cunningham Trust to WCD, a Postdoctoral Fellowship for Research Abroad from the Japan Society for the Promotion of Science and the Suntory Foundation for Life Sciences to J.N.-K.; W.C.D. is supported by an MRC Centre Grant G1002033 and a Scottish Senior Clinical Fellowship. The authors have nothing to disclose.

Context: Inadequate progesterone production from the corpus luteum is associated with pregnancy loss. Data available in model species suggest important roles of microRNAs (miRNAs) in luteal development and maintenance. Objective: To comprehensively investigate the involvement of miRNAs during the ovarian follicle-luteal transition. Design: The effects of specific miRNAs on survival and steroid production by human luteinized granulosa cells (hLGCs) were tested using specific miRNA inhibitors. Candidate miRNAs were identified through microarray analyses of follicular and luteal tissues in a bovine model. Setting: An academic institution in the United Kingdom associated with a teaching hospital. hLGCs were obtained by standard transvaginal follicular-fluid aspiration from 35 women undergoing assisted conception. Intervention(s): Inhibition of candidate miRNAs in vitro. Main outcome measure(s): Levels of miRNAs, mRNAs, FOXO1 protein, apoptosis, and steroids were measured in tissues and/or cultured cells. Results: Two specific miRNA clusters, miR-183-96-182 and miR-212-132, were dramatically increased in luteal relative to follicular tissues. miR-96 and miR-132 were the most upregulated miRNAs within each cluster. Database analyses identified FOXO1 as a putative target of both these miRNAs. In cultured hLGCs, inhibition of miR-96 increased apoptosis and FOXO1 protein levels, and decreased progesterone production. These effects were prevented by small interfering RNA-mediated downregulation of FOXO1. In bovine luteal cells, miR-96 inhibition also led to increases in apoptosis and FOXO1 protein levels. Conclusions: miR-96 targets FOXO1 to regulate luteal development through effects on cell survival and steroid production. The miR-183-96-182 cluster could provide a novel target for the manipulation of luteal function., The involvement of miRNAs in the follicle-luteal transition was investigated. miR-96 was found to promote luteal cell survival and progesterone synthesis by luteinized granulosa cells.

The corpus luteum of women is remarkable. The dominant follicle shows high levels of cellular proliferation, and its transition into the corpus luteum involves intense angiogenesis, higher than that seen in the most aggressive solid tumors. The corpus luteum, however, does not undergo malignant change. Its formation and resolution involve acute inflammatory responses and yet these processes are completed with no evidence of scarring. It is the most active endocrine gland in the body with a huge capacity for steroid synthesis, making up to 40 mg progesterone each day, and there are no disorders of clinical function. It has an essential role in human reproduction and the establishment of pregnancy, and yet we still do not fully understand how it works at a molecular level. The corpus luteum is fundamental to women’s health but research into the human corpus luteum is decreasing. However, although increased molecular understanding of the corpus luteum in women may facilitate the development of novel contraceptive paradigms and strategies to reduce the incidence and impact of ovarian hyperstimulation syndrome in assisted conception, it is likely that understanding the corpus luteum will provide generic insights into processes that affect the health of men and women throughout and beyond their reproductive years.

One in three women will present with problems, usually bleeding and/or pain, in early pregnancy prior to their planned maternity booking appointment at the end of the first trimester. The mainstay of investigation is pelvic ultrasonography. However, often the diagnosis is not clear and further ultrasound scans or repeated measurements of serum human chorionic gonadotropin concentrations are required. Most women with bleeding in early pregnancy will have an ongoing pregnancy and can be reassured. However, around 15% of pregnancies will miscarry in the first trimester. Management of miscarriage is often conservative but there are both surgical and medical, using misoprostol, management options. Importantly, approximately 1 in 80 pregnancies are ectopic pregnancies. These can be difficult to diagnose and are associated with increased maternal morbidity and mortality. Management is usually by laparoscopic salpingectomy or systemic methotrexate administration. As ectopic pregnancy can present to disparate medical practitioners it is important to have an awareness and suspicion of this condition as well as considering the possibility of pregnancy in all women of reproductive age.

Cationic host defence peptides (CHDP; also known as antimicrobial peptides) are key components of the immune response in the female reproductive tract. The role of the placental trophoblast in ovine host defence remains poorly understood. This study characterises expression of genes for cathelicidin and defensin peptides in primary ovine placental tissues, the ovine trophoblast cell line (AH-1) and in response to the TLR-4 ligand LPS, the abortifacient organism Waddlia chondrophila and 1alpha,25-dihydroxyvitamin D3. Using RT-PCR, expression of the CHDP SMAP-29, sBD-1 and sBD-2 was assessed in the AH-1 cell line in response to LPS, 1alpha,25-dihydroxyvitamin D3 exposure (a known stimulator of cathelicidin gene expression), or W. chondrophila infection. Expression of cathelicidin in the trophoblast compartment of the ovine placenta and in the ovine trophoblast cell line (AH-1) was also established. AH-1 cells did not upregulate expression of CHDP in response to LPS, but sBD-1 and sBD-2 expression was significantly increased in response to W. chondrophila infection. SMAP-29 expression was not altered by in vitro exposure to 1alpha,25-dihydroxyvitamin D3. This study demonstrates that the ovine trophoblast expresses cathelicidins, but does not upregulate expression of CHDP in response to LPS. Ovine trophoblasts are shown to differentially regulate expression of CHDP and lack a demonstrable vitamin D-mediated cathelicidin response.

This study assessed the effect of pre-natal social stress and post-natal pain on the reproductive development of young (approximately day 40) pigs. Male pigs carried by sows that were stressed by mixing with unfamiliar older sows for two 1-week periods during mid-pregnancy had lower plasma testosterone (0.54 vs 0.86 ng/ml, s.e.d.=0.11; P=0.014) and oestradiol (E2; 22.9 vs 38.7 pg/ml, s.e.d.=7.80; P=0.021) concentrations compared with males carried by unstressed control sows. Although there was no effect of pre-natal stress on female E2 concentrations, female pigs carried by stressed sows had fewer primordial ovarian follicles (log −4.32/μm2 vs −4.00/μm2, s.e.d.=0.136; P=0.027). Tail amputation on day 3 after birth reduced E2 concentrations in female (4.78 vs 6.84 pg/ml, s.e.d.=0.86; P=0.03) and in male (25.6 vs 34.9 pg/ml, s.e.d.=3.56; P=0.021) pigs and reduced both testis weight (0.09% of body weight vs 0.10% of body weight, s.e.d.=0.003; P=0.01) and the percentage of proliferating Leydig cells (1.97 vs 2.12, s.e.d.=0.114; P=0.036) compared with sham-amputated littermate controls. There was a significant (P=0.036) interaction between the effects of pre-natal stress and post-natal pain on testicular expression of the steroidogenic enzyme 17α-hydroxylase, such that amputation increased expression in pigs born to control sows, but reduced expression in animals born to stressed sows. This study shows that stressful procedures associated with routine animal husbandry can disrupt the developing reproductive axis.

Macrophages are the most abundant immune cell within the ovary. Their dynamic distribution throughout the ovarian cycle and heterogenic array of functions suggest the involvement in various ovarian processes, but their functional role has yet to be fully established. The aim was to induce conditional macrophage ablation to elucidate the putative role of macrophages in maintaining the integrity of ovarian vasculature. Using the CD11b-diphtheria toxin receptor (DTR) mouse, in which expression of human DTR is under the control of the macrophage-specific promoter sequence CD11b, ovarian macrophages were specifically ablated in adult females by injections of diphtheria toxin (DT). CD11b-DTR mice were given DT treatment or vehicle and ovaries collected at 2, 8, 16, 24 and 48 h. Histochemical stains were employed to characterise morphological changes, immunohistochemistry for F4/80 to identify macrophages and the endothelial cell marker CD31 used to quantify vascular changes. In normal ovaries, macrophages were detected in corpora lutea and in the theca layer of healthy and atretic follicles. As macrophage ablation progressed, increasing amounts of ovarian haemorrhage were observed affecting both luteal and thecal tissue associated with significant endothelial cell depletion, increased erythrocyte accumulation and increased follicular atresia by 16 h. These events were followed by necrosis and profound structural damage. Changes were limited to the ovary, as DT treatment does not disrupt the vasculature of other tissues likely reflecting the unique cyclical nature of the ovarian vasculature and heterogeneity between macrophages within different tissues. These results show that macrophages play a critical role in maintaining ovarian vascular integrity.

Gonadotrophin-induced ovulation (GnOI) is generally an effective treatment when anti-estrogen therapy is unsuccessful. It remains uncertain how appropriate it is in older and obese women. We carried out an analysis of a GnOI programme between 2000 and 2008, including 514 treatment cycles in 140 women with World Health Organisation Group II anovulation. A low-dose step-up stimulation protocol using recombinant follicle stimulating hormone (FSH) (with natural intercourse) was used throughout in the treatment of women aged up to 40 years and with body mass index (BMI) up to 34 kg/m(2), with a rigorously-applied cancellation criterion. The livebirth rates in first stimulated cycle and cumulatively over six cycles (or until pregnancy) were 19.2% and 74.2%, respectively and the multiple livebirth rate was 2.5%. There was no significant relationship between age and BMI with pregnancy rates, although higher BMI was associated with higher threshold dose and longer duration of stimulation. Anti-estrogen resistant patients attained significantly higher cumulative ongoing pregnancy rates than those who were anti-estrogen responsive. These data demonstrate that judicious administration of gonadotrophins to women with WHO Group II anovulatory subfertility can result in a good pregnancy rate with low risk of multiple pregnancy in women aged up to 40 years and with BMI of up to 34 kg/m(2).

Inhibitor of differentiation (Id) proteins act during embryogenesis and development to repress gene transcription required for lineage commitment, while promoting cell growth. Growth factors belonging to the TGF beta superfamily of signaling molecules, notably the bone morphogenetic proteins (BMPs) and activin, can regulate Id expression in these tissues. Id expression and function in adult physiology is less well determined, and we hypothesized a role for Id proteins in the adult mammalian ovary. Immunohistochemistry for Id1, Id2, Id3, and Id4 in the sheep ovary revealed consistent expression in granulosa and thecal cells of ovarian follicles throughout development. In atretic follicles, Id proteins were selectively down-regulated in thecal cells (P < 0.0001). Additionally, Id1 was universally up-regulated in the cumulus cells adjacent to the oocyte. Immunohistochemistry for phospho (p)-smad 1/5/8 signaling components (stimulated by BMPs) showed a punctate pattern of expression whereas p-smad 2/3 (stimulated by activin) was ubiquitously expressed in follicles. Neither pathway, however, displayed differential staining in line with Id1 cumulus-specific expression, suggesting a more complex relationship between Id1 expression and TGF beta signaling in these cells. Nevertheless, in vitro, stimulation of ovine granulosa cells with BMP6 or activin A led to a respective increase and decrease in Id1 (P < 0.0001), Id2 (P < 0.0001), Id3 (P < 0.0001), and Id4 (P < 0.05) transcripts, and Id1 gene expression was further manipulated by the oocyte-secreted factors BMP15 and growth differentiation factor 9 (P < 0.001). These data confirm that TGF beta signaling can regulate Id gene expression in the sheep ovarian follicle and suggest a functional role for the Id family in the mammalian ovary. (Endocrinology 151: 1247-1256, 2010)

The Renin-Angiotensin-System (RAS) has been suspected not only to control vascular tone but also to regulate angiogenesis. Angiotensin II has been shown to influence angiogenic factors such as vascular endothelial growth factor (VEGF). The Corpus luteum undergoes intense VEGF-dependent angiogenesis, regulated by luteinising hormone (LH) and human chorionic gonadotrophin (hCG). We therefore hypothesised, that locally produced Angiotensin II could act as a physiological co-regulator with hCG in luteal VEGF expression.We investigated the expression of RAS components and their regulation by hCG in human granulosa lutein cells using RT-PCR, immunocytochemistry and Western blotting. In addition, we studied the effect of Angiotensin II on basal and hCG-stimulated VEGF expression using TaqMan-analysis, ELISA, and Western blot analysis.Human granulosa lutein cells express angiotensinogen and angiotensin converting enzyme (ACE) and synthesise Angiotensin. In addition, they express both Angiotensin receptors. Angiotensin II stimulated VEGF mRNA (p0.05) and protein expression (p0.05). However, hCG decreased angiotensinogen (p0.05) and Angiotensin II (p0.05). Both, the addition of Angiotensin II and its inhibition using Candesartan did not change the magnitude of hCG-increased VEGF expression.These findings suggest a role for locally synthesised Angiotensin II in the regulation of luteal VEGF expression. However, Angiotensin II does not appear to have a major contribution in the presence of hCG when the RAS pathway is down-regulated.

Summary Background and objective There are currently no sensitive and specific assays for activin B that could be utilized to study human biological fluids. The aim of this project was to develop and validate a ‘total’ activin B ELISA for use with human biological fluids and establish concentrations of activin B in the circulation and fluids from the reproductive organs. Design The new ELISA was validated and then used to measure activin B levels in the circulation of healthy participants, IVF patients, pregnant women and in ovarian follicular fluid and seminal plasma. Patients and measurements Healthy adult subjects (n = 143), subjects from an IVF clinic (n = 27) and pregnancy groups (n = 29) were sampled. Results The sensitivity of the assay was 0·019 ng/ml. Validation of the activin B ELISA showed good recovery (90·7 ± 9·8%) and linearity in biological fluid and cell culture media and low cross-reactivity with related analytes (inhibin B = 0·077% and activin A = 0·0034%). There was a negative correlation between activin B concentration (r = −0·281, P

The transition of the dominant follicle into the corpus luteum is of fundamental reproductive importance. Luteinisation involves disparate changes in the gene expression of follicular granulosa cells that differentiate into the granulosa-lutein cells of the corpus luteum after the gonadotrophin surge. We have shown that activin and human chorionic gonadotropin (hCG) have opposing effects during luteolysis. Therefore, we hypothesised that activin A was an inhibitor of luteinisation that was blocked during the pre–ovulatory gonadotrophin surge. Ovarian tissue and cells were collected from women with regular cycles having hysterectomy and women undergoing oocyte retrieval for assisted conception. Genes that changes during luteinisation were investigated in primary cultures of luteinised granulosa cells exposed to activin A and hCG in vitro. hCG promotes a luteinised granulosa cell phenotype, while activin A promotes a more follicular phenotype in luteinised cells by upregulating granulosa cells markers such as FSHR, HSD11B2 and downregulating LHCGR. In addition, activin A blocked hCG upregulation of STAR, HSD3B1 and HSD11B1 and downregulation of oestrogen receptor α. Activin A antagonised hCG effects in a dose-dependent manner and could block the hCG-stimulated molecular inhibitors of activin action (inhibin α-subunit, follistatin and TGFBR3). These studies show that hCG and activin A have opposing effects on luteinised granulosa cells and some effects of activin are seen only in the presence of hCG. While hCG can inhibit activin action in granulosa cells to facilitate luteinisation, activin A can promote an unluteinised phenotype in luteinised granulosa cells. This confirms the importance of adequate activin withdrawal during luteinisation in women.

Vascular endothelial growth factor (VEGF)-dependent angiogenesis is essential for normal luteal development. Although it is believed that hypoxia is the primary inducer of VEGF, in the corpus luteum it is up-regulated by human chorionic gonadotrophin (hCG). As hypoxia-inducible factor (HIF)1A has been shown to regulate VEGFA under ligand-stimulated conditions, we hypothesized that the effect of hCG on luteal VEGFA was mediated through HIF1A. We studied the effect of hCG on VEGFA and HIF1A expression in human luteinized granulosa cells in vitro and in human corpora lutea in vivo. HCG up-regulated VEGFA (P < 0.05) and HIF1A (P < 0.001) in vitro and VEGFA (P < 0.05) and HIF1A (P < 0.05) in vivo. There was a correlation between HIF1A and VEGFA in vivo (P < 0.005) and in vitro (P < 0.05). Nuclear HIF1A in granulosa-lutein cells was highest during luteal formation and absent from the fully functional corpus luteum (P < 0.05). Both VEGFA (P < 0.001) and HIF1A (P < 0.01) were up-regulated by dibutyryl-cAMP, through a PKA pathway. Hypoxia increased VEGFA (P < 0.001) and HIF1A (P < 0.05) expression and hCG further augmented VEGFA (P < 0.001) and HIF1A (P < 0.01) under hypoxic conditions. However, progesterone increased hCG-stimulated VEGFA but had no effect on HIF1A expression. The expression of HIF1A is therefore hormonally regulated in luteal cells in vitro and in vivo and may regulate VEGFA expression under normoxic and hypoxic conditions. However, the differential effects of progesterone suggest that not all regulation of VEGFA is associated with an up-regulation of HIF1A.