Combination of gefitinib and methotrexate to treat tubal ectopic pregnancy (GEM3): a multicentre, randomised, double-blind, placebo-controlled trial.

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.

Early (Days 1-4) post-treatment serum hCG level changes predict single-dose methotrexate treatment success in tubal ectopic pregnancy.

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 <30 IU/l following single-dose methotrexate treatment without additional treatment. Patient characteristics of the treatment success and failure groups were compared. Changes in Days 1-4, 1-7, and 4-7 serum hCG were evaluated as predictors of treatment success through receiver operating characteristic curve analysis. Test performance characteristics were calculated for percentage change ranges and thresholds including optimal classification thresholds. 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 <18% was identified as an optimal test threshold that predicted treatment success with 79% sensitivity and 74% specificity, resulting in 82% positive predictive value and 69% negative predictive value. 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 (<18%) rise in Days 1-4 serum hCG levels, that their treatment will likely be effective. 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).

A Randomized Trial of Progesterone in Women with Bleeding in Early Pregnancy.

BACKGROUND: Bleeding in early pregnancy is strongly associated with pregnancy loss. Progesterone is essential for the maintenance of pregnancy. Several small trials have suggested that progesterone therapy may improve pregnancy outcomes in women who have bleeding in early pregnancy. METHODS: We conducted a multicenter, randomized, double-blind, placebo-controlled trial to evaluate progesterone, as compared with placebo, in women with vaginal bleeding in early pregnancy. Women were randomly assigned to receive vaginal suppositories containing either 400 mg of progesterone or matching placebo twice daily, from the time at which they presented with bleeding through 16 weeks of gestation. The primary outcome was the birth of a live-born baby after at least 34 weeks of gestation. The primary analysis was performed in all participants for whom data on the primary outcome were available. A sensitivity analysis of the primary outcome that included all the participants was performed with the use of multiple imputation to account for missing data. RESULTS: A total of 4153 women, recruited at 48 hospitals in the United Kingdom, were randomly assigned to receive progesterone (2079 women) or placebo (2074 women). The percentage of women with available data for the primary outcome was 97% (4038 of 4153 women). The incidence of live births after at least 34 weeks of gestation was 75% (1513 of 2025 women) in the progesterone group and 72% (1459 of 2013 women) in the placebo group (relative rate, 1.03; 95% confidence interval [CI], 1.00 to 1.07; P = 0.08). The sensitivity analysis, in which missing primary outcome data were imputed, resulted in a similar finding (relative rate, 1.03; 95% CI, 1.00 to 1.07; P = 0.08). The incidence of adverse events did not differ significantly between the groups. CONCLUSIONS: Among women with bleeding in early pregnancy, progesterone therapy administered during the first trimester did not result in a significantly higher incidence of live births than placebo. (Funded by the United Kingdom National Institute for Health Research Health Technology Assessment program; PRISM Current Controlled Trials number, ISRCTN14163439.).

Doppler ultrasound can be used to monitor umbilical arterial blood flow in lightly sedated pigs at multiple gestational ages.

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.

Loss of luteotropic prostaglandin E plays an important role in the regulation of luteolysis in women.

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 3ß-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 ßA- and ßB-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.

ID2 mediates the transforming growth factor-ß1-induced Warburg-like effect seen in the peritoneum of women with endometriosis.

STUDY QUESTION: Is inhibitor of DNA-binding protein 2 (ID2) a mediator of the transforming growth factor (TGF)-ß1-induced Warburg-like effect seen in the peritoneum of women with endometriosis? SUMMARY ANSWER: The TGF-ß1-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-ß1 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-ß1. STUDY DESIGN, SAMPLES/MATERIALS, 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-ß1 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 localized 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-ß1 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-ß1. 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 TGFß/ID pathway offer a potential role in the treatment of endometriosis. LARGE SCALE DATA: None. STUDY FUNDING AND COMPETING INTERESTS: This work was funded by a Wellbeing of Women research grant (R42533) awarded to A.W.H., J.K.B. and W.C.D.; and an MRC Centre Grant G1002033. V.J.Y. 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.

Cigarette smoking alters sialylation in the Fallopian tube of women, with implications for the pathogenesis of ectopic pregnancy.

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 ß-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 < 0.0001), whereas ST6GAL1 (P < 0.0001), ST3GAL3 (P = 0.0029), ST3GAL5 (P = 0.0089), and ST3GAL6 (P = 0.0018) were all lower in Fallopian tubes with ectopic implantations. α2,3- and α2,6-sialic acids, however, both remained enriched on the surface of Fallopian tube epithelium. Cigarette smoking, a major risk factor for tubal ectopic pregnancy, was associated with reduced mid-secretory-phase expression of ST6GAL1 (P = 0.0298), but elevated expression of ST3GAL5 (P = 0.0006), an enzyme known to be involved in ciliogenesis. Indeed, sialic acid-containing ciliated inclusion cysts, which are associated with abnormal ciliogenesis, were observed within the epithelium at a higher frequency in women who smoked (P = 0.0177), suggesting that abnormal ciliogenesis is associated with smoking. Thus, cigarette smoking alters sialylation in the Fallopian tube epithelium, and is potentially a source of decreased tubal transport and increased receptivity for blastocyst in the human Fallopian tube. Mol. Reprod. Dev. 83: 1083-1091, 2016. © 2016 Wiley Periodicals, Inc.

Regulated C-C motif ligand 2 (CCL2) in luteal cells contributes to macrophage infiltration into the human corpus luteum during luteolysis.

Intense macrophage infiltration is observed during luteolysis in various animals including women; however, we still do not know how macrophage infiltration into the human corpus luteum (CL) during luteolysis is regulated. In this study, we examined the expression, localization and regulation of an important chemokine for the recruitment of monocyte/macrophage lineages, C-C motif ligand 2 (CCL2), in the human CL across the luteal phase and in cultured human luteinized granulosa cells (LGCs), with special reference to the number of infiltrating macrophages and luteal cell function. CCL2 mRNA increased in the non-functional regressing CL during menstruation (P < 0.01), corresponding to an elevated mRNA expression of a macrophage-derived cytokine, tumor necrosis factor (TNF), and an increased number of infiltrating macrophages positively stained with a macrophage marker, CD68. CCL2 protein was immunohistochemically localized to the cytoplasm of granulosa-lutein and theca-lutein cells, and CCL2 mRNA was significantly reduced by hCG both in vivo (P < 0.05) and in vitro (P < 0.01). CCL2 was also down-regulated by luteotrophic prostaglandin (PG) E (P < 0.0001), but up-regulated by luteolytic PGF (P < 0.05) in vitro. Administration of TNF significantly enhanced the CCL2 mRNA expression in cultured LGCs (P < 0.01). A greater abundance of infiltrating macrophages were found around granulosa-lutein cells lacking 3ß-HSD or PGE synthase (PGES) immunostaining. CCL2 mRNA expression was negatively correlated with both HSD3B1 and PGES, suggesting that locally produced progesterone and PGE suppress macrophage infiltration into the CL. Taken together, the infiltration of macrophages in the human CL is regulated by endocrine and paracrine molecules via regulation of the CCL2 expression in luteal cells.

The Expression and Cellular Localization of Galectin-1 and Galectin-3 in the Fallopian Tube Are Altered in Women with Tubal Ectopic Pregnancy.

Galectin-1 and galectin-3 are abundantly expressed at implantation sites in the uterus, suggesting their involvement in the establishment of pregnancy. In this study, we examined the expression and localization of galectin-1 and galectin-3 in fallopian tubes from nonpregnant women, and in those presenting with tubal ectopic pregnancy. There was no significant difference in the expression of either galectin-1 (LGALS1) or galectin-3 (LGALS3) transcripts in the fallopian tube across the menstrual cycle. Their expressions in the fallopian tube were inversely correlated to each other (r = -0.5134, p < 0.0001) and differentially localized. Galectin-1 protein was abundant in the stroma of nonpregnant fallopian tubes, whereas galectin-3 was mainly localized to the epithelium, notably to the cilia of ciliated cells and the apical cytoplasm of secretory cells. In ectopic pregnancies, LGALS3 expression was significantly reduced (p < 0.0001), but LGALS1 expression did not change when compared to nonpregnant fallopian tubes collected during the mid-secretory phase. The percentage of fallopian tube epithelial cells expressing galectin-3 in cilia tended to be reduced (p = 0.0685), with an accompanying loss of a normal ciliary structure, while nuclear galectin-3 increased (p < 0.05) in ectopic pregnancies. Epithelial immunostaining for galectin-1 tended to be elevated in fallopian tubes from women with ectopic pregnancy. Coculture of human trophoblast origin SW71 cells significantly increased LGALS1 expression in human fallopian tube epithelial OE-E6/E7 cells, suggesting that trophoblast-derived products regulate LGALS1 expression in the oviductal epithelium. These findings imply a differential contribution of galectin-1 and galectin-3 in the homeostasis of human fallopian tubes and in the pathophysiology of ectopic pregnancy.

Expression and localization of inhibitor of differentiation (ID) proteins during tissue and vascular remodelling in the human corpus luteum.

Members of the transforming growth factor-ß (TGF-ß) superfamily are likely to have major roles in the regulation of tissue and vascular remodelling in the corpus luteum (CL). There are four inhibitor-of-differentiation (ID1-4) genes that are regulated by members of the TGF-ß superfamily and are involved in the transcriptional regulation of cell growth and differentiation. We studied their expression, localization and regulation in dated human corpora lutea from across the luteal phase (n = 22) and after human chorionic gonadotrophin (hCG) administration in vivo (n = 5), and in luteinized granulosa cells (LGCs), using immunohistochemistry and quantitative RT-PCR. ID1-4 can be localized to multiple cell types in the CL across the luteal phase. Endothelial cell ID3 (P < 0.05) and ID4 (P < 0.05) immunostaining intensities peak at the time of angiogenesis but overall ID1 (P < 0.05) and ID3 (P < 0.05) expression peaks at the time of luteolysis, and luteal ID3 expression is inhibited by hCG in vivo (P < 0.01). In LGC cultures in vitro, hCG had no effect on ID1, down-regulated ID3 (P < 0.001), and up-regulated ID2 (P < 0.001) and ID4 (P < 0.01). Bone morphogenic proteins (BMPs) had no effect on ID4 expression but up-regulated ID1 (P < 0.01 to P < 0.005). BMP up-regulation of ID2 (P < 0.05) was additive to the hCG up-regulation of ID2 expression (P < 0.001), while BMP cancelled out the down regulative effect of hCG on ID3 regulation. As well as documenting regulation patterns specific for ID1, ID2, ID3 and ID4, we have shown that IDs are located and differentially regulated in the human CL, suggesting a role in the transcriptional regulation of luteal cells during tissue and vascular remodelling.

Targeting angiogenesis in the pathological ovary.

The ovary is a key tissue in the study of physiological neo-vascularisation in the adult and its study has highlighted important molecules involved in the regulation of angiogenesis in vivo. These include vascular endothelial growth factor, delta-like ligand 4, thrombospondin-1, prokineticin-1 and prostaglandin E2. Targeting these molecular pathways has therapeutic potential and their manipulation has an increasing preclinical and clinical role in the management of the pathological ovary. Targeting angiogenic pathways has utility in the promotion of ovarian angiogenesis to improve tissue and follicle survival and function as well as the prevention and management of ovarian hyperstimulation syndrome. There is a theoretical possibility that targeting angiogenesis may improve the function of the polycystic ovary and a real role for targeting angiogenesis in ovarian cancer.

The financial costs to patients of diagnosing and excluding ectopic pregnancy.

OBJECTIVES: Health care costs are one of the greatest challenges in modern medicine. In gynaecology, diagnosing and excluding ectopic pregnancy (EP) has been shown to be a financial burden to health services because it commonly requires multiple investigations and hospital visits. However, the full economic costs are not captured by an analysis of health care costs alone. This study therefore aimed to assess the indirect costs to patients of diagnosing and excluding EP. METHODS: Patients presenting to a Pregnancy Support Centre in a large UK teaching hospital with abdominal pain and/or bleeding and a positive pregnancy test were recruited during the period June 2010-February 2011. Patients were provided with questionnaires to be completed at home and designed to record and quantify costs that they had incurred until a final diagnosis of their condition was made. A cost-description analysis was performed. RESULTS: 52/203 (26%) recruited patients returned completed questionnaires. The mean cost to patients of diagnosing or excluding EP was £135.13±£51.60 (median £20.70). The main cost drivers identified were hospital visits, holiday cancellations, income loss and household help. CONCLUSIONS: Quantification of the indirect costs of diagnosing and excluding EP is challenging because it relies on questionnaire feedback from patients at a time when they have suffered from the emotional impact of pregnancy loss. However, initial estimates suggest that such costs are significant due to diagnostic delays. This further highlights the importance of the development of potential biomarkers of EP to allow prompt diagnosis.

Recent advances in the understanding of tubal ectopic pregnancy.

Ectopic pregnancy (EP) is described as the implantation of an embryo outside the normal uterine cavity. It most commonly occurs in the fallopian tube, hence termed a tubal ectopic pregnancy (tEP). It is a gynaecological emergency and remains the leading cause of direct maternal mortality related to the first trimester of pregnancy worldwide. This article explores the emergence of additional risk factors for tEP, showing new evidence for identifying patient risk factors and highlighting potential areas of research. Additionally, we discuss the up-to-date patient-centred approach for the diagnosis, management and counselling of patients with tEP and ongoing clinical trials for the improvement of medical management.

Subsequent pregnancy outcomes among women with tubal ectopic pregnancy treated with methotrexate.

Lay summary: An ectopic pregnancy occurs when an embryo implants outside of the uterus, usually in a fallopian tube. When detected early, treatment is often with a medication called methotrexate. When methotrexate does not work, surgery is required. A recent clinical trial of ectopic pregnancy treatment (called GEM3) found that adding a drug called gefitinib to methotrexate did not reduce the need for surgery. We have used data from the GEM3 trial, combined with data collected 12 months after the trial finished, to investigate post-methotrexate pregnancy outcomes. We found no difference in pregnancy rates, pregnancy loss rates and recurrent ectopic pregnancy rates between those treated medically only and those who subsequently also needed surgery. The surgical technique used also did not affect pregnancy rates. This research provides reassurance that women with ectopic pregnancies treated medically who need surgery have similar post-treatment pregnancy outcomes to those treated successfully medically.

Development of an acute ovine model of polycystic ovaries to assess the effect of ovarian denervation.

Introduction: Polycystic ovary syndrome (PCOS) seems to be associated with increased ovarian sympathetic nerve activity and in rodent models of PCOS reducing the sympathetic drive to the ovary, through denervation or neuromodulation, improves ovulation rate. We hypothesised that sympathetic nerves work with gonadotropins to promote development and survival of small antral follicles to develop a polycystic ovary phenotype. Methods: Using a clinically realistic ovine model we showed a rich sympathetic innervation to the normal ovary and reinnervation after ovarian transplantation. Using needlepoint diathermy to the nerve plexus in the ovarian vascular pedicle we were able to denervate the ovary resulting in reduced intraovarian noradrenaline and tyrosine hydroxylase immunostained sympathetic nerves. We developed an acute polycystic ovary (PCO) model using gonadotrophin releasing hormone (GnRH) agonist followed infusion of follicle stimulating hormone (FSH) with increased pulsatile luteinising hormone (LH). This resulted in increased numbers of smaller antral follicles in the ovary when compared to FSH infusion suggesting a polycystic ovary. Results: Denervation had no effect of the survival or numbers of follicles in the acute PCO model and did not impact on ovulation, follicular and luteal hormone profiles in a normal cycle. Discussion: Although the ovary is richly inervated we did not find evidence for a role of sympathetic nerves in ovarian function or small follicle growth and survival.

Has increased clinical experience with methotrexate reduced the direct costs of medical management of ectopic pregnancy compared to surgery?

BACKGROUND: There is a debate about the cost-efficiency of methotrexate for the management of ectopic pregnancy (EP), especially for patients presenting with serum human chorionic gonadotrophin levels of >1500 IU/L. We hypothesised that further experience with methotrexate, and increased use of guideline-based protocols, has reduced the direct costs of management with methotrexate. METHODS: We conducted a retrospective cost analysis on women treated for EP in a large UK teaching hospital to (1) investigate whether the cost of medical management is less expensive than surgical management for those patients eligible for both treatments and (2) to compare the cost of medical management for women with hCG concentrations 1500-3000 IU/L against those with similar hCG concentrations that elected for surgery. Three distinct treatment groups were identified: (1) those who had initial medical management with methotrexate, (2) those who were eligible for initial medical management but chose surgery ('elected' surgery) and (3) those who initially 'required' surgery and did not meet the eligibility criteria for methotrexate. We calculated the costs from the point of view of the National Health Service (NHS) in the UK. We summarised the cost per study group using the mean, standard deviation, median and range and, to account for the skewed nature of the data, we calculated 95% confidence intervals for differential costs using the nonparametric bootstrap method. RESULTS: Methotrexate was £1179 (CI 819-1550) per patient cheaper than surgery but there were no significant savings with methotrexate in women with hCG >1500 IU/L due to treatment failures. CONCLUSIONS: Our data support an ongoing unmet economic need for better medical treatments for EP with hCG >1500 IU/L.

Did the NICE guideline for progesterone treatment of threatened miscarriage get it right?

In November 2021, NICE updated its clinical guideline that covers the management of threatened miscarriage in the first trimester. They recommended offering vaginal micronised progesterone twice daily until 16 completed weeks of pregnancy in those with a previous miscarriage. However, the duration of treatment is not evidence based. In the major clinical trial that informed the guideline, there was no benefit in starting progesterone after 9 weeks and the full effect of progesterone was present at 12 weeks of pregnancy. There are theoretical risks impacting offspring health in later life after maternal pharmaceutical progesterone treatment. As the effect of progesterone seems to be complete by 12 weeks of gestation, we should consider carefully whether to follow the guidance and treat up to 16 weeks of pregnancy. Lay summary: In November 2021, new guidelines were published about the management of bleeding in early pregnancy. If someone who has had a previous miscarriage starts bleeding, they should now be treated with progesterone as this slightly reduces the chance of miscarriage. The guideline says progesterone should be given if the pregnancy is in the womb, and potentially normal, until 16 weeks of pregnancy. However, in the big studies looking at progesterone's effect in reducing miscarriage the beneficial effects of progesterone were complete by 12 weeks of pregnancy. At that stage, it is the placenta and not the mother's ovary that makes the progesterone to support the pregnancy. We do not know the long-term effects of giving extra progesterone during pregnancy on the offspring. Some research has raised the possibility that there might be some adverse effects if progesterone is given for too long. Maybe the guidance should have suggested stopping at 12 weeks rather than 16 weeks of pregnancy.

Hepatic Mitochondrial Dysfunction and Risk of Liver Disease in an Ovine Model of "PCOS Males".

First-degree male relatives of polycystic ovary syndrome (PCOS) sufferers can develop metabolic abnormalities evidenced by elevated circulating cholesterol and triglycerides, suggestive of a male PCOS equivalent. Similarly, male sheep overexposed to excess androgens in fetal life develop dyslipidaemia in adolescence. Dyslipidaemia, altered lipid metabolism, and dysfunctional hepatic mitochondria are associated with the development of non-alcoholic liver disease (NAFLD). We therefore dissected hepatic mitochondrial function and lipid metabolism in adolescent prenatally androgenized (PA) males from an ovine model of PCOS. Testosterone was directly administered to male ovine fetuses to create prenatal androgenic overexposure. Liver RNA sequencing and proteomics occurred at 6 months of age. Hepatic lipids, glycogen, ATP, reactive oxygen species (ROS), DNA damage, and collagen were assessed. Adolescent PA males had an increased accumulation of hepatic cholesterol and glycogen, together with perturbed glucose and fatty acid metabolism, mitochondrial dysfunction, with altered mitochondrial transport, decreased oxidative phosphorylation and ATP synthesis, and impaired mitophagy. Mitochondrial dysfunction in PA males was associated with increased hepatic ROS level and signs of early liver fibrosis, with clinical relevance to NAFLD progression. We conclude that excess in utero androgen exposure in male fetuses leads to a PCOS-like metabolic phenotype with dysregulated mitochondrial function and likely lifelong health sequelae.

Q fever and early pregnancy failure: a Scottish case-control study.

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.

Conditional ablation of macrophages disrupts ovarian vasculature.

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.