Inferring biallelism of two FSH receptor mutations associated with spontaneous ovarian hyperstimulation syndrome by evaluating FSH, LH and HCG cross-activity.

RESEARCH QUESTION: What is the cumulative effect of two follicle-stimulating hormone receptor (FSHR) mutations in spontaneous ovarian hyperstimulation syndrome (sOHSS) pathogenesis? Are these mutations in the mono- or biallelic state? DESIGN: Two FSHR mutations were found in a pregnant patient affected by sOHSS with no predisposing conditions. While the p.Asn106His mutation is novel, the p.Ser128Tyr mutation has been associated with sOHSS previously. The patient's FSHR gene was analysed by Sanger sequencing, and FSHR cDNAs carrying a single or both point mutations were created by mutagenesis in vitro. cAMP activation by recombinant FSH, luteinizing hormone (LH), human chorionic gonadotropin (HCG) and thyroid-stimulating hormone (TSH) was evaluated in transfected HEK293 cells by bioluminescence resonance energy transfer. RESULTS: All mutations decreased the 50% effective concentration of FSH calculated for cAMP (P < 0.05, n = 6), resulting in two- to 10-fold lower ligand potency. TSH failed to induce an FSHR-mediated increase in intracellular cAMP, while LH was approximately four-fold more potent than HCG in p.Ser128Tyr FSHR-expressing HEK293 cells despite lower cAMP plateau levels (P < 0.05, n = 5). The p.Ser128Tyr FSHR mutation was found to be responsible for an LH-/HCG-induced increase in cAMP when it was in the biallelic heterozygous state with p.Asn106His, but no increase in cAMP was induced in the monoallelic state. CONCLUSION: In-vitro data support that, in pregnant patients with sOHSS, the two FSHR mutations have an opposing effect on the pathogenesis of sOHSS and are in the biallelic heterozygous form, allowing HCG to induce a p.Ser128Tyr FSHR-mediated increase in cAMP.

Introduction and Evaluation of an Electronic Tool for Improved Data Quality and Data Use during Malaria Case Management Supportive Supervision.

Although on-site supervision programs are implemented in many countries to assess and improve the quality of care, few publications have described the use of electronic tools during health facility supervision. The President's Malaria Initiative-funded MalariaCare project developed the MalariaCare Electronic Data System (EDS), a custom-built, open-source, Java-based, Android application that links to District Health Information Software 2, for data storage and visualization. The EDS was used during supervision visits at 4,951 health facilities across seven countries in Africa. The introduction of the EDS led to dramatic improvements in both completeness and timeliness of data on the quality of care provided for febrile patients. The EDS improved data completeness by 47 percentage points (42-89%) on average when compared with paper-based data collection. The average time from data submission to a final data analysis product dropped from over 5 months to 1 month. With more complete and timely data available, the Ministry of Health and the National Malaria Control Program (NMCP) staff could more effectively plan corrective actions and promptly allocate resources, ultimately leading to several improvements in the quality of malaria case management. Although government staff used supervision data during MalariaCare-supported lessons learned workshops to develop plans that led to improvements in quality of care, data use outside of these workshops has been limited. Additional efforts are required to institutionalize the use of supervision data within ministries of health and NMCPs.

Fever case management at private health facilities and private pharmacies on the Kenyan coast: analysis of data from two rounds of client exit interviews and mystery client visits.

BACKGROUND: Private sector availability and use of malaria rapid diagnostic tests (RDTs) lags behind the public sector in Kenya. Increasing channels through which quality malaria diagnostic services are available can improve access to testing and help meet the target of universal diagnostic testing. Registered pharmacies are currently not permitted to perform blood tests, and evidence of whether malaria RDTs can be used by non-laboratory private providers in line with the national malaria control guidelines is required to inform ongoing policy discussions in Kenya. METHODS: Two rounds of descriptive cross-sectional exit interviews and mystery client surveys were conducted at private health facilities and registered pharmacies in 2014 and 2015, 6 and 18 months into a multi-country project to prime the private sector market for the introduction of RDTs. Data were collected on reported RDT use, medicines received and prescribed, and case management of malaria test-negative mystery clients. Analysis compared outcomes at facilities and pharmacies independently for the two survey rounds. RESULTS: Across two rounds, 534 and 633 clients (including patients) from 130 and 120 outlets were interviewed, and 214 and 250 mystery client visits were completed. Reported testing by any malaria diagnostic test was higher in private health facilities than registered pharmacies in both rounds (2014: 85.6% vs. 60.8%, p < 0.001; 2015: 85.3% vs. 56.3%, p < 0.001). In registered pharmacies, testing by RDT was 52.1% in 2014 and 56.3% in 2015. At least 75% of test-positive patients received artemisinin-based combination therapy (ACT) in both rounds, with no significant difference between outlet types in either round. Provision of any anti-malarial for test-negative patients ranged from 0 to 13.9% across outlet types and rounds. In 2015, mystery clients received the correct (negative) diagnosis and did not receive an anti-malarial in 75.5% of visits to private health facilities and in 78.4% of visits to registered pharmacies. CONCLUSIONS: Non-laboratory staff working in registered pharmacies in Kenya can follow national guidelines for diagnosis with RDTs when provided with the same level of training and supervision as private health facility staff. Performance and compliance to treatment recommendations are comparable to diagnostic testing outcomes recorded in private health facilities.

Quality issues with malaria rapid diagnostic test accessories and buffer packaging: findings from a 5-country private sector project in Africa.

BACKGROUND: Use of antigen-detecting malaria rapid diagnostic tests (RDTs) has increased exponentially over the last decade. WHO's Global Malaria Programme, FIND, and other collaborators have established a quality assurance scheme to guide product selection, lot verification, transport, storage, and training procedures. Recent concerns over the quality of buffer packaging and test accessories suggest a need to include these items in product assessments. This paper describes quality problems with buffer and accessories encountered in a project promoting private sector RDT use in five African countries and suggests steps to avoid or more rapidly identify and resolve such problems. METHODS: Private provider complaints about RDT buffer vials and kit accessories were collected during supervisory visits, and a standard assessment process was developed. Using 100 tests drawn from six different lots produced by two manufacturers, lab technicians visually assessed alcohol swab packaging, blood transfer device (BTD) usability, and buffer appearance, then calculated mean blood volume from 10 BTD transfers and mean buffer volume from 10 individual buffer vials. WHO guided complaint reporting and follow-up with manufacturers. RESULTS: Supervisory visits confirmed user reports of dry alcohol swabs, poorly functioning BTDs, and non-uniform volumes of buffer. Lot testing revealed further evidence of quality problems, leading one manufacturer to replace buffer vials and accessories for 40,000 RDTs. In December 2014, WHO issued an Information Notice for Users regarding variable buffer volumes in single-use vials and recommended against procurement of these products until defects were addressed. DISCUSSION: Though not necessarily comprehensive or generalizable, the findings presented here highlight the need for extending quality assessment to all malaria RDT test kit contents. Defects such as those described in this paper could reduce test accuracy and increase probability of invalid, false positive, or false negative results. Such deficiencies could undermine provider confidence in RDTs, prompting a return to presumptive treatment or reliance on poor quality microscopy. In partial response to this experience, WHO, FIND, and other project partners have developed guidance on documenting, troubleshooting, reporting, and resolving such problems when they occur.

Impact of introducing subsidized combination treatment with artemether-lumefantrine on sales of anti-malarial monotherapies: a survey of private sector pharmacies in Huambo, Angola.

BACKGROUND: Artemisinin-based combination therapies (ACTs) against malaria are subsidized in many African countries, but the impact of subsidy programs in reducing the sales of concomitantly available antimalarial monotherapies is poorly defined. METHODS: Data from The MENTOR initiative, that introduced subsidized artemether-lumefantrine (sAL) in the private sector of Huambo province, Angola, were used. The main response variable was represented by sales of sAL and of monotherapies, measured as number of treatment courses. Sales in private pharmacies of sAL and four antimalarial monotherapies between 2009 and 2013 were organized in four time-periods, and analyzed using generalized linear models for repeated measures. A secondary analysis evaluated changes in relative market share. RESULTS: We analyzed data from 34 pharmacies at four time points, taken from a larger survey that involved 165 pharmacies between June 2009 and March 2013. The sAL, following its introduction, became the dominant antimalarial treatment in the private sector, usually exceeding the total sales of all antimalarial monotherapies combined (1480/2800 total treatment courses, 52.8% of all sales in March 2013). Sales of monotherapies decreased significantly, but did not stop, representing 36.7% (1028/2800) of sales at the end of the survey. CONCLUSIONS: Subsidized ACTs can attain rapidly a high relative market share. Their introduction reduced, but did not eliminate the demand for less effective monotherapies, that might favor parasite resistance.

Towards subsidized malaria rapid diagnostic tests. Lessons learned from programmes to subsidise artemisinin-based combination therapies in the private sector: a review.

The idea of a private sector subsidy programme of artemisinin-based combination therapies (ACTs) was first proposed in 2004. Since then, several countries around the world have hosted pilot projects or programmes on subsidized ACTs and/or the Affordable Medicines Facility-malaria programme (AMFm). Overall the private sector subsidy programmes of ACTs have been effective in increasing availability of ACTs in the private sector and driving down average prices but struggled to crowd out antimalarial monotherapies. The results obtained from this ambitious strategy should inform policy makers in the designing of future interventions aimed to control malaria morbidity and mortality. Among the interventions recently proposed, a subsidy of rapid diagnostic tests (RDTs) in the private sector has been recommended by governments and international donors to cope with over-treatment with ACTs and to delay the emergence of resistance to artemisinin. In order to improve the cost-effectiveness of co-paid RDTs, we should build on the lessons we learned from almost 10 years of private sector subsidy programmes of ACTs in malaria-endemic countries.

Effectiveness of a prevention of mother-to-child HIV transmission programme in an urban hospital in Angola.

BACKGROUND: Antiretroviral therapy is effective in reducing rates of mother-to child transmission of HIV to low levels in resource-limited contexts but the applicability and efficacy of these programs in the field are scarcely known. In order to explore such issues, we performed a descriptive study on retrospective data from hospital records of HIV-infected pregnant women who accessed in 2007-2010 the Luanda Municipal Hospital service for prevention of mother-to-child transmission (PMTCT). The main outcome measure was infant survival and HIV transmission. Our aim was to evaluate PMTCT programme in a local hospital setting in Africa. RESULTS: Data for 104 pregnancies and 107 infants were analysed. Sixty-eight women (65.4%) had a first visit before or during pregnancy and received combination antiretroviral treatment (ART) in pregnancy. The remaining 36 women (34.6%) presented after delivery and received no ART during pregnancy. Across a median cohort follow-up time of 73 weeks, mortality among women with and without ART in pregnancy was 4.4% and 16.7%, respectively (death hazard ratio: 0.30, 95% CI 0.07-1.20, p = 0.089). The estimated rates of HIV transmission or death in the infants over a median follow up time of 74 weeks were 8.5% with maternal ART during pregnancy and 38.9% without maternal ART during pregnancy. Following adjustment for use of oral zidovudine in the newborn and exposure to maternal milk, no ART in pregnancy remained associated with a 5-fold higher infant risk of HIV transmission or death (adjusted odds ratio: 5.13, 95% CI: 1.31-20.15, p = 0.019). CONCLUSIONS: Among the women and infants adhering to the PMTCT programme, HIV transmission and mortality were low. However, many women presented too late for PMTCT, and about 20% of infants did not complete follow up. This suggests the need of targeted interventions that maintain the access of mothers and infants to prevention and care services for HIV.

Predictors of bacterial meningitis in resource-limited contexts: an Angolan case.

BACKGROUND: Despite the great morbidity and mortality that childhood bacterial meningitis (BM) is experiencing in Africa, diagnosis of BM in resource-limited contexts is still a challenge. Several algorithms and clinical predictors have been proposed to help physicians in decision-making but a lot of these markers used variables that are calculable only in well-equipped laboratories. Predictors or algorithm based on parameters that can be easily performed in basic laboratories can help significantly in BM diagnosis, even in resource-limited settings, rural hospitals or health centers. RESULTS: This retrospective study examined 145 cerebral-spinal fluid (CSF) specimens from children from 2 months to 14 years. CSF specimens were divided into two groups, according to the presence or not of a clinical diagnosis of BM. For each specimen, CSF aspect, CSF white blood cells (WBC) count, CSF glucose and protein concentration were analyzed and statistical analysis were performed. CSF WBC count ≥10/µl is no more a valuable predictor of BM. CSF protein concentration ≥50 mg/dl has a better sensitivity for BM diagnosis and when used with CSF glucose concentration ≤40 mg/dl, can help to diagnose correctly almost all the BM cases. An algorithm including CSF protein concentration, glucose concentration and WBC count has been proposed to rule out BM and to correctly diagnose it. CONCLUSIONS: In resource-limited health centers, the availability of a combination of easy-to-obtain parameters can significantly help physicians in BM diagnosis. The prompt identification of a BM case can be rapid treated or transferred to adequate structures and can modify the outcome in the patient.

FSH-receptor Ala307Thr polymorphism is associated to polycystic ovary syndrome and to a higher responsiveness to exogenous FSH in Italian women.

OBJECTIVE: Herein we analyzed FSH-R polymorphism at position 307 aiming (a) to assess the prevalence of the three allelic variants (Ala307Ala, Ala307Thr and Thr307Thr) in relation to the type of ovary and (b) to clarify if the allelic variant could influence the responsiveness to exogenous FSH. STUDY DESIGN: We prospectively studied a group of 106 Italian women undergoing in vitro fertilization (IVF), among which 40 were subjects with polycystic ovary syndrome (PCOS) and 66 were normo-ovulatory women with a normal ovarian morphology at transvaginal ultrasound. DNA extraction, denaturing high-performance liquid chromatography (dHPLC) and DNA sequencing were used to detect the FSH-R 307 polymorphic genotype and the whole exon 10 was analyzed. RESULTS: The heterozygote variant Ala307Thr was significantly more frequent than the homozygote variants in women with PCOS, whereas in normo-ovulatory women with normal ovary the three allelic variants had a comparable prevalence. Women bearing the Ala307Thr variant showed a higher ovarian responsiveness to exogenous FSH than normo-ovulatory subjects. CONCLUSIONS: The heterozygote FSH-R polymorphism Ala307Thr is significantly more frequent in women with PCOS than in normo-ovulatory subjects and is more frequently associated with a higher ovarian responsiveness to exogenous FSH.

The pentose phosphate pathway plays an essential role in supporting human sperm capacitation.

In swim-up-selected spermatozoa of 38 normozoospermic patients, capacitated spermatozoa exhibited enhanced pentose phosphate pathway (PPP) activity and increased expression of glucose-6-phosphate dehydrogenase (G6PD). The G6PD inhibitor DHEA and the inhibitors of NADPH oxidase apocynin and diphenylene iodonium (DPI) prevented both superoxide generation and capacitation in human spermatozoa, but whereas DPI and DHEA inhibited PPP, apocynin did not influence it, suggesting that PPP activation during capacitation is not a response to increased oxidative stress but exerts a role by supplying reducing equivalents to oxygen.

Ovarian hyper-stimulation syndrome after spontaneous conception.

Ovarian hyperstimulation syndrome (OHSS) is rather frequent (1-5%) in women submitted to superovulation with gonadotropins for in vitro fertilisation (IVF), whereas it is very rare in case of spontaneous ovulation. Spontaneous OHSS (sOHSS) was previously described to be associated to hydatiform mole, multiple conception, hypothyroidism in pregnancy. It may also depend on activating mutations of the FSH receptor (FSHR) gene that cause ovarian hyper-responsiveness to circulating FSH or even cross-responsiveness of FSHR to hormones having a structure similar to FSH, such as hCG or TSH. We report, herein, a case of sOHSS in a woman who conceived spontaneously. We checked the presence of all possible factors that could explain the onset of the syndrome, and we evidenced hypothyroidism and abnormally elevated hCG levels in the second trimester of pregnancy. The thorough molecular biology study of FSHR gene did not detect exonic mutations, but revealed the presence of intronic mutations whose role in the onset of sOHSS is still uncertain.

Mutations and polymorphisms of the FSH receptor (FSHR) gene: clinical implications in female fecundity and molecular biology of FSHR protein and gene.

UNLABELLED: The portion of chromosome 2, including the gene codifying the receptor of FSH (FSHR gene), can display point mutations that cause variations in the amino acid sequence of the receptor protein (FSHR protein). Some of these structural changes affect the receptor functional properties that may be enhanced (activating mutations) or impaired (inactivating mutations). Activating mutations confer to FSHR a higher responsiveness to FSH, making it constitutively active even in the absence of the ligand, or render it able to nonspecifically respond to other tropic hormones (e.g., TSH). Inactivating mutations reduce the receptor's function up to a total block, altering either the formation of the receptor-ligand complex, or FSH signal transduction. FSHR inactivating mutations may cause primary or secondary amenorrhea, infertility, and premature ovarian failure (POF), whereas activating mutations can predispose to ovarian hyperstimulation syndrome (OHSS) as a consequence of exogenous FSH administration, or with a spontaneous onset. Beside point mutations, FSHR gene polymorphisms at specific sites (e.g., codons 307 and 680) may influence FSHR protein responsiveness to exogenous FSH, and finally affect the effectiveness of in vitro fertilization (IVF) treatment as well as the likelihood of developing a severe OHSS as a consequence of superovulation. This review summarizes the current knowledge about the FSHR gene mutations and polymorphisms, illustrating in the first part their clinical consequences for female reproductive function. In the second part, it describes the techniques to study the FSHR gene sequence, and gives more details about the molecular biology of FSHR protein, of FSHR gene and its mutations. TARGET AUDIENCE: Obstetricians & Gynecologists, Family Physicians. LEARNING OBJECTIVES: After reading this article, the reader should be able to list clinical disorders related to mutations in the follicle stimulating hormone receptor (FSHR) gene, explain the principles behind the study of FSHR mutations, and state possible future applications of knowledge of mutations in the FSHR gene in reproductive medicine.

Classical inhibitors of NOX NAD(P)H oxidases are not specific.

NAD(P)H oxidases (NOXs) are a family of enzymes catalyzing the univalent reduction of oxygen to produce the superoxide anion radical, which in turn can be converted in other reactive oxygen species (ROS) and may participate to the formation of reactive nitrogen derivatives, such as peroxynitrite. By virtue of their activity, NOXs may represent a double-edged sword for the organism's homeostasis. On one hand ROS participate in host defence by killing invading microbes and may regulate several important physiological functions, such as cell signalling, regulation of cell growth and differentiation, oxygen sensing, angiogenesis, fertilization and control of vascular tone. On the other hand ROS may play an important role in pathological processes such as hypertension, atherosclerosis, diabetes, cancer, ischemia/reperfusion injury, neurodegenerative diseases. Many roles suggested for NOXs in various tissues and physiopathological situations have been inferred by the in vitro and in vivo effects of several NOX inhibitors. In particular, most studies are based on the use of two compounds, diphenyleneiodonium and apocynin. Aim of this review is to describe the main features of these two compounds, to show that they cannot be used as specific NOX inhibitors and to solicit researchers to find other tools for investigating the role of NOXs.