Whole exome sequencing, a hypothesis-free approach to investigate recurrent early miscarriage.

RESEARCH QUESTION: Are there genetic determinants shared by unrelated women with unexplained recurrent early miscarriage (REM)? DESIGN: Thirty REM cases and 30 controls were selected with extreme phenotype among women from Eastern Brittany (France), previously enrolled in an incident case-control study on thrombophilic mutations. Cases and controls were selected based on the number of early miscarriages or live births, respectively. Peripheral blood was collected for DNA extraction at initial visit. The burden of low-frequency variants in the coding part of the genes was compared using whole exome sequencing (WES). RESULTS: Cases had 3 to 17 early miscarriages (20 cases: ≥5 previous losses). Controls had 1 to 4 live births (20 controls: ≥3 previous live births) and no miscarriages. WES data were available for 29 cases and 30 controls. A total of 209,387 variants were found (mean variant per patient: 59,073.05) with no difference between groups (P = 0.68). The top five most significantly associated genes were ABCA4, NFAM1, TCN2, AL078585.1 and EPS15. Previous studies suggest the involvement of vitamin B12 deficiency in REM. TCN2 encodes for vitamin B12 transporter into cells. Therefore, holotranscobalamin (active vitamin B12) was measured for both cases and controls (81.2 ± 32.1 versus 92.9 ± 34.3 pmol/l, respectively, P = 0.186). Five cases but no controls were below 50 pmol/l (P = 0.052). CONCLUSIONS: This study highlights four new genes of interest in REM, some of which belong to known networks of genes involved in embryonic development (clathrin-mediated endocytosis and ciliary pathway). The study also confirms the involvement of TCN2 (vitamin B12 pathway) in the early first trimester of pregnancy.

Hydroxychloroquine may be beneficial in preeclampsia and recurrent miscarriage.

Recurrent miscarriage (RM) and vasculoplacental disorders, such as preeclampsia (PE), affect women of childbearing age worldwide. Vascular endothelial dysfunction and immunological impairment are associated with both RM and PE. To date, there is no effective or optimal therapeutic approach for these conditions. Notably, aspirin use is only partially effective in the prevention of PE. Hydroxychloroquine (HCQ) has demonstrated beneficial effects on disease flares, pregnancy outcomes and cardiovascular impairment in systemic erythaematosus lupus (SLE) through its immunomodulatory, vasculoprotective and antithrombotic properties. Here, in the context of the underlying physiological dysregulation associated with PE and RM, the beneficial properties and potential therapeutic efficacy of HCQ are reviewed in anticipation of the results of current and future trials. Two related trials addressing RM in the absence of maternal autoimmune disease are ongoing. Other trials addressing pregnancy outcomes in the presence of maternal autoimmune disease are forthcoming. In this review, we hypothesise that the immunological and endothelial effects of HCQ may be beneficial in the context of PE and RM, regardless of the maternal autoimmune status.

Blood anti-Müllerian hormone is a possible determinant of recurrent early miscarriage, yet not conclusive in predicting a further miscarriage.

RESEARCH QUESTION: Is blood anti-Müllerian hormone (AMH) concentration a strong determinant of unexplained recurrent early miscarriage (REM)? DESIGN: In the first part of the study, AMH concentrations measured using an Immunotech ELISA Kit were compared between 188 unselected (mostly fertile) women consecutively referred for three or more miscarriages in the first trimester of pregnancy and 376 age-matched parous women without pregnancy loss. Cases and controls were previously enrolled in an incident case-control study on thrombophilic mutations. Blood samples were collected >2 months after any recognized obstetric event or hormonal treatment. In the second part of the study, a prospective 2-year follow-up of cases was performed. RESULTS: When considering all women irrespective of age, AMH concentration did not significantly differ between cases and controls. However, in the subgroup ≥25 years old (176 cases versus 358 controls of ∼33.5 years), the cases had significantly lower AMH concentrations than the controls (median [interquartile range]: 2.8 [1.4-4.7] versus 3.25 [1.7-5.5], P = 0.046) and the proportion of cases with an AMH concentration <1 ng/ml was significantly higher (17.6% versus 10.6%; odds ratio 1.80; 95% confidence interval 1.07-3.00, P = 0.028). With regard to the subsequent pregnancy, AMH concentration was not correlated with either the conception delay or the miscarriage occurrence. However, increased age and number of previous miscarriages were significantly predictive of a subsequent miscarriage (P = 0.046 and 0.03, respectively). CONCLUSION: An altered ovarian reserve is a possible determinant of unexplained REM. However, AMH blood concentration predicts neither the delay nor the outcome of a subsequent pregnancy.

Clinical Characteristics of Pruritus in Systemic Sclerosis Vary According to the Autoimmune Subtype.

Pruritus is a frequent symptom in systemic sclerosis (SSc), with a prevalence of 40-65%, but its pathophysiology is poorly understood. This study investigated the immunological component of pruritus. Fifty-six patients with SSc responded to a standardized questionnaire regarding both SSc disease and pruritus characteristics. Among patients with SSc, those with pruritus did not display a particular immunological profile (inflammatory, humoral, and/or cellular factors), but pruritus was, in most cases, concomitant with the development of SSc. Thus, pruritus characteristics were evaluated further, according to the detection of anti-centromere autoantibodies (ACA), into ACA+ (n = 17) and ACA- (n = 19). The ACA+ subgroup was characterized by a longer evolution of SSc and pruritus, pruritus present outside the sclerotic area, and a shorter daily duration of pruritus. In conclusion, the concomitant appearance of the 2 processes and the differences observed between ACA+ and ACA- subgroups support the presence of an immunological component in pruritus.

Enoxaparin for prevention of unexplained recurrent miscarriage: a multicenter randomized double-blind placebo-controlled trial.

It is common practice in many centers to offer antithrombotic medications to women with unexplained recurrent miscarriage, in the presence or absence of inherited thrombophilia. Although no benefit of aspirin vs placebo has been clearly demonstrated, a double-blind placebo-controlled trial on the effect of low-molecular-weight heparin is lacking. We enrolled 258 pregnant women with a history of unexplained recurrent miscarriage (≥2 consecutive miscarriages before 15 weeks' gestation) and a negative thrombophilia workup. They were randomly assigned to receive one daily subcutaneous injection of enoxaparin 40 mg or placebo until 35 weeks' gestation. We included 256 women (mean age 32 years, ≥3 miscarriages: 72%; mean gestational age 39 days of amenorrhea) in the intention-to-treat analysis; 66.6% of 138 who received enoxaparin had a live birth vs 72.9% of 118 who received placebo. The absolute difference was -6% (95% CI, -17.1 to 5.1), excluding a 10% increase in the rate of live-birth on enoxaparin (P = .34). In this first randomized, double-blind, placebo-controlled trial, enoxaparin (40 mg once daily) did not improve the chance of a live birth in nonthrombophilic women with unexplained recurrent miscarriage. This trial is registered at www.ClinicalTrials.gov as #NCT00740545 and the French National Health and Drug Safety Agency (EudraCT #2006-003350-18).

History of pre-eclampsia does not appear to be a risk factor for vascular phenotype in women with systemic sclerosis.

BACKGROUND: Vascular phenotype is associated with a poor prognosis in systemic sclerosis (SSc). The identification of its risk factors could facilitate its early detection. OBJECTIVES: To explore risk factors for a vascular phenotype of SSc, among them a history of pre-eclampsia. METHODS: This observational multicentre case-control study enrolled adult women fulfilling European Alliance of Associations for Rheumatology 2013 diagnosis criteria for SSc and having a pregnancy history≥6 months before SSc diagnosis in 14 French hospital-based recruiting centres from July 2020 to July 2022. Cases had specific vascular complications of SSc defined as history of digital ischaemic ulcers, pulmonary arterial hypertension, specific cardiac involvement or renal crisis. Women with SSc were included during their annual follow-up visit and filled in a self-administered questionnaire about pregnancy. A case report form was completed by their physician, reporting data on medical history, physical examination, clinical investigations and current medication. The main outcome was the presence/absence of a personal history of pre-eclampsia before SSc diagnosis, according to the validated pre-eclampsia questionnaire. RESULTS: 378 women were included: 129 cases with a vascular phenotype and 249 matched controls. A history of pre-eclampsia was reported in 5 (3.9%) cases and 12 (4.8%) controls and was not associated with a vascular phenotype (OR=0.96, 95% CI 0.28 to 3.34, p=0.9). Besides, Rodnan skin score and disease duration≥5 years were risk factors for vascular phenotype. CONCLUSIONS: In women with SSc and a pregnancy history≥6 months before SSc, a history of pre-eclampsia is not associated with a vascular phenotype.

Characterisation of a high-risk profile for maternal thrombotic and severe haemorrhagic complications in pregnant women with antiphospholipid syndrome in France (GR2): a multicentre, prospective, observational study.

BACKGROUND: Prospective data about the risks of thrombotic and severe haemorrhagic complications during pregnancy and post partum are unavailable for women with antiphospholipid syndrome. We aimed to assess thrombotic and haemorrhagic events in a prospective cohort of pregnant women with antiphospholipid syndrome. METHODS: This multicentre, prospective, observational study was done at 76 centres in France. To be eligible for this study, women had to have diagnosis of antiphospholipid syndrome; have conceived before April 17, 2020; have an ongoing pregnancy that had reached 12 weeks of gestation; and be included in the study before 18 weeks of gestation. Exclusion criteria were active systemic lupus erythematosus nephropathy, or a multifetal pregnancy. Severe haemorrhage was defined as the need for red blood cell transfusion or maternal intensive care unit admission because of bleeding or invasive procedures, defined as interventional radiology or surgery, to control bleeding. The GR2 study is registered with ClinicalTrials.gov, NCT02450396. FINDINGS: Between May 26, 2014, and April 17, 2020, 168 pregnancies in 27 centres met the inclusion criteria for the study. 89 (53%) of 168 women had a history of thrombosis. The median term at inclusion was 8 weeks gestation. 16 (10%) of 168 women (95%CI 5-15) had a thrombotic (six [4%] women; 95% CI 1-8) or severe haemorrhagic event (12 [7%] women; 95% CI 4-12). There were no deaths during the study. The main risk factors for thrombotic events were lupus anticoagulant positivity at inclusion (six [100%] of six women with thrombosis vs 78 [51%] of 152 of those with no thrombosis; p=0·030) and placental insufficiency (four [67%] of six women vs 28 [17%] of 162 women; p=0·013). The main risk factors for severe haemorrhagic events were pre-existing maternal hypertension (four [33%] of 12 women vs 11 [7%] of 156 women; p=0·014), lupus anticoagulant positivity at inclusion (12 [100%] of 12 women vs 72 [49%] of 146 women; p<0·0001) and during antiphospholipid history (12 [100%] of 12 women vs 104 [67%] of 156 women; p=0·019), triple antiphospholipid antibody positivity (eight [67%] of 12 women vs 36 [24%] of 147 women; p=0·0040), placental insufficiency (five [42%] of 12 women vs 27 [17%] of 156 women; p=0·038), and preterm delivery at 34 weeks or earlier (five [45%] of 11 women vs 12 [8%] of 145 women; p=0·0030). INTERPRETATION: Despite treatment adhering to international recommendations, a proportion of women with antiphospholipid syndrome developed a thrombotic or severe haemorrhagic complication related to pregnancy, most frequently in the post-partum period. Lupus anticoagulant and placental insufficiency were risk factors for these life-threatening complications. These complications are difficult to prevent, but knowledge of the antenatal characteristics associated with them should increase awareness and help physicians manage these high-risk pregnancies. FUNDING: Lupus France, association des Sclérodermiques de France, association Gougerot Sjögren, Association Francophone contre la Polychondrite chronique atrophiante, AFM-Telethon, the French Society of Internal Medicine and Rheumatology, Cochin Hospital, the French Health Ministry, FOREUM, the Association Prix Veronique Roualet, and UCB.

Hydroxychloroquine for prevention of recurrent miscarriage: study protocol for a multicentre randomised placebo-controlled trial BBQ study.

INTRODUCTION: Recurrent miscarriage (RM), defined by three or more consecutive losses during the first trimester of pregnancy, affects 1%-2% of fertile couples. Standard investigations fail to reveal any apparent cause in ~50% of couples. However, on the basis of animal models and clinical studies, several hypotheses have been put forward concerning underlying mechanisms of RM: altered ovarian reserve, progesterone defect, thrombotic and/or endothelial dysfunction and immunological disturbances. Nonetheless, no study has yet reached conclusive beneficial clinical evidence for a potential treatment in unexplained RM. Hydroxychloroquine (HCQ) is a molecule with extensive safety data during pregnancy. The pharmacological properties of HCQ (eg, antithrombotic, vascular protective, immunomodulatory, improved glucose tolerance, lipidlowering and anti-infectious) could be effective against some mechanisms of unexplained RM. Furthermore, eventhough clinical benefit of HCQ is suggested in prevention of thrombotic and late obstetric events in antiphospholipid (APL) syndrome, there are no data suggesting the benefit of HCQ in RM in the presence of APL antibodies. METHODS AND ANALYSIS: Taken all together and given the low cost of HCQ, the aim of this multicentre, randomised, placebo-controlled, double-blind study is to investigate whether HCQ would improve the live birth rate in women with RM, irrespective of maternal thrombophilic status: (1) no known thrombophilia, (2) inherited thrombophilia or (3) APL antibodies. The primary end point is a live and viable birth. After confirming eligibility and obtaining consent, 300 non-pregnant women will be randomised into two parallel groups for a daily oral treatment (HCQ 400 mg or placebo), initiated before conception and stopped at 10 weeks' gestation. If pregnancy does not occur after 1 year, the treatment will be stopped. ETHICS AND DISSEMINATION: Agreement from the French National Public Health and Drug Security Agency (160765A-22) and ethical approval from the Committee for the Protection of Persons of NORD-OUEST I (2016-001330-97) have been obtained. TRIAL REGISTRATION NUMBERS: NCT0316513; Pre-results.

Minisci-Photoredox-Mediated α-Heteroarylation of N-Protected Secondary Amines: Remarkable Selectivity of Azetidines.

The development of a general, mild, and functional-group-tolerant direct functionalization of N-heteroarenes by C-H functionalization with N-protected amines, including azetidines under Minisci-mediated photoredox conditions, is reported. A broad scope of substituted azetidines, including spirocyclic derivatives, and heterocycles were explored. This reaction enables the production of sp3-rich complex druglike structures in one step from unactivated feedstock amines and heterocycles.

Estrogen receptor alpha polymorphism and venous thromboembolism in male and female: data from the EDITH study.

BACKGROUND: Association between estrogen receptor (ER) alpha polymorphism c.454-397 T>C and venous thromboembolism (VTE) has been reported in postmenopausal women. Comprehensive data are lacking. We herein evaluated the risk for VTE related to c.454-397 T>C ER alpha gene polymorphism in both men and women. PATIENTS/METHODS: The EDITH Study enrolled consecutive patients, aged over 18 years, hospitalized between May 2000 and December 2004 in Brest University Hospital with an objectively proven symptomatic VTE. For each case, one control who matched the case for age within a five-year age band, gender and major acquired risk factors, was selected. The present analysis was restricted to 677 cases with a VTE event not related to major acquired risk factors and their matched controls. RESULTS AND CONCLUSIONS: The relationship between VTE and c.454-397 T>C ER alpha polymorphism was consistent with a dominant model in women and a recessive model in men. Adjusted conditional odds ratios (95% CI) were 1.37 (1.05-1.78) and 1.29 (0.85-1.94) for CT/CC genotypes in women and CC genotype in men respectively compared to TT genotype. Among women hormone use did not substantially modify effect-measure estimate. Our results further extend results from previous studies. Other investigations are required to precise underlying mechanisms.

Synthesis of novel diarylpyrimidine analogues of TMC278 and their antiviral activity against HIV-1 wild-type and mutant strains.

Novel diarylpyrimidines (DAPY), which represent next generation of non-nucleoside reverse transcriptase inhibitors (NNRTIs), were synthesized and their activities against human immunodeficiency virus type I (HIV-1) assessed. Modulations at positions 2 and 6 of the left phenyl ring generated interesting derivatives of TMC278 displaying high potency against wild-type and mutant viruses compared to nevirapine and efavirenz. The pharmacokinetic profile of the best newly synthesized DAPY was evaluated and compared with TMC278 now in phase II clinical trials.

Lymphocyte Disturbances in Primary Antiphospholipid Syndrome and Application to Venous Thromboembolism Follow-Up.

Among patients with venous thromboembolism (VTE), the persistent detection of antiphospholipid (aPL) antibodies (Ab) represents an independent high risk factor for recurrence. However, oral anticoagulation vitamin K antagonist therapy, frequently used in these patients, is problematic in assessing and/or confirming a diagnosis of primary aPL syndrome (pAPS), suggesting use of alternative strategies. For this reason, and by analogy with other autoimmune diseases, a flow cytometer approach testing peripheral T cell subsets (CD3, CD4, and CD8), B cell subsets (B1, transitional, naive, and memory), and NK cells can be proposed. As an example and to validate the concept, pAPS patients selected from the monocentric VTE case-control EDITH's cohort were selected during their follow-up. As suspected and in contrast to non-APS VTE patients, other autoimmune diseases, and controls, pAPS VTE patients displayed specific lymphocyte disturbances. Quantitative and qualitative modifications were related to total CD4+ T cell reduction, a lower CD4/CD8 ratio, and disturbance in B cell homeostasis with increased proportions of B1 cells, transitional B cells (CD24++CD38++), and naive B cells (IgD+CD27-), while memory B cells (IgD+CD27+ and IgD-CD27+) were reduced. Interestingly, the absolute number of CD4+ T cells positively correlated with IgG anti-cardiolipin Ab levels. Altogether, disturbances of T and B cell homeostasis characterized pAPS VTE patients during their follow-up. This suggests a means of profiling that could be used in addition to existing criteria to characterize them.

Premature atherosclerosis in HIV positive patients and cumulated time of exposure to antiretroviral therapy (SHIVA study).

BACKGROUND: With the advent of antiretroviral therapy regimens in HIV positive patients, it is crucial to consider their long-term benefits to risk ratios. The responsibility of treatment in premature atherosclerosis is not clear. Thus, the aim of this study is to evaluate the impact of exposure to reverse transcriptase inhibitors (nucleosidic and non-nucleosidic) and to protease inhibitors on the cardiovascular status of an entire hospital based cohort of patients. METHODS: 154 patients were included. Using a linear analysis, we sought an association between the cumulative time of exposure to these three classes of antiretroviral drugs and the carotid intima-media thickness measured by ultrasonography and a cardiovascular composite score. RESULTS: The study confirms premature atherosclerosis, which not only correlates with the usual risk factors, such as triglyceride level, but also with protease inhibitor exposure, especially that of lopinavir. Nevertheless as regards current drug exposure, the clinical impact was low: five clinical complications of atherosclerosis and only one out of 35 scintigraphic and ECG exercise tests warranted a coronary angiography which was negative. CONCLUSION: These data should not lead to the rejection of protease inhibitors but should strengthen the prevention of cardiovascular diseases as an integral part of the management of HIV patients.

Asymptomatic carotid occlusion in a young HIV-infected patient receiving HAART.

The discovery of an asymptomatic and spontaneous internal carotid occlusion in a young HIV-infected patient, without atherosclerosis, asks the question of cardio-vascular disease's mechanism. A pro-atherogenic profile HAART-associated does not fully explain the high cardio-vascular disease's incidence among the HIV infected population. Carotid stenosis and/or thrombophilic conditions are emergent problems among HIV-infected persons.

[Bosentan for treatment of active digital ulcers in patients with systemic sclerosis]. / Le bosentan dans le traitement des ulcères digitaux évolutifs au cours de la sclérodermie systémique (9 cas).

OBJECTIVES: To describe the effect of bosentan and its dual inhibition of endothelin-1 ETA and ETB receptors on digital ulcers in patients with systemic sclerosis (SSc). METHODS: Patients receiving bosentan for SSc-related digital ulcers were identified in eight centers, and their characteristics and follow-up were recorded. RESULTS: Nine (six with diffuse and three with limited cutaneous forms of SSc) patients (median age: 54 years) had received bosentan for digital ulcers. Complete healing occurred in seven (median time to improvement: 4 weeks). Another experienced a significant decrease in the number of ulcers (from 22 to 5) in 8 weeks, while one had no improvement. After a median follow-up of 24.3 months, only one recurrence was observed. Raynaud phenomenon improved in all but one patient. DISCUSSION: These data suggest that some patients may benefit from bosentan to treat digital ulcers. The short time to healing in these patients with rather chronic ulcers argues strongly in favor of its use. These results also strengthen the evidence that endothelin-1 plays an important role in the vascular manifestations of SSc. CONCLUSION: Bosentan can be effective in the treatment of digital ulcers in some SSc patients with SSc, probably especially those involving substantial ischemia. Bosentan is not a first-line drug in this indication yet and must be carefully used by specialists in SSc. Forthcoming results from the international RAPIDS-2 study should clarify the indications for bosentan in the treatment of SSc-related digital ulcers.

In search of a novel anti-HIV drug: multidisciplinary coordination in the discovery of 4-[[4-[[4-[(1E)-2-cyanoethenyl]-2,6-dimethylphenyl]amino]-2- pyrimidinyl]amino]benzonitrile (R278474, rilpivirine).

Ideally, an anti-HIV drug should (1) be highly active against wild-type and mutant HIV without allowing breakthrough; (2) have high oral bioavailability and long elimination half-life, allowing once-daily oral treatment at low doses; (3) have minimal adverse effects; and (4) be easy to synthesize and formulate. R278474, a new diarylpyrimidine (DAPY) non-nucleoside reverse transcriptase inhibitor (NNRTI), appears to meet these criteria and to be suitable for high compliance oral treatment of HIV-1 infection. The discovery of R278474 was the result of a coordinated multidisciplinary effort involving medicinal chemists, virologists, crystallographers, molecular modelers, toxicologists, analytical chemists, pharmacists, and many others.

Synthesis of novel diarylpyrimidine analogues and their antiviral activity against human immunodeficiency virus type 1.

This paper reports the synthesis and the antiviral properties of new diarylpyrimidine (DAPY) compounds as nonnucleoside reverse transcriptase inhibitors (NNRTIs). The synthesis program around this new DAPY series was further optimized to produce compounds displaying improved activity against a panel of eight clinically relevant single and double mutant strains of human immunodeficiency virus type 1 (HIV-1).