Expectant management versus IUI in unexplained subfertility and a poor pregnancy prognosis (EXIUI study): a randomized controlled trial.

STUDY QUESTION: For couples with unexplained subfertility and a poor prognosis for natural conception, is 6 months expectant management (EM) inferior to IUI with ovarian stimulation (IUI-OS), in terms of live births? SUMMARY ANSWER: In couples with unexplained subfertility and a poor prognosis for natural conception, 6 months of EM is inferior compared to IUI-OS in terms of live births. WHAT IS KNOWN ALREADY: Couples with unexplained subfertility and a poor prognosis are often treated with IUI-OS. In couples with unexplained subfertility and a relatively good prognosis for natural conception (>30% in 12 months), IUI-OS does not increase the live birth rate as compared to 6 months of EM. However, in couples with a poor prognosis for natural conception (<30% in 12 months), the effectiveness of IUI-OS is uncertain. STUDY DESIGN, SIZE, DURATION: We performed a non-inferiority multicentre randomized controlled trial within the infrastructure of the Dutch Consortium for Healthcare Evaluation and Research in Obstetrics and Gynaecology. We intended to include 1091 couples within 3 years. The couples were allocated in a 1:1 ratio to 6 months EM or 6 months IUI-OS with either clomiphene citrate or gonadotrophins. PARTICIPANTS/MATERIALS, SETTING, METHODS: We studied heterosexual couples with unexplained subfertility and a poor prognosis for natural conception (<30% in 12 months). The primary outcome was ongoing pregnancy leading to a live birth. Non-inferiority would be shown if the lower limit of the one-sided 90% risk difference (RD) CI was less than minus 7% compared to an expected live birth rate of 30% following IUI-OS. We calculated RD, relative risks (RRs) with 90% CI and a corresponding hazard rate for live birth over time based on intention-to-treat and per-protocol (PP) analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Between October 2016 and September 2020, we allocated 92 couples to EM and 86 to IUI-OS. The trial was halted pre-maturely owing to slow inclusion. Mean female age was 34 years, median duration of subfertility was 21 months. Couples allocated to EM had a lower live birth rate than couples allocated to IUI-OS (12/92 (13%) in the EM group versus 28/86 (33%) in the IUI-OS group; RR 0.40 90% CI 0.24 to 0.67). This corresponds to an absolute RD of minus 20%; 90% CI: -30% to -9%. The hazard ratio for live birth over time was 0.36 (95% CI 0.18 to 0.70). In the PP analysis, live births rates were 8 of 70 women (11%) in the EM group versus 26 of 73 women (36%) in the IUI-OS group (RR 0.32, 90% CI 0.18 to 0.59; RD -24%, 90% CI -36% to -13%) in line with inferiority of EM. LIMITATIONS, REASONS FOR CAUTION: Our trial did not reach the planned sample size, therefore the results are limited by the number of participants. WIDER IMPLICATIONS OF THE FINDINGS: This study confirms the results of a previous trial that in couples with unexplained subfertility and a poor prognosis for natural conception, EM is inferior to IUI-OS. STUDY FUNDING/COMPETING INTEREST(S): The trial was supported by a grant of the SEENEZ healthcare initiative. The subsidizing parties were The Dutch Organisation for Health Research and Development (ZonMW 837004023, www.zonmw.nl) and the umbrella organization of 10 health insurers in The Netherlands. E.R.G. receives personal fees from Titus Health care outside the submitted work. M.G. declares unrestricted research and educational grants from Guerbet, Merck and Ferring not related to the presented work, paid to their institution VU medical centre. A.B.H. reports receiving travel and speakers fees from Nordic Pharma and Merck and he is member of the Nordic Pharma ANGEL group and of the Safety Monitoring Board of Womed. C.B.L. reports speakers fee from Inmed and Yingming, and his department receives research grants from Ferring, Merck and Guerbet paid to VU medical centre. B.W.J.M. is supported by a NHMRC Investigator grant (GNT1176437) and reports consultancy for ObsEva and Merck. M.v.W. received a grant from the Netherlands Organisation for Health Research and Development ZonMW (80-8520098-91072). F.M. received two grants from the Netherlands Organisation for Health Research and Development ZonMW (NTR 5599 and NTR 6590). The other authors report no competing interest. TRIAL REGISTRATION NUMBER: Dutch Trial register NL5455 (NTR5599). TRIAL REGISTRATION DATE: 18 December 2015. DATE OF FIRST PATIENT'S ENROLMENT: 26 January 2017.

Ovarian stimulation strategies for intrauterine insemination in couples with unexplained infertility: a systematic review and individual participant data meta-analysis.

BACKGROUND: Intrauterine insemination with ovarian stimulation (IUI-OS) is a first-line treatment for unexplained infertility. Gonadotrophins, letrozole and clomiphene citrate (CC) are commonly used agents during IUI-OS and have been compared in multiple aggregate data meta-analyses, with substantial heterogeneity and no analysis on time-to-event outcomes. Individual participant data meta-analysis (IPD-MA) is considered the gold standard for evidence synthesis as it can offset inadequate reporting of individual studies by obtaining the IPD, and allows analyses on treatment-covariate interactions to identify couples who benefit most from a particular treatment. OBJECTIVE AND RATIONALE: We performed this IPD-MA to compare the effectiveness and safety of ovarian stimulation with gonadotrophins, letrozole and CC and to explore treatment-covariate interactions for important baseline characteristics in couples undergoing IUI. SEARCH METHODS: We searched electronic databases including MEDLINE, EMBASE, CENTRAL, CINAHL, and PsycINFO from their inception to 28 June 2021. We included randomized controlled trials (RCTs) comparing IUI-OS with gonadotrophins, letrozole and CC among couples with unexplained infertility. We contacted the authors of eligible RCTs to share the IPD and established the IUI IPD-MA Collaboration. The primary effectiveness outcome was live birth and the primary safety outcome was multiple pregnancy. Secondary outcomes were other reproductive outcomes, including time to conception leading to live birth. We performed a one-stage random effects IPD-MA. OUTCOMES: Seven of 22 (31.8%) eligible RCTs provided IPD of 2495 couples (62.4% of the 3997 couples participating in 22 RCTs), of which 2411 had unexplained infertility and were included in this IPD-MA. Six RCTs (n = 1511) compared gonadotrophins with CC, and one (n = 900) compared gonadotrophins, letrozole and CC. Moderate-certainty evidence showed that gonadotrophins increased the live birth rate compared to CC (6 RCTs, 2058 women, RR 1.30, 95% CI 1.12-1.51, I2 = 26%). Low-certainty evidence showed that gonadotrophins may also increase the multiple pregnancy rate compared to CC (6 RCTs, 2058 women, RR 2.17, 95% CI 1.33-3.54, I2 = 69%). Heterogeneity on multiple pregnancy could be explained by differences in gonadotrophin starting dose and choice of cancellation criteria. Post-hoc sensitivity analysis on RCTs with a low starting dose of gonadotrophins (≤75 IU) confirmed increased live birth rates compared to CC (5 RCTs, 1457 women, RR 1.26, 95% CI 1.05-1.51), but analysis on only RCTs with stricter cancellation criteria showed inconclusive evidence on live birth (4 RCTs, 1238 women, RR 1.15, 95% CI 0.94-1.41). For multiple pregnancy, both sensitivity analyses showed inconclusive findings between gonadotrophins and CC (RR 0.94, 95% CI 0.45-1.96; RR 0.81, 95% CI 0.32-2.03, respectively). Moderate certainty evidence showed that gonadotrophins reduced the time to conception leading to a live birth when compared to CC (6 RCTs, 2058 women, HR 1.37, 95% CI 1.15-1.63, I2 = 22%). No strong evidence on the treatment-covariate (female age, BMI or primary versus secondary infertility) interactions was found. WIDER IMPLICATIONS: In couples with unexplained infertility undergoing IUI-OS, gonadotrophins increased the chance of a live birth and reduced the time to conception compared to CC, at the cost of a higher multiple pregnancy rate, when not differentiating strategies on cancellation criteria or the starting dose. The treatment effects did not seem to differ in women of different age, BMI or primary versus secondary infertility. In a modern practice where a lower starting dose and stricter cancellation criteria are in place, effectiveness and safety of different agents seem both acceptable, and therefore intervention availability, cost and patients' preferences should factor in the clinical decision-making. As the evidence for comparisons to letrozole is based on one RCT providing IPD, further RCTs comparing letrozole and other interventions for unexplained infertility are needed.

Birthweight and other perinatal outcomes of singletons conceived after assisted reproduction compared to natural conceived singletons in couples with unexplained subfertility: follow-up of two randomized clinical trials.

STUDY QUESTION: Does assisted reproduction, such as ovarian stimulation and/or laboratory procedures, have impact on perinatal outcomes of singleton live births compared to natural conception in couples with unexplained subfertility? SUMMARY ANSWER: Compared to natural conception, singletons born after intrauterine insemination with ovarian stimulation (IUI-OS) had a lower birthweight, while singletons born after IVF had comparable birthweights, in couples with unexplained subfertility. WHAT IS KNOWN ALREADY: Singletons conceived by assisted reproduction have different perinatal outcomes such as low birthweight and a higher risk of premature birth than naturally conceived singletons. This might be due to the assisted reproduction, such as laboratory procedures or the ovarian stimulation, or to an intrinsic factor in couples with subfertility. STUDY DESIGN, SIZE, DURATION: We performed a prospective cohort study using the follow-up data of two randomized clinical trials performed in couples with unexplained subfertility. We evaluated perinatal outcomes of 472 live birth singletons conceived after assisted reproduction or after natural conception within the time horizon of the studies. PARTICIPANTS/MATERIALS, SETTING, METHODS: To assess the possible impact of ovarian stimulation we compared the singletons conceived after IUI with FSH or clomiphene citrate (CC) and IVF in a modified natural cycle (IVF-MNC) or standard IVF with single embryo transfer (IVF-SET) to naturally conceived singletons in the same cohorts. To further look into the possible effect of the laboratory procedures, we put both IUI and IVF groups together into IUI-OS and IVF and compared both to singletons born after natural conception. We only included singletons conceived after fresh embryo transfers. The main outcome was birthweight presented as absolute weight in grams and gestational age- and gender-adjusted percentiles. We calculated differences in birthweight using regression analyses adjusted for maternal age, BMI, smoking, parity, duration of subfertility and child gender. MAIN RESULTS AND THE ROLE OF CHANCE: In total, there were 472 live birth singletons. Of the 472 singleton pregnancies, 209 were conceived after IUI-OS (136 with FSH and 73 with CC as ovarian stimulation), 138 after IVF (50 after IVF-MNC and 88 after IVF-SET) and 125 were conceived naturally.Singletons conceived following IUI-FSH and IUI-CC both had lower birthweights compared to naturally conceived singletons (adjusted difference IUI-FSH -156.3 g, 95% CI -287.9 to -24.7; IUI-CC -160.3 g, 95% CI -316.7 to -3.8). When we compared IVF-MNC and IVF-SET to naturally conceived singletons, no significant difference was found (adjusted difference IVF-MNC 75.8 g, 95% CI -102.0 to 253.7; IVF-SET -10.6 g, 95% CI -159.2 to 138.1). The mean birthweight percentile was only significantly lower in the IUI-FSH group (-7.0 percentile, 95% CI -13.9 to -0.2). The IUI-CC and IVF-SET group had a lower mean percentile and the IVF-MNC group a higher mean percentile, but these groups were not significant different compared to the naturally conceived group (IUI-CC -5.1 percentile, 95% CI -13.3 to 3.0; IVF-MNC 4.4 percentile, 95% CI -4.9 to 13.6; IVF-SET -1.3 percentile, 95% CI -9.1 to 6.4).Looking at the laboratory process that took place, singletons conceived following IUI-OS had lower birthweights than naturally conceived singletons (adjusted difference -157.7 g, 95% CI -277.4 to -38.0). The IVF group had comparable birthweights with the naturally conceived group (adjusted difference 20.9 g, 95% CI -110.8 to 152.6). The mean birthweight percentile was significantly lower in the IUI-OS group compared to the natural group (-6.4 percentile, 95% CI -12.6 to -0.1). The IVF group was comparable (0.7 percentile, 95% CI -6.1 to 7.6). LIMITATIONS, REASONS FOR CAUTION: The results are limited by the number of cases. The data were collected prospectively alongside the randomized controlled trials, but analyzed as treated. WIDER IMPLICATIONS OF THE FINDINGS: Our data suggest IUI in a stimulated cycle may have a negative impact on the birthweight of the child and possibly on pre-eclampsia. Further research should look into the effect of different methods of ovarian stimulation on placenta pathology and pre-eclampsia in couples with unexplained subfertility using naturally conceived singletons in the unexplained population as a reference. STUDY FUNDING/COMPETING INTEREST(S): Both initial trials were supported by a grant from ZonMW, the Dutch Organization for Health Research and Development (INeS 120620027, SUPER 80-83600-98-10192). The INeS study also had a grant from Zorgverzekeraars Nederland, the Dutch association of healthcare insurers (09-003). B.W.J.M. is supported by an NHMRC investigator Grant (GNT1176437) and reports consultancy for ObsEva, Merck Merck KGaA, Guerbet and iGenomix, outside the submitted work. A.H. reports grants from Ferring Pharmaceutical company (the Netherlands), outside the submitted work. F.J.M.B. receives monetary compensation as a member of the external advisory board for Merck Serono (the Netherlands), Ferring Pharmaceutics BV (the Netherlands) and Gedeon Richter (Belgium), he receives personal fees from educational activities for Ferring BV (the Netherlands) and for advisory and consultancy work for Roche and he receives research support grants from Merck Serono and Ferring Pharmaceutics BV, outside the submitted work. The remaining authors have nothing to disclose. TRIAL REGISTRATION NUMBER: INeS study Trial NL915 (NTR939); SUPER Trial NL3895 (NTR4057).

Investigation into the Mechanism of Homo- and Heterodimerization of Angiotensin-Converting Enzyme.

Angiotensin-converting enzyme (ACE) plays a central role in the renin-angiotensin system (RAS), which is primarily responsible for blood pressure homeostasis. Studies have shown that ACE inhibitors yield cardiovascular benefits that cannot be entirely attributed to the inhibition of ACE catalytic activity. It is possible that these benefits are due to interactions between ACE and RAS receptors that mediate the protective arm of the RAS, such as angiotensin II receptor type 2 (AT2R) and the receptor MAS. Therefore, in this study, we investigated the molecular interactions of ACE, including ACE homodimerization and heterodimerization with AT2R and MAS, respectively. Molecular interactions were assessed by fluorescence resonance energy transfer and bimolecular fluorescence complementation in human embryonic kidney 293 cells and Chinese hamster ovary-K1 cells transfected with vectors encoding fluorophore-tagged proteins. The specificity of dimerization was verified by competition experiments using untagged proteins. These techniques were used to study several potential requirements for the germinal isoform of angiotensin-converting enzyme expressed in the testes (tACE) dimerization as well as the effect of ACE inhibitors on both somatic isoforms of angiotensin-converting enzyme expressed in the testes (sACE) and tACE dimerization. We demonstrated constitutive homodimerization of sACE and of both of its domains separately, as well as heterodimerization of both sACE and tACE with AT2R, but not MAS. In addition, we investigated both soluble sACE and the sACE N domain using size-exclusion chromatography-coupled small-angle X-ray scattering and we observed dimers in solution for both forms of the enzyme. Our results suggest that ACE homo- and heterodimerization does occur under physiologic conditions.

Recent advances in targeting coenzyme A biosynthesis and utilization for antimicrobial drug development.

The biosynthesis and utilization of CoA (coenzyme A), the ubiquitous and essential acyl carrier in all organisms, have long been regarded as excellent targets for the development of new antimicrobial drugs. Moreover, bioinformatics and biochemical studies have highlighted significant differences between several of the bacterial enzyme targets and their human counterparts, indicating that selective inhibition of the former should be possible. Over the past decade, a large amount of structural and mechanistic data has been gathered on CoA metabolism and the CoA biosynthetic enzymes, and this has facilitated the discovery and development of several promising candidate antimicrobial agents. These compounds include both target-specific inhibitors, as well as CoA antimetabolite precursors that can reduce CoA levels and interfere with processes that are dependent on this cofactor. In the present mini-review we provide an overview of the most recent of these studies that, taken together, have also provided chemical validation of CoA biosynthesis and utilization as viable targets for antimicrobial drug development.

Turnover-dependent covalent inactivation of Staphylococcus aureus coenzyme A-disulfide reductase by coenzyme A-mimetics: mechanistic and structural insights.

Disruption of the unusual thiol-based redox homeostasis mechanisms in Staphylococcus aureus represents a unique opportunity to identify new metabolic processes and new targets for intervention. Targeting uncommon aspects of CoASH biosynthetic and redox functions in S. aureus, the antibiotic CJ-15,801 has recently been demonstrated to be an antimetabolite of the CoASH biosynthetic pathway in this organism; CoAS-mimetics containing α,ß-unsaturated sulfone and carboxyl moieties have also been exploited as irreversible inhibitors of S. aureus coenzyme A-disulfide reductase (SaCoADR). In this work we have determined the crystal structures of three of these covalent SaCoADR-inhibitor complexes, prepared by inactivation of wild-type enzyme during turnover. The structures reveal the covalent linkage between the active-site Cys43-S(γ) and C(ß) of the vinyl sulfone or carboxyl moiety. The full occupancy of two inhibitor molecules per enzyme dimer, together with kinetic analyses of the wild-type/C43S heterodimer, indicates that half-sites-reactivity is not a factor during normal catalytic turnover. Further, we provide the structures of SaCoADR active-site mutants; in particular, Tyr419'-OH plays dramatic roles in directing intramolecular reduction of the Cys43-SSCoA redox center, in the redox asymmetry observed for the two FAD per dimer in NADPH titrations, and in catalysis. The two conformations observed for the Ser43 side chain in the C43S mutant structure lend support to a conformational switch for Cys43-S(γ) during its catalytic Cys43-SSCoA/Cys43-SH redox cycle. Finally, the structures of the three inhibitor complexes provide a framework for design of more effective inhibitors with therapeutic potential against several major bacterial pathogens.

The antibiotic CJ-15,801 is an antimetabolite that hijacks and then inhibits CoA biosynthesis.

The natural product CJ-15,801 is an inhibitor of Staphylococcus aureus, but not other bacteria. Its close structural resemblance to pantothenic acid, the vitamin precursor of coenzyme A (CoA), and its Michael acceptor moiety suggest that it irreversibly inhibits an enzyme involved in CoA biosynthesis or utilization. However, its mode of action and the basis for its specificity have not been elucidated to date. We demonstrate that CJ-15,801 is transformed by the uniquely selective S. aureus pantothenate kinase, the first CoA biosynthetic enzyme, into a substrate for the next enzyme, phosphopantothenoylcysteine synthetase, which is inhibited through formation of a tight-binding structural mimic of its native reaction intermediate. These findings reveal CJ-15,801 as a vitamin biosynthetic pathway antimetabolite with a mechanism similar to that of the sulfonamide antibiotics and highlight CoA biosynthesis as a viable antimicrobial drug target.