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STUDY QUESTIONWhat are the long-term outcomes after allocation to use of gonadotrophins versus clomiphene citrate (CC) with or without IUI in women with normogonadotropic anovulation and clomiphene failure?SUMMARY ANSWERAbout four in five women with normogonadotropic anovulation and CC failure had a live birth, with no evidence of a difference in pregnancy outcomes between the allocated groups.WHAT IS KNOWN ALREADYCC has long been used as first line treatment for ovulation induction in women with normogonadotropic anovulation. Between 2009 and 2015, a two-by-two factorial multicentre randomized clinical trial in 666 women with normogonadotropic anovulation and six cycles of CC failure was performed (M-ovin trial). This study compared a switch to gonadotrophins with continued treatment with CC for another six cycles, with or without IUI within 8 months. Switching to gonadotrophins increased the chance of conception leading to live birth by 11% over continued treatment with CC after six failed ovulatory cycles, at a cost of €15 258 per additional live birth. The addition of IUI did not significantly increase live birth rates.STUDY DESIGN, SIZE, DURATIONIn order to investigate the long-term outcomes of switching to gonadotrophins versus continuing treatment with CC, and undergoing IUI versus continuing with intercourse, we conducted a follow-up study. The study population comprised all women who participated in the M-ovin trial.PARTICIPANTS/MATERIALS, SETTING, METHODSThe participating women were asked to complete a web-based questionnaire. The primary outcome of this study was cumulative live birth. Secondary outcomes included clinical pregnancies, multiple pregnancies, miscarriage, stillbirth, ectopic pregnancy, fertility treatments, neonatal outcomes and pregnancy complications.MAIN RESULTS AND THE ROLE OF CHANCEWe approached 564 women (85%), of whom 374 (66%) responded (184 allocated to gonadotrophins; 190 to CC). After a median follow-up time of 8 years, 154 women in the gonadotrophin group had a live birth (83.7%) versus 150 women in the CC group (78.9%) (relative risk (RR) 1.06, 95% CI 0.96–1.17). A second live birth occurred in 85 of 184 women (49.0%) in the gonadotrophin group and in 85 of 190 women (44.7%) in the CC group (RR 1.03, 95% CI 0.83–1.29). Women allocated to gonadotrophins had a third live birth in 6 of 184 women (3.3%) and women allocated to CC had a third live birth in 14 of 190 women (7.4%). There were respectively 12 and 11 twins in the gonadotrophin and CC groups. The use of fertility treatments in the follow-up period was comparable between both groups. In the IUI group, a first live birth occurred in 158 of 192 women (82.3%) and while in the intercourse group, 146 of 182 women (80.2%) reached at least one live birth (RR: 1.03 95% CI 0.93–1.13; 2.13%, 95% CI −5.95, 10.21).LIMITATIONS, REASONS FOR CAUTIONWe have complete follow-up results for 57% of the women.There were 185 women who did not respond to the questionnaire, while 102 women had not been approached due to missing contact details. Five women had not started the original trial.WIDER IMPLICATIONS OF THE FINDINGSWomen with normogonadotropic anovulation and CC failure have a high chance of reaching at least one live birth. In terms of pregnancy rates, the long-term differences between initially switching to gonadotrophins are small compared to continuing treatment with CC.STUDY FUNDING/COMPETING INTEREST(S)The original study received funding from the Dutch Organization for Health Research and Development (ZonMw number: 80-82310-97-12067). A.H. reports consultancy for development and implementation of a lifestyle App, MyFertiCoach, developed by Ferring Pharmaceutical Company. M.G. receives unrestricted grants for scientific research and education from Ferring, Merck and Guerbet. B.W.M. is supported by an NHMRC Investigatorgrant (GNT1176437). B.W.M. reports consultancy for ObsEva and Merck and travel support from Merck. All other authors have nothing to declare.TRIAL REGISTRATION NUMBERThis follow-up study was registered in the OSF Register, https://osf.io/pf24m. The original M-ovin trial was registered in the Netherlands Trial Register, number NTR1449.

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)

Increasing evidence links changes in epigenetic systems, such as DNA methylation, histone modification, and non-coding RNA expression, to the occurrence of cardiovascular disease (CVD). These epigenetic modifications can change genetic function under influence of exogenous stimuli and can be transferred to next generations, providing a potential mechanism for inheritance of behavioural intervention effects. The benefits of exercise and nutritional interventions in the primary and secondary prevention of CVD are well established, but the mechanisms are not completely understood. In this review, we describe the acute and chronic epigenetic effects of physical activity and dietary changes. We propose exercise and nutrition as potential triggers of epigenetic signals, promoting the reshaping of transcriptional programmes with effects on CVD phenotypes. Finally, we highlight recent developments in epigenetic therapeutics with implications for primary and secondary CVD prevention., European Journal of Preventive Cardiology, 29 (17), ISSN:2047-4873, ISSN:2047-4881

BACKGROUND: Observational studies suggest that high dairy intake is associated with a lower blood pressure (BP).OBJECTIVE: We aimed to investigate the effect of a high-dairy diet (HDD) as compared with a low-dairy diet (LDD) on BP in overweight middle-aged adults.METHODS: Fifty-two overweight men and women were included in a randomized crossover intervention study. Each subject consumed 2 isocaloric diets for 6 wk, an LDD (≤1 dairy portion per day) and an HDD (6 or 5 reduced-fat dairy portions for men and women, respectively), with a 4-wk washout period in between the diets during which the subjects consumed their habitual diet. BP was measured at the start and at the end of the intervention diets. The effect of the intervention study was evaluated by 2-sample t tests. Mixed-model analyses were used for adjustment for the potential influence of changes in dietary protein and mineral intake and risk factors for hypertension including body weight and plasma cholesterol.RESULTS: Consumption of an HDD as compared with an LDD resulted in a reduction of both systolic BP (mean ± SD: 4.6 ± 11.2 mm Hg, P < 0.01) and diastolic BP (3.0 ± 6.7 mm Hg, P < 0.01). In further analyses, these reductions appeared dependent on the concomitant increase in calcium intake.CONCLUSIONS: This intervention study shows that an HDD results in a reduction of both systolic and diastolic BP in overweight middle-aged men and women. If the results of our study are reproduced by other studies, advice for high dairy intake may be added to treatment and prevention of high BP. This trial was registered at trialregister.nl as NTR4899.

Background: The global prevalence of obesity in women keeps increasing. The preconception period may be a window of opportunity to improve lifestyle, reduce obesity and improve cardiometabolic health. This study assessed the effect of a preconception lifestyle intervention on long-term cardiometabolic health in two randomized controlled trials (RCTs).Methods: Participants of the LIFEstyle and RADIEL preconception lifestyle intervention studies with a baseline body mass index (BMI) ≥29 kg/m2 were eligible for this follow-up study. Both studies randomized between a lifestyle intervention targeting physical activity, diet and behaviour modification or usual care. We assessed cardiometabolic health 6 years after randomization.Results: In the LIFEstyle study (n = 111) and RADIEL study (n = 39), no statistically significant differences between the intervention and control groups were found for body composition, blood pressure, arterial stiffness, fasting glucose, homeostasis model assessment of insulin resistance, HbA1c, lipids and high sensitive C-reactive protein levels 6 years after randomization. Participants of the LIFEstyle study who successfully lost ≥5% bodyweight or reached a BMI Conclusion: We found no evidence of improved cardiometabolic health 6 years after a preconception lifestyle intervention among overweight and obese women in two RCTs. Women who successfully lost weight during the intervention had better cardiometabolic health 6 years later, emphasizing the potential of successful preconception lifestyle improvement.

STUDY QUESTION: Does Day-3 cleavage-stage PGS affect neurodevelopment of 9-year-old IVF offspring?SUMMARY ANSWER: We did not find evidence of adverse consequences of Day-3 cleavage-stage PGS on neurodevelopment of 9-year-old IVF offspring, although children born after IVF with or without PGS often had a non-optimal neurological condition.WHAT IS KNOWN ALREADY: Knowledge on long-term sequelae for development and health of children born following PGS is lacking. This is striking as evidence accumulates that IVF itself is associated with increased risk for impaired health and development in the offspring.STUDY DESIGN SIZE, DURATION: This prospective, assessor-blinded, multicentre, follow-up study evaluated development and health of 9-year-old IVF children born to women who were randomly assigned to IVF with PGS (PGS group) or without PGS (control group). The follow-up examination at 9 years took place between March 2014 and May 2016.PARTICIPANTS/MATERIALS, SETTING, METHODS: In total, 408 women were included and randomly assigned to IVF with or without Day-3 cleavage-stage PGS. This resulted in 52 ongoing pregnancies in the PGS group and 74 in the control group. In the PGS group, 59 children were born alive; in the control group, 85 children were born alive. At the age of 9 years, 43 children born after PGS and 56 control children participated in the study. Our primary outcome was the neurological optimality score, a sensitive measure of neurological condition assessed with a standardized, age-specific test (Touwen test). Secondary outcomes were adverse neurological condition (neurologically abnormal and the complex form of minor neurological dysfunction), cognitive development (intelligence quotient and specific domains), behaviour (parental and teacher's questionnaires), blood pressure and anthropometrics.MAIN RESULTS AND THE ROLE OF CHANCE: Neurodevelopmental outcome of PGS children did not differ from that of controls; the neurological optimality scores (mean values [(95% CI]: PGS children 51.5 [49.3; 53.7], control children 53.1 [50.5; 55.7]) were not significantly different. The prevalences of adverse neurological outcome (in all but one child implying the presence of the complex form of minor neurological dysfunction) did not differ between the groups (PGS group 17/43 [40%], control group 19/56 [34%]), although the prevalence of complex minor neurological dysfunction in both groups was rather high. Also intelligence quotient scores of the two groups were not significantly different (PGS group 114 [108; 120]); control group 117 [109; 125]), and the behaviour, blood pressure and anthropometrics of both groups did not differ. Mean blood pressures of both groups were above the 60th percentile.LIMITATIONS REASONS FOR CAUTION: The power analysis of the study was not based on the number of children needed for the follow-up study, but on the number of women who were needed to detect an increase in ongoing pregnancy rates after PGS. In addition, our study evaluated embryo biopsy in the form of PGS at cleavage stage (Day-3 embryo biopsy), while currently PGS at blastocyst stage (Day-5 embryo biopsy) is recommended and increasingly being used.WIDER IMPLICATIONS OF THE FINDINGS: Our findings indicate that PGS in cleavage stage embryos is not associated with adverse effects on neurological, cognitive and behavioural development, blood pressure and anthropometrics of offspring at 9 years. This is a reassuring finding as embryo biopsy in the forms of PGS and PGD is increasingly applied. However, both groups of IVF offspring showed high prevalences of the clinically relevant form of minor neurological dysfunction, which is a point of concern for the IVF community. In addition, our study confirms findings of others that IVF offspring may be at risk of an unfavourable cardiovascular outcome. These findings are alarming and highlight the importance of research on the underlying mechanisms of unfavourable neurodevelopmental and cardiovascular outcomes in IVF offspring.STUDY FUNDING/COMPETING INTEREST(S): The randomized controlled trial was financially supported by the Organization for Health Research and Development (ZonMw), The Netherlands (Grant number 945-03-013). The follow-up was financially supported by the University Medical Center Groningen (Grant number: 754510), the Cornelia Foundation, and the graduate schools BCN and Share, Groningen, The Netherlands. The sponsors of the study had no role in study design, data collection, data analysis, data interpretation or writing of the report. There are no conflicts of interest.TRIAL REGISTRATION NUMBER: ISRCTN76355836.

STUDY QUESTION: Does an increased FSH dose result in higher cumulative live birth rates in women with a predicted poor ovarian response, apparent from a low antral follicle count (AFC), scheduled for IVF or ICSI? SUMMARY ANSWER: In women with a predicted poor ovarian response (AFC < 11) undergoing IVF/ICSI, an increased FSH dose (225/ 450 IU/day) does not improve cumulative live birth rates as compared to a standard dose (150 IU/day). WHAT IS KNOWN ALREADY: In women scheduled for IVF/ICSI, an ovarian reserve test (ORT) can predict ovarian response to stimulation. The FSH starting dose is often adjusted based on the ORT from the belief that it will improve live birth rates. However, the existing RCTs on this topic, most of which show no benefit, are underpowered. STUDY DESIGN, SIZE, DURATION: Between May 2011 and May 2014, we performed an open-label multicentre RCT in women with an AFC < 11 (Dutch Trial Register NTR2657). The primary outcome was ongoing pregnancy achieved within 18 months after randomization and resulting in a live birth. We needed 300 women to assess whether an increased dose strategy would increase the cumulative live birth rate from 25 to 40% (two-sided alpha-error 0.05, power 80%). PARTICIPANTS/MATERIALS, SETTING, METHODS: Women with an AFC ≤ 7 were randomized to an FSH dose of 450 IU/day or 150 IU/day, and women with an AFC 8–10 were randomized to 225 IU or 150 IU/day. In the standard group, dose adjustment was allowed in subsequent cycles based on pre-specified criteria. Both effectiveness and cost-effectiveness of the strategies were evaluated from an intention-to-treat perspective. MAIN RESULTS AND THE ROLE OF CHANCE: In total, 511 women were randomized, 234 with an AFC ≤ 7 and 277 with an AFC 8–10. The cumulative live birth rate for increased versus standard dosing was 42.4% (106/250) versus 44.8% (117/261), respectively [relative risk (RR): 0.95 (95%CI, 0.78–1.15), P = 0.58]. As an increased dose strategy was more expensive [delta costs/woman: €1099 (95%CI, 562–1591)], standard FSH dosing was the dominant strategy in our economic analysis. LIMITATIONS, REASONS FOR CAUTION: Despite our training programme, the AFC might have suffered from inter-observer variation. As this open study permitted small dose adjustments between cycles, potential selective cancelling of cycles in women treated with 150 IU could have influenced the cumulative results. However, since first cycle live birth rates point in the same direction we consider it unlikely that the open design masked a potential benefit for the individualized strategy. WIDER IMPLICATIONS OF THE FINDINGS: Since an increased dose in women scheduled for IVF/ICSI with a predicted poor response (AFC < 11) does not improve live birth rates and is more expensive, we recommend using a standard dose of 150 IU/day in these women. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by The Netherlands Organisation for Health Research and Development (ZonMW number 171102020). T.C.T., H.L.T. and S.C.O. received an unrestricted personal grant from Merck BV. H.R.V. receives monetary compensation as a member on an external advisory board for Ferring pharmaceutical BV. B.W.J.M. is supported by a NHMRC Practitioner Fellowship (GNT1082548) and reports consultancy for OvsEva, Merck and Guerbet. F.J.M.B. receives monetary compensation as a member of the external advisory board for Ferring pharmaceutics BV (the Netherlands) and Merck Serono (the Netherlands) for consultancy work for Gedeon Richter (Belgium) and Roche Diagnostics on automated AMH assay development (Switzerland) and for a research cooperation with Ansh Labs (USA). All other authors have nothing to declare.

STUDY QUESTION: Does a reduced FSH dose in women with a predicted hyper response, apparent from a high antral follicle count (AFC), who are scheduled for IVF/ICSI lead to a different outcome with respect to cumulative live birth rate and safety?SUMMARY ANSWER: Although in women with a predicted hyper response (AFC > 15) undergoing IVF/ICSI a reduced FSH dose (100 IU per day) results in similar cumulative live birth rates and a lower occurrence of any grade of ovarian hyperstimulation syndrome (OHSS) as compared to a standard dose (150 IU/day), a higher first cycle cancellation rate and similar severe OHSS rate were observed.WHAT IS KNOWN ALREADY: Excessive ovarian response to controlled ovarian stimulation (COS) for IVF/ICSI may result in increased rates of cycle cancellation, the occurrence of OHSS and suboptimal live birth rates. In women scheduled for IVF/ICSI, an ovarian reserve test (ORT) can be used to predict response to COS. No consensus has been reached on whether ORT-based FSH dosing improves effectiveness and safety in women with a predicted hyper response.STUDY DESIGN SIZE, DURATION: Between May 2011 and May 2014, we performed an open-label, multicentre RCT in women with regular menstrual cycles and an AFC > 15. Women with polycystic ovary syndrome (Rotterdam criteria) were excluded. The primary outcome was ongoing pregnancy achieved within 18 months after randomization and resulting in a live birth. Secondary outcomes included the occurrence of OHSS and cost-effectiveness. Since this RCT was embedded in a cohort study assessing over 1500 women, we expected to randomize 300 predicted hyper responders.PARTICIPANTS/MATERIALS, SETTING, METHODS: Women with an AFC > 15 were randomized to an FSH dose of 100 IU or 150 IU/day. In both groups, dose adjustment was allowed in subsequent cycles (maximum 25 IU in the reduced and 50 IU in the standard group) based on pre-specified criteria. Both effectiveness and cost-effectiveness were evaluated from an intention-to-treat perspective.MAIN RESULTS AND THE ROLE OF CHANCE: We randomized 255 women to a daily FSH dose of 100 IU and 266 women to a daily FSH dose of 150 IU. The cumulative live birth rate was 66.3% (169/255) in the reduced versus 69.5% (185/266) in the standard group (relative risk (RR) 0.95 [95%CI, 0.85-1.07], P = 0.423). The occurrence of any grade of OHSS was lower after a lower FSH dose (5.2% versus 11.8%, RR 0.44 [95%CI, 0.28-0.71], P = 0.001), but the occurrence of severe OHSS did not differ (1.3% versus 1.1%, RR 1.25 [95%CI, 0.38-4.07], P = 0.728). As dose reduction was not less expensive (€4.622 versus €4.714, delta costs/woman €92 [95%CI, -479-325]), there was no dominant strategy in the economic analysis.LIMITATIONS, REASONS FOR CAUTION: Despite our training programme, the AFC might have suffered from inter-observer variation. Although strict cancellation criteria were provided, selective cancelling in the reduced dose group (for poor response in particular) cannot be excluded as observers were not blinded for the FSH dose and small dose adjustments were allowed in subsequent cycles. However, as first cycle live birth rates did not differ from the cumulative results, the open design probably did not mask a potential benefit for the reduced dosing group. As this RCT was embedded in a larger cohort study, the power in this study was unavoidably lower than it should be. Participants had a relatively low BMI from an international perspective, which may limit generalization of the findings.WIDER IMPLICATIONS OF THE FINDINGS: In women with a predicted hyper response scheduled for IVF/ICSI, a reduced FSH dose does not affect live birth rates. A lower FSH dose did reduce the incidence of mild and moderate OHSS, but had no impact on severe OHSS. Future research into ORT-based dosing in women with a predicted hyper response should compare various safety management strategies and should be powered on a clinically relevant safety outcome while assessing non-inferiority towards live birth rates.STUDY FUNDING/COMPETING INTEREST(S): This trial was funded by The Netherlands Organization for Health Research and Development (ZonMW, Project Number 171102020). SCO, TCvT and HLT received an unrestricted research grant from Merck Serono (the Netherlands). CBL receives grants from Merck, Ferring and Guerbet. BWJM is supported by a NHMRC Practitioner Fellowship (GNT1082548) and reports consultancy for OvsEva, Merck and Guerbet. FJMB receives monetary compensation as a member of the external advisory board for Ferring pharmaceutics BV and Merck Serono for consultancy work for Gedeon Richter (Belgium) and Roche Diagnostics (Switzerland) and for a research cooperation with Ansh Labs (USA). All other authors have nothing to declare.TRIAL REGISTRATION NUMBER: Registered at the ICMJE-recognized Dutch Trial Registry (www.trialregister.nl). Registration number: NTR2657.TRIAL REGISTRATION DATE: 20 December 2010.DATE OF FIRST PATIENT’S ENROLMENT: 12 May 2011.

STUDY QUESTION: Are bile acids (BA) and their respective subspecies present in human follicular fluid (FF) and do they relate to embryo quality in modified natural cycle IVF (MNC-IVF)?SUMMARY ANSWER: BAconcentrations are 2-fold higher in follicular fluid than in serum and ursodeoxycholic acid (UDCA) derivatives were associated with development of top quality embryos on Day 3 after fertilization.WHAT IS KNOWN ALREADY: Granulosa cells are capable of synthesizing BA, but a potential correlation with oocyte and embryo quality as well as information on the presence and role of BA subspecies in follicular fluid have yet to be investigated.STUDY DESIGN, SIZE, DURATION: Between January 2001 and June 2004, follicular fluid and serum samples were collected from 303 patients treated in a single academic centre that was involved in a multicentre cohort study on the effectiveness of MNC-IVF.PARTICIPANTS/MATERIALS, SETTING, METHODS: Material from patients who underwent a first cycle of MNC-IVF was used. Serum was not stored from all patients, and the available material comprised 156 follicular fluid and 116 matching serum samples. Total BA and BA subspecies were measured in follicular fluid and in matching serum by enzymatic fluorimetric assay and liquid chromatography-mass spectrometry, respectively. The association of BA in follicular fluid with oocyte and embryo quality parameters, such as fertilization rate and cell number, presence of multinucleated blastomeres and percentage of fragmentation on Day 3, was analysed.MAIN RESULTS AND THE ROLE OF CHANCE: Embryos with eight cells on Day 3 after oocyte retrieval were more likely to originate from follicles with a higher level of UDCA derivatives than those with fewer than eight cells (P LIMITATIONS, REASONS FOR CAUTION: Only samples originating from first cycle MNC-IVF were used, which resulted in 14 samples only from women with an ongoing pregnancy, therefore further prospective studies are required to confirm the association of UDCA with IVF pregnancy outcomes. The inter-cycle variability of BA levels in follicular fluid within individuals has yet to be investigated. We checked for macroscopic signs of contamination of follicular fluid by blood but the possibility that small traces of blood were present within the follicular fluid remains. Finally, although BA are considered stable when stored at -20 degrees C, there was a time lag of 10 years between the collection and analysis of follicular fluid and serum samples.WIDER IMPLICATIONS OF THE FINDINGS: The favourable relation between UDCA derivatives in follicular fluid and good embryo development and quality deserves further prospective research, with live birth rates as the end-point.

Is there an association between trisomic pregnancy, a marker for decreased oocyte quality, and the reduced oocyte quantity that follows ovarian surgery?Previous ovarian surgery is not associated with an increased risk for a subsequent trisomic pregnancy.Ovarian surgery diminishes the number of oocytes. The risk for a trisomic pregnancy is suggested to be higher in women with fewer oocytes, independent of their chronological age.This is a matched case-control study. Cases are women with a confirmed trisomic pregnancy occurring between 1 January 2000 and 31 December 2010 regardless of pregnancy outcome and controls are women that had a live born child without a trisomy. In total, there were 8573 participants in the study; 1723 cases and 6850 controls.Data were obtained from Danish medical registries. Matching criteria were maternal age and year of conception. Number of controls matched per case ranged from one to four. Among cases and controls with a trisomic pregnancy, 2.7% (46/1723) versus 2.5% (172/6850) had undergone ovarian surgery before pregnancy.History of ovarian surgery is not associated with a higher risk for a subsequent trisomic pregnancy (odds ratio = 1.00, 95% confidence interval 0.99-1.01). Subgroup analyses by indication of surgery and interval between ovarian surgery and pregnancy do not show an effect on trisomic pregnancy risk.The medical registries used to select cases and controls did not contain information on surgical technique nor volume of ovarian tissue resected, previous trisomic pregnancy prior to the ovarian surgery or long-term use of oral contraceptives. Therefore, correction for these factors was not performed.We did not confirm the hypothesis that ovarian surgery, a marker for decreased oocyte quantity, is related to trisomic pregnancy, a marker for decreased oocyte quality. This suggests that ovarian surgery, which has a direct reductive effect on the size of the follicle pool, may affect oocyte quality differently when compared with the reduction in follicle pool size due to ageing.The study was supported by grants from the Gratama Stichting, University of Groningen and the University Medical Center Groningen, The Netherlands. Ø.L. has within the last 3 years received honoraria for speeches in pharmacoepidemiological issues, not related to this study. The Department of Obstetrics and Gynaecology receives unrestricted educational grants from Ferring Pharmaceuticals. A.H. received a grant from ZonMW (i.e. National Dutch Scientific funding) for a RCT not related to this publication. Dr A.H. received speakers fee from MSD for an educational presentation. All other authors have no conflict of interest.

Dietary fiber intake is associated with lower incidence and mortality from disease, but the underlying mechanisms of these protective effects are unclear.We hypothesized that β2→1-fructan dietary fibers confer protection on intestinal epithelial cell barrier function via Toll-like receptor 2 (TLR2), and we studied whether β2→1-fructan chain-length differences affect this process. T84 human intestinal epithelial cell monolayers were incubated with 4 β2→1-fructan formulations of different chain-length compositions and were stimulated with the proinflammatory phorbol 12-myristate 13-acetate (PMA). Transepithelial electrical resistance (TEER) was analyzed by electric cell substrate impedance sensing (ECIS) as a measure for tight junction-mediated barrier function. To confirm TLR2 involvement in barrier modulation by β2→1-fructans, ECIS experiments were repeated using TLR2 blocking antibody. After preincubation of T84 cells with short-chain β2→1-fructans, the decrease in TEER as induced by PMA (62.3 ± 5.2%, P < 0.001) was strongly attenuated (15.2 ± 8.8%, P < 0.01). However, when PMA was applied first, no effect on recovery was observed during addition of the fructans. By blocking TLR2 on the T84 cells, the protective effect of short-chain β2→1-fructans was substantially inhibited. Stimulation of human embryonic kidney human TLR2 reporter cells with β2→1-fructans induced activation of nuclear factor kappa-light-chain-enhancer of activated B cells, confirming that β2→1-fructans are specific ligands for TLR2. To conclude, β2→1-fructans exert time-dependent and chain length-dependent protective effects on the T84 intestinal epithelial cell barrier mediated via TLR2. These results suggest that TLR2 located on intestinal epithelial cells could be a target of β2→1-fructan-mediated health effects. © 2014 American Society for Nutrition.Chemicals/CAS: inulinase, 9025-67-6; phorbol 13 acetate 12 myristate, 16561-29-8; toll like receptor 2, 203811-81-8

STUDY QUESTION: Does embryo biopsy inherent to preimplantation genetic screening (PGS) affect neurological, cognitive and behavioural development of 4-year-old children?SUMMARY ANSWER: PGS does not seem to affect neurological, cognitive and behavioural development of 4-year-old singletons; however, our data suggest that it may be associated with altered neurodevelopment in twins.WHAT IS KNOWN ALREADY: Evidence concerning the safety of PGS on neurodevelopmental outcome in offspring is scarce. The present study provides information on neurodevelopmental, cognitive and behavioural outcome of 4-year-old PGS offspring.STUDY DESIGN, SIZE, DURATION: A prospective, assessor-blinded follow-up study of children born to women who participated in a multi-centre RCT on the effect of IVF with or without PGS.PARTICIPANTS/MATERIALS, SETTING, METHODS: At 4 years, 49 children (31 singletons, 9 sets of twins) born following IVF with PGS and 64 children (42 singletons, 11 sets of twins) born following IVF without PGS (controls) were assessed (post-natal attrition 18). Neurological development was evaluated with the standardized, age-specific and sensitive neurological examination according to Hempel, resulting in a neurological optimality score (NOS), a fluency score and the rate of adverse neurological outcome. Primary outcome was the fluency score, as fluency of movements is easily reduced by subtle dysfunction of the brain. Cognitive development was evaluated with the Kaufman Assessment Battery for Children; behavioural development was evaluated with the Child Behavior Checklist. The effect of PGS was analysed with a mixed effects model.MAIN RESULTS AND THE ROLE OF CHANCE: Based on the intention to treat analysis, neurodevelopmental outcome of PGS children was similar to that of controls. However, additional analyses indicated that PGS affected neurodevelopmental outcome of twins in a different way than that of singletons. The fluency score of singletons born following PGS was similar to that of control singletons [mean values, 95 confidence intervals (CIs): 12.2 (11.5;12.8) and 12.2 (11.6;12.8)], respectively, P 0.977) that was also true for the other neurodevelopmental parameters. The fluency score of PGS twins was significantly lower than that of control twins [mean values, 95 CIs: 10.6 (9.8;11.3) and 12.3 (11.5;13.1)], respectively, P 0.001); the same was true for the NOS. In addition, PGS in twins was associated with a higher sequential intelligence quotient score. On the other hand, other neurodevelopmental parameters were similar for PGS twins and control twins. Post hoc sample size calculation for the primary outcome parameter, the fluency score, indicated that the study groups, including the subgroups of singletons and twins, were adequately powered.LIMITATIONS, REASONS FOR CAUTION: We assessed singletons and twins who contributed to the generalizability of the study. A limitation of our study is the relative small size of our study groups and the selective dropout in both groups (dropouts PGS group: higher gestational age; control group: less well-educated parents). These preclude the conclusion that PGS per se is not associated with neurodevelopmental, cognitive and behavioural problems in singletons and the conclusion that PGS is associated with altered neurodevelopmental outcome in twins.WIDER IMPLICATIONS OF THE FINDINGS: The need for careful long-term monitoring of children born following embryo biopsy remains, as it is still applied in the form of PGD and it is still unknown whether embryo biopsy affects long-term neurodevelopmental outcome.STUDY FUNDING/COMPETING INTEREST(S): The RCT was financially supported by the Organization for Health Research and Development (ZonMw), The Netherlands (grant number 945-03-013). The follow-up at 4 years was financially supported by the University Medical Center Groningen (grant number: 754510), the Cornelia Foundation, the Junior Scientific Master Class and the Postgraduate School of Behavioural and Cognitive Neurosciences, Groningen, The Netherlands. The sponsors of the study had no role in study design, data collection, data analysis, data interpretation or writing of the report. There are no conflicts of interest.

BACKGROUND: Knowledge of patients' preferences for elective single embryo transfer (eSET) or double embryo transfer (DET) and for singletons or twins is of great importance in counselling for embryo transfer (ET) strategies. In this study, the stability of IVF patients' preferences over time for either a healthy single child or healthy twins was measured and we investigated which factors could explain preference shifts.METHODS: Infertile women (n = 177) who participated in an RCT comparing one cycle eSET with one cycle DET were included. A satisfaction questionnaire was developed to measure patient preferences and attitudes at two moments in time, i.e. at 2 weeks before ET and at 2 weeks following ET, after the results of the pregnancy test. Regression analysis examined the effect of several variables on preference shifts.RESULTS: Before ET, most patients expressed a preference for a singleton, whereas most patients were indifferent 2 weeks after ET, resulting in an overall preference shift towards twins (P = 0.002; n = 145). Overall, 62% of patients showed a preference shift. Preference shifts were explained by patients' global satisfaction of the information given by the fertility clinic staff received by the fertility clinic staff, and an interaction between the occurrence of pregnancy and transfer policy (eSET or DET).CONCLUSIONS: In general, patients' preferences for a singleton or twins are not stable during IVF treatment. Possible explanations of a shift in preference are that pregnant patients attuned their preferences to what they expect their pregnancy to result in, whereas non-pregnant patients shifted towards a preference for twins in order to be able to fulfil their ultimate child wish.

Aims To investigate the yield of cardiogenetic screening of relatives of young sudden cardiac death (SCD) and sudden unexplained death (SUD) victims in a population-based setting.Methods and results A population-based study was carried out between 2000 and 2006. Records of the hospital, death declaration certificates, and resuscitation records were reviewed for SCD and SUD cases (1-40 years). Information on autopsy results and cardiogenetic screening of the victims' first-degree relatives was collected. Relatives were invited for additional cardiogenetic screening when this had not yet been performed. The search led to 16 cases of SCD/SUD and 4 cases of aborted SCD/SUD. Causes of SCD/SUD were myocardial infarction (n = 3), arrhythmogenic right ventricular cardiomyopathy (ARVC) (n = 2), long-QT syndrome (n = 1), hypertrophic cardiomyopathy (n = 2), left ventricular hypertrophy due to aortic stenosis (n = 1), and unknown cause of death (n = 7). Causes of aborted SCD/SUD were myocardial infarction (n = 2), idiopatic ventricular fibrillation (n = 1), and the Brugada syndrome (n = 1). The cardiogenetic screening of 37 relatives of 12 victims led to a diagnosis of Brugada syndrome in 3 relatives and the suspicion of ARVC in 2 relatives. The yield of screening of these relatives was 14% (95% confidence interval: 3-25%).Conclusion In the usual care, relatives of (aborted) SCD and SUD victims are often not referred for cardiogenetic screening. Screening is often not performed according to a systematic approach, and the detection rate of inherited diseases in relatives of (aborted) SCD and SUD victims in a population-based setting, although substantial, is lower than expected based on previous studies.

Guidelines on karyotyping infertile men before ICSI treatment are not consistent. Most guidelines recommend chromosomal screening in azoospermic and severe oligozoospermic men, because they are assumed to have the highest risk of abnormalities. We performed a retrospective cohort study in azoospermic men and men eligible for ICSI. We determined the prevalence of chromosomal abnormalities in relation to sperm concentration and compared our data to studies in the literature. A high prevalence of chromosomal abnormalities in azoospermic men was found, but no difference in the prevalence of abnormalities was seen between different sperm concentration categories in non-azoospermic men. This raises the question of who should be screened for chromosomal abnormalities before ICSI treatment. Considering the costs and benefits, we would propose limiting screening to infertile couples with non-obstructive azoospermia.

BACKGROUND: The predictive value of ovarian reserve tests (ORTs) for spontaneous pregnancy is unclear. Our study aimed to determine whether ORTs have added value to previously identified prognostic factors for spontaneous pregnancy in subfertile ovulatory couples. METHODS: A prospective cohort study was performed on 474 subfertile ovulatory couples in two hospitals in Groningen, The Netherlands. The ORTs performed were: antral follicle count (AFC), follicle-stimulating hormone (FSH), inhibin B (basal levels and after stimulation with clomiphene citrate) and the clomiphene citrate challenge test. For each couple, the probability of spontaneous pregnancy was retrospectively calculated using the validated Hunault prediction model which includes the main known prognostic factors for spontaneous pregnancy. Outcome measure was time to spontaneous pregnancy resulting in a live birth. RESULTS: When added to the Hunault model, only basal FSH and AFC significantly improved the prediction of spontaneous pregnancy (P-values of 0.05 and 0.04). Absolute changes in predicted probabilities after adding basal FSH or AFC were small: the predicted probability of spontaneous pregnancy shifted >= 10% in only 3.8% and 7.9% of the couples, respectively. CONCLUSIONS: Although basal FSH and AFC significantly improved the validated prediction model for spontaneous pregnancy, the clinical relevance of this finding is limited. We recommend that none of the ORTs studied should be used routinely in the subfertility evaluation of ovulatory couples to predict spontaneous pregnancy chances.

Background: Gender differences have been reported in patients with the congenital long QT syndrome (LQTS). We analyzed whether electrocardiographic differences existed in females, males, girls and boys in response to beta-adrenoceptor blockade. Methods: 12-lead ECGs before and during beta-adrenoceptor blockade were collected in 87 genotyped LQTS patients (48 women, 14 men, 12 girls and 13 boys). Up to three QTc intervals were determined in each lead of the ECG.V4 was used for QT/QTc analysis. Difference between longest and shortest QT interval was taken as a measure for dispersion of QT intervals. Results: (1) Adult males had the greatest shortening of the QTc interval upon treatment with P-adrenoceptor blockade. During treatment, adult males with LQTS(1) (mutation in the KCNQ1 gene, affecting I-Ks current) were found to have shorter QTc intervals than adult females; this difference did not exist in LQTS(2) patients (mutation in the HERG gene, affecting I-Kr current). (2) Female LQTS(2) patients had a 50% larger dispersion than female LQTS(1) patients both before and during treatment. (3) Adult male LQTS(1) patients, constitute the only patient group with a marked decrease in QTc intervals and dispersion associated with a 100% efficacy of treatment in response to beta-adrenoceptor blockade. Conclusions: These findings indicate that, in addition to underlying differences in repolarization between men and women, cardiae electrophysiological responses to beta-adrenoceptor blockade can be modulated by gender-related factors. (C) 2002 Elsevier Science B.V. All rights reserved