Obese outbred mice only partially benefit from diet normalization or calorie restriction as preconception care interventions to improve metabolic health and oocyte quality.

STUDY QUESTION: Can diet normalization or a calorie-restricted diet for 2 or 4 weeks be used as a preconception care intervention (PCCI) in Western-type diet-induced obese Swiss mice to restore metabolic health and oocyte quality? SUMMARY ANSWER: Metabolic health and oocyte developmental competence was already significantly improved in the calorie-restricted group after 2 weeks, while obese mice that underwent diet normalization showed improved metabolic health after 2 weeks and improved oocyte quality after 4 weeks. WHAT IS KNOWN ALREADY: Maternal obesity is linked with reduced metabolic health and oocyte quality; therefore, infertile obese women are advised to lose weight before conception to increase pregnancy chances. However, as there are no univocal guidelines and the specific impact on oocyte quality is not known, strategically designed studies are needed to provide fundamental insights in the importance of the type and duration of the dietary weight loss strategy for preconception metabolic health and oocyte quality. STUDY DESIGN, SIZE, DURATION: Outbred female Swiss mice were fed a control (CTRL) or high-fat/high-sugar (HF/HS) diet. After 7 weeks, some of the HF mice were put on two different PCCIs, resulting in four treatment groups: (i) only control diet for up to 11 weeks (CTRL_CTRL), (ii) only HF diet for up to 11 weeks (HF_HF), (iii) switch at 7 weeks from an HF to an ad libitum control diet (HF_CTRL) and (iv) switch at 7 weeks from an HF to a 30% calorie-restricted control diet (HF_CR) for 2 or 4 weeks. Metabolic health and oocyte quality were assessed at 2 and 4 weeks after the start of the intervention (n = 8 mice/treatment/time point). PARTICIPANTS/MATERIALS, SETTING, METHODS: Changes in body weight were recorded. To study the impact on metabolic health, serum insulin, glucose, triglycerides, total cholesterol and alanine aminotransferase concentrations were measured, and glucose tolerance and insulin sensitivity were analyzed at PCCI Weeks 2 and 4. The quality of in vivo matured oocytes was evaluated by assessing intracellular lipid droplet content, mitochondrial activity and localization of active mitochondria, mitochondrial ultrastructure, cumulus cell targeted gene expression and oocyte in vitro developmental competence. MAIN RESULTS AND THE ROLE OF CHANCE: Significant negative effects of an HF/HS diet on metabolic health and oocyte quality were confirmed (P < 0.05). HF_CTRL mice already showed restored body weight, serum lipid profile and glucose tolerance, similar to the CTRL_CTRL group after only 2 weeks of PCCI (P < 0.05 compared with HF_HF) while insulin sensitivity was not improved. Oocyte lipid droplet volume was reduced at PCCI Week 2 (P < 0.05 compared with HF_HF), while mitochondrial localization and activity were still aberrant. At PCCI Week 4, oocytes from HF_CTRL mice displayed significantly fewer mitochondrial ultrastructural abnormalities and improved mitochondrial activity (P < 0.05), while lipid content was again elevated. The in vitro developmental capacity of the oocytes was improved but did not reach the levels of the CTRL_CTRL mice. HF_CR mice completely restored cholesterol concentrations and insulin sensitivity already after 2 weeks. Other metabolic health parameters were only restored after 4 weeks of intervention with clear signs of fasting hypoglycemia. Although all mitochondrial parameters in HF_CR oocytes stayed aberrant, oocyte developmental competence in vitro was completely restored already after 2 weeks of intervention. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: In this study, we applied a relevant HF/HS Western-type diet to induce obesity in an outbred mouse model. Nevertheless, physiological differences should be considered when translating these results to the human setting. However, the in-depth study and follow-up of the metabolic health changes together with the strategic implementation of specific PCCI intervals (2 and 4 weeks) related to the duration of the mouse folliculogenesis (3 weeks), should aid in the extrapolation of our findings to the human setting. WIDER IMPLICATIONS OF THE FINDINGS: Our study results with a specific focus on oocyte quality provide important fundamental insights to be considered when developing preconception care guidelines for obese metabolically compromised women wishing to become pregnant. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Flemish Research Fund (FWO-SB grant 1S25020N and FWO project G038619N). The authors declare there are no conflicts of interest.

Peak retrograde flow a potential objective management tool to identify young adults with varicocele 'at risk' for a high sperm DNA fragmentation.

INTRODUCTION: Determining which patients are negatively affected by varicocele would enable clinicians to better select those men who would benefit most from surgery. Since conventional semen parameters, have been limited in their ability to evaluate the negative effects of varicocele on fertility, specialized laboratory tests have emerged. OBJECTIVE: To identify clinical and ultrasound parameters (including PRF) which would negatively influence standard and functional semen variables in young adults with a varicocele. DESIGN: Prospective, cross-sectional observational study. SETTING: Antwerp University Hospital, Belgium. PATIENT(S): Young volunteers between 16 and 26 years, Tanner 5, were recruited. INTERVENTION(S): Every participant had a scrotal ultrasound to calculate testicular volumes. If a varicocele was present, the grade, vein diameter, peak retrograde flow (PRF) in supine position and spontaneous reflux in standing position were measured. All participants provided a semen sample. Standard semen parameters were analyzed and sperm DNA fragmentation. MAIN OUTCOME MEASURE(S): Of all clinical and ultrasound parameters tested, PRF was an objective tool identifying young adults with a varicocele. PRF was highlighted by the prevalence of SDF, both in the total and vital fractions of the spermatozoa, providing opportunities to manage such 'at-risk' adolescents/young adults. RESULT(S): Total SDF was significantly increased in grade 3 varicocele compared to grade 1 and 2 but no significant difference with vital SDF or standard descriptive semen parameters was seen. Total and vital SDF on the other hand were significantly increased when PRF was above 38.4 cm/s. Standard semen analysis showed no difference with PRF as an independent predictor. Testicular atrophy index, varicocele vein diameter and spontaneous reflux revealed no significant differences in both the descriptive and functional semen variables. DISCUSSION: Descriptive semen parameters showed no significant difference between the non-varicocele controls and the varicocele group with low and high PRF. Increased PRF negatively influenced sperm quality via increased DNA fragmentation both in the total as in the vital fractions of the semen. CONCLUSION(S): Of all clinical and ultrasound parameters tested, PRF was an objective non-invasive tool to identify varicocele patients at risk for a high SDF.

Systematic development of a mobile preconception lifestyle programme for couples undergoing IVF: the PreLiFe-programme.

STUDY QUESTION: Can we develop a preconception lifestyle programme for couples undergoing IVF that is in line with their needs. SUMMARY ANSWER: A mobile preconception lifestyle programme was systematically developed based on expert opinion, literature and needs of IVF-patients. WHAT IS KNOWN ALREADY: A healthy lifestyle prior to conception is not only beneficial for the general health of couples, but evidence on its importance for their reproductive health and the health of their children is also emerging. So far, the vast majority of fertility clinics do not offer a lifestyle programme for couples undergoing IVF. Therefore, the present study aimed to develop a lifestyle programme for IVF-couples. STUDY DESIGN, SIZE, DURATION: The development of the PreLiFe-programme was guided by the steps of the Medical Research Council (MRC) framework for developing complex interventions, a systematic approach for developing theory- and evidence-based health promotion interventions. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: First, the evidence base on lifestyle programmes for IVF-couples was reviewed. Second, several iterations between an expert panel, the literature, and quantitative and qualitative data from IVF-patients identified the content, the format, behaviour change techniques and theory of the programme. Third, the PreLiFe-programme was produced and the expected process and outcomes of a randomized controlled trial assessing it were modelled. Finally, user tests among experts and patients and pilot tests among patients were conducted. MAIN RESULTS AND ROLE OF CHANCE: The finally developed PreLiFe-programme is a mobile application to be used autonomously by both partners of IVF-couples during the first year of IVF, in combination with motivational interviewing over the telephone every three months (i.e. blended care). The PreLiFe-programme provides advice and skills training on physical activity, diet and mindfulness based stress reduction and is in part tailored based on monitoring and tracking the lifestyle of patients. Based on the literature the expert panel considers it plausible that all three components contribute to IVF-success rates. The PreLiFe-programme is likely to be acceptable to patients as it meets the need of patients for lifestyle advice and treatment information. LIMITATIONS, REASON FOR CAUTION: The pilot in IVF-couples had a 3-month duration. The feasibility of the PreLiFe-programme in other infertile populations and/or upon longer use is yet to be examined. Whether the PreLiFe-programme effectively improves lifestyle and IVF-success rates is currently being examined in a trial randomizing heterosexual couples starting IVF to the PreLiFe-programme or an attention-control group for 12 months. WIDER IMPLICATIONS OF THE FINDINGS: If the PreLiFe-programme improves lifestyle and the chance of IVF-success, it will be a powerful tool and provide guidance for implementing lifestyle programmes in fertility clinics. STUDY FUNDING/COMPETING INTEREST(S): Funded by the Research Foundation Flanders (FWO-TBM (Applied Biomedical Research with a Primary Social finality); reference: T005417N). The authors have no conflict of interest to report. TRIAL REGISTRATION NUMBER: NCT03790449.

Time intervals between semen production, initiation of analysis, and IUI significantly influence clinical pregnancies and live births.

PURPOSE: Does IDEF mapping help monitor the technical process of IUI and explore the potential improvements which might contribute to increased pregnancy and live birth rates? METHOD: Retrospective analysis of 1729 homologous IUI cycles of couples attending a fertility clinic in a university hospital setting. Standardized conventional semen parameters were analyzed and the semen samples prepared via discontinuous density gradient centrifugation. RESULTS: There was no significant association between sperm concentration, motility and morphology (analysis phase), and pregnancy outcome. Only female and male ages were significantly associated with the pregnancy outcome. There was a significant difference in the odds on clinical pregnancies and live births when analysis was ≤ 21 min initiated, and < 107 min between sample production and IUI, adjusted for male and female age. CONCLUSIONS: Adjusting for the couple's age, we could show that time intervals between semen production and analysis and IUI when kept low significantly influenced clinical pregnancies and live births.

DNA fragmentation in concert with the simultaneous assessment of cell viability in a subfertile population: establishing thresholds of normality both before and after density gradient centrifugation.

PURPOSE: TUNEL assay is the most common, direct test for sperm chromatin integrity assessment. But, lack of standardized protocols makes interlaboratory comparisons impossible. Consequently, clinical thresholds to predict the chance of a clinical pregnancy also vary with the technique adopted. This prospective study was undertaken to assess the incidence of sperm DNA fragmentation in a subfertile population and to establish threshold values of normality as compared to a fertile cohort, both before and after density gradient centrifugation in the total and vital fractions. METHOD: Men presenting at a university hospital setup for infertility treatment. DNA damage via TUNEL assay was validated on fresh semen samples, as conventional semen parameters, to reduce variability of results. RESULTS: Total DNA fragmentation in the neat semen was significantly higher in the subfertile group, but the vital fraction was not significantly different between the two cohorts. After gradient centrifugation, DNA fragmentation increased significantly in the total fraction of the subfertile group but decreased significantly in the vital fraction. In the fertile cohort, there was a non-significant increase in total fragmentation and in the vital fraction the trend was unclear. CONCLUSIONS: Estimating total and vital sperm DNA fragmentation, after density gradient centrifugation, increased both the sensitivity and the specificity, thereby lowering the number of false negatives and false positives encountered. These findings provide opportunities to investigate the significance of the total and the vital fractions after different assisted reproductive technologies.

Sperm DNA fragmentation in the total and vital fractions before and after density gradient centrifugation: Significance in male fertility diagnosis.

BACKGROUND: Sperm DNA fragmentation measured by different techniques make comparisons impossible due to lack of standardization. Induction of DNA damage after sperm preparation in the entire fraction has been observed on independent occasions but findings are not consistent. METHODS: Men presenting at a University hospital setup for infertility treatment. DNA damage via TUNEL assay was validated on fresh semen samples, as conventional semen parameters, to reduce variability of results. RESULTS: Sperm motility in neat semen inversely correlated with sperm DNA fragmentation in the total fraction, but, total count, leukocytes and immature germ cells significantly affected the vital fraction. Sperm DNA fragmentation was observed both in normal and subnormal semen samples, but was significantly different in the total fraction of astheno-, asthenoterato- and oligoteratozoospermic men. After density gradient centrifugation, sperm DNA fragmentation increased significantly in the total but decreased in the vital fraction. Advancing male age significantly influenced damage in the total but not in the vital population. CONCLUSIONS: These findings provide opportunities to investigate the significance of the total and the vital fractions both in natural conception and after different assisted reproductive technologies.

Validating semen processing for an intrauterine program should take into consideration the inputs, actions and the outputs of the process.

To validate semen preparation via density gradient centrifugation, we took into account the input via the semen sample, the action generated by technical and equipment characteristics and the output measured by the level of performance. Longer periods of abstinence reduced % yield, but increased viscosity and incomplete samples collected had no effect. Under controlled technical and equipment characteristics, precision and reproducibility were validated for density gradient. Additionally, as a good laboratory practice, internal and external quality control measures were implemented to guarantee the level of performance. Inseminating motile sperm count is an important predictive parameter for IUI success. In our group of patients, a yield of an absolute lower limit of 2 million motile spermatozoa was sufficient to contemplate IUI. Pregnancy rate of 13.8% where >2 million rapid progressive spermatozoa were inseminated was significantly higher than the pregnancies (4.4%) obtained with <2 million rapid progressive spermatozoa. This percentage was even higher than the national data registered for IUI (12.2%). To make IUI an attractive first-line treatment, standardization and proper validation of semen preparation procedure are mandatory.

How do cumulative live birth rates and cumulative multiple live birth rates over complete courses of assisted reproductive technology treatment per woman compare among registries?

STUDY QUESTION: How do the national cumulative (multiple) live birth rates over complete assisted reproduction technology (ART) courses of treatment per woman in Belgium compare to those in other registries? SUMMARY ANSWER: Cumulative live birth rates (CLBRs) remain high with a low cumulative multiple live birth rate when compared with other registries and publications. WHAT IS KNOWN ALREADY: In ART, a reduction in the multiple live birth rate could be achieved by reducing the number of embryos transferred. It has been shown that by doing so, live birth rates per cycle were maintained, particularly when the augmentation effect of attached frozen-thawed cycles was considered. STUDY DESIGN, SIZE, DURATION: A retrospective cohort study included all patients with a Belgian national insurance number who were registered in the national ART registry (Belrap) and who started a first fresh ART cycle between 1 July 2009 until 31 December 2011 with follow up until 31 December 2012. We analysed 12 869 patients and 38 008 cycles (both fresh and attached frozen cycles). PARTICIPANTS, MATERIALS, SETTINGS, METHODS: CLBRs per patient who started a first ART cycle including fresh and consecutive frozen cycles leading to a live birth. Conservative estimates of cumulative live birth assumed that patients who did not return for treatment had no chance of achieving an ART-related live birth, whereas optimal estimates assumed that women discontinuing treatment would have the same chance of achieving a live birth as those continuing treatment. A maximum of six fresh ART cycles with corresponding frozen cycles was investigated and compared with other registries and publications. MAIN RESULTS AND ROLE OF CHANCE: The CLBR was age dependent and declined from 62.9% for women <35 years, to 51.4% for women 35-37 years, to 34.1% for women 38-40 years and 17.7% for women 41-42 years in the conservative analysis after six cycles. In the optimal estimate, the CLBR declined from 85.9% for women <35 years, to 72.0% for women 35-37 years, to 50.4% for women 38-40 years and 36.4% for women 41-42 years. The cumulative multiple live birth rates for the whole population were 5.1 and 8.6% for the conservative and optimal estimate, respectively. LIMITATIONS, REASONS FOR CAUTION: Conservative and optimal estimates use assumptions for the whole ART population and do not take the individual patient into account. WIDER IMPLICATIONS OF THE FINDINGS: These data reinforce the validity of the Belgian model of coupling reimbursement of ART costs to a restriction in the number of embryos transferred. Our data can improve decision-making in medical ART practice both on the patient level and for society at large and could provide health care takers and insurance companies with a valid model. STUDY FUNDING COMPETING INTERESTS: none.

Vitrification of cleavage stage day 3 embryos results in higher live birth rates than conventional slow freezing: a RCT.

STUDY QUESTION: Is the live birth rate (LBR) per embryo thawed/warmed higher when Day 3 cleavage stage embryos are cryopreserved by vitrification compared with slow freezing? SUMMARY ANSWER: The LBR per embryo thawed/warmed was higher after vitrification than after slow freezing on Day 3, based on better embryo survival, quality and availability of embryos in the vitrification group. WHAT IS KNOWN ALREADY: Post-thawing survival rate of cleavage-stage embryos has been reported to be higher after vitrification than after slow freezing. STUDY DESIGN, SIZE, DURATION: This RCT was performed in an academic tertiary center between September 2011 and March 2013. If supernumerary embryos were available on Day 3, patients were randomized at the time of cryopreservation using a computerized system to determine a simple allocation to the vitrification group or the slow freezing group and all embryos were frozen with the same technique. The primary outcome of this study was the LBR per embryo thawed/warmed. Power calculation revealed that 184 thawed embryos were needed in each group (ß = 0.8, α < 0.05) to test the hypothesis that the LBR per embryo thawed/warmed was significantly higher (16%) after vitrification than after slow freezing (6%). PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients <40 years old undergoing their first oocyte retrieval (OR), with embryo transfer and with supernumerary embryos on Day 3, were randomized. Day 3 embryos with ≥6 cells, <25% fragmentation and morphologically equal blastomeres were cryopreserved by slow freezing (using 1,2-propanediol and 0.1 M sucrose as cryoprotectant) or by closed vitrification using commercially available freezing/vitrification media. Survival was defined as ≥50% cells were intact after thawing. Thawed embryos were further cultured overnight. In total, 307 patients were randomized to slow freezing (155 patients, 480 embryos) or vitrification (152 patients, 495 embryos). MAIN RESULTS AND THE ROLE OF CHANCE: By March 2013, 200 embryos were thawed after slow freezing in 95 cycles for 79 patients and 217 embryos were warmed after vitrification in 121 cycles in 90 patients. The LBR per embryo thawed/warmed was significantly higher after vitrification (16.1% (35/217)) than after slow freezing (5.0% (10/200); P < 0.0022; relative risk (RR) 3.23; 95% confidence interval (CI) 1.64-6.35). Similarly, the implantation rate per embryo thawed/warmed was higher after vitrification (20.7% (45/217) than after slow freezing (7.5% (15/200); P = 0.0012; RR 2.76; CI 1.59-4.81). The survival rate was significantly higher after vitrification (84.3% (183/217) than after slow freezing (52.5% (105/200); P < 0.0001). Significantly more embryos were fully intact after vitrification (75.4% (138/183) than after slow freezing (28.6% (30/105); P < 0.0001). The number of transfers was significantly higher after vitrification (90.1% (109/121)) than after slow freezing (73.7% (70/95); P = 0.0024). LIMITATIONS, REASONS FOR CAUTION: Survival rates in the slow freezing group were low in this study. Additional RCTs are needed to compare reproductive outcome after vitrification and after slow freezing with 1,2-propanediol and 0.2 M sucrose, since this method has been reported to have better survival than the method used in our study. Our findings are only applicable to the specific slow freezing cryopreservation medium used in our study, and not to any other commercially available media. WIDER IMPLICATIONS OF THE FINDINGS: When compared with slow freezing using 1,2-propanediol and 0.1 M sucrose as cryoprotectant, vitrification of Day 3 cleavage stage embryos resulted in a higher LBR per embryo warmed, and may therefore result into a higher cumulative delivery rate after one oocyte retrieval. STUDY FUNDING/COMPETING INTERESTS: None. TRIAL REGISTRATION NUMBER: NCT02013024.

Low-dose human menopausal gonadotrophin versus clomiphene citrate in subfertile couples treated with intrauterine insemination: a randomized controlled trial.

STUDY QUESTION: Can controlled ovarian stimulation with low-dose human menopausal gonadotrophin (hMG) improve the clinical pregnancy rate when compared with ovarian stimulation with clomiphene citrate (CC) in an intrauterine insemination (IUI) programme for subfertile couples? SUMMARY ANSWER: Ovarian stimulation with low-dose hMG is superior to CC in IUI cycles with respect to clinical pregnancy rate. WHAT IS KNOWN ALREADY: IUI after ovarian stimulation is an effective treatment for mild male subfertility, unexplained subfertility and minimal-mild endometriosis, but it is unclear which medication for ovarian stimulation is more effective. STUDY DESIGN, SIZE, DURATION: A total of 330 women scheduled for IUI during 657 cycles (September 2004-December 2011) were enrolled in an open-label randomized clinical trial to ovarian stimulation with low-dose hMG subcutaneous (n = 334, 37.5-75 IU per day) or CC per oral (n = 323, 50 mg/day from Day 3-7). Assuming a difference of 10% in 'clinical pregnancy with positive fetal heart beat', we needed 219 cycles per group (alpha-error 0.05, power 0.80). PARTICIPANTS/MATERIALS, SETTING, METHODS: We studied subfertile couples with mild male subfertility, unexplained subfertility or minimal-mild endometriosis. Further inclusion criteria were failure to conceive for ≥12 months, female age ≤42 years, at least one patent Fallopian tube and a total motility count (TMC) ≥5.0 million spermatozoa after capacitation. The primary end-point was clinical pregnancy. Analysis was by intention to treat and controlled for the presence of multiple measures, as one couple could have more randomizations in multiple cycles. Linear mixed models were used for continuous measures. For binary outcomes we estimated the relative risk using a Poisson model with log link and using generalized estimating equations. MAIN RESULTS AND THE ROLE OF CHANCE: When compared with ovarian stimulation with CC, hMG stimulation was characterized by a higher clinical pregnancy rate (hMG 48/334 (14.4%) versus CC 29/323 (9.0%), relative risk (RR) 1.6 (95% confidence interval (CI) 1.1-2.4)), higher live birth rate (hMG 46/334 (13.8%) versus CC 28/323 (8.7%), RR 1.6 (95% CI 1.0-2.4)), low and comparable multiple live birth rate (hMG 3/46 (6.5%) versus CC 1/28 (3.6%), P > 0.99), lower number of preovulatory follicles (hMG 1.2 versus CC 1.5, P < 0.001), increased endometrial thickness (hMG 8.5 mm versus CC 7.5 mm, P < 0.001), and a lower cancellation rate per started cycle (hMG 15/322 (4.7%) versus CC 46/298 (15.4%), P < 0.001). LIMITATIONS, REASONS FOR CAUTION: We randomized patients at a cycle level, and not at a strategy over multiple cycles. WIDER IMPLICATIONS OF THE FINDINGS: This study showed better reproductive outcome after ovarian stimulation with low-dose gonadotrophins. A health economic analysis of our data is planned to test the hypothesis that ovarian stimulation with low-dose hMG combined with IUI is associated with increased cost-effectiveness when compared with ovarian stimulation with CC. STUDY FUNDING/ COMPETING INTERESTS: T.M.D. and K.P. were supported by the Clinical Research Foundation of UZ Leuven, Belgium. This study was also supported by the Ferring company (Copenhagen, Denmark) which provide free medication (Menopur) required for the group of patients who were randomized in the hMG COS group. The Ferring company was not involved in the study design, data analysis, writing and submission of the paper. TRIAL REGISTRATION NUMBER: NCT01569945 (ClinicalTrials.gov).

The impact of legally restricted embryo transfer and reimbursement policy on cumulative delivery rate after treatment with assisted reproduction technology.

STUDY QUESTION: What is the impact of the Belgian legislation (1 July 2003), coupling reimbursement of six assisted reproduction technology (ART) cycles per patient to restricted embryo transfer policy, on cumulative delivery rate (CDR) per patient? SUMMARY ANSWER: The introduction of Belgian legislation in ART had no negative impact on the CDR per patient based on realistic estimates within six cycles or 36 months. WHAT IS KNOWN ALREADY: The introduction of Belgian legislation limiting the number of embryos for transfer resulted in a reduction of the multiple pregnancy rate (MPR) per cycle by 50%. STUDY DESIGN, SIZE, DURATION: A retrospective cohort study with a study group after implementation of the new ART legislation (July 2003 to June 2006) and the control group, before legislation (July 1999 to June 2002). PARTICIPANTS/MATERIALS, SETTING, METHODS: CDR was compared in an academic tertiary setting between a study group after legislation (n = 795 patients, 1927 fresh and 383 frozen-thawed embryo transfer (FET) cycles) and a control group before legislation (n = 463 patients, 876 fresh and 185 FET cycles) within six cycles or 36 months, delivery or discontinuation of treatment. The CDR was estimated using life table analysis considering pessimistic, optimistic and realistic scenarios and compared after adjustment for confounding variables. In the realistic scenario we included information on embryo quality to define the prognosis of each patient discontinuing treatment. MAIN RESULTS AND THE ROLE OF CHANCE: In the realistic scenario, CDR within 36 months was comparable (all ages, P = 0.221) in study group (60.8%) and control group (65.6%), as well as in different age groups (<36 years, P = 0.242; 36-39 years, P = 0.851; 40-42 years, P = 0.840). In the realistic scenario applied to six cycles, we found lower CDRs in the study group than in the control group within the two first cycles (all ages, P = 0.009; <36 years, P = 0.007) but no difference in CDRs between the two groups within the four subsequent cycles (all ages P = 0.232; <36 years, P = 0.198). The CDR within six cycles was 60 and 65.3% for study group and control group, respectively, for all ages, and 65.8 and 70.4%, respectively, in the subgroup younger than 36 years. In women ≥36 years, CDR within six cycles was comparable in both groups (36-39 years, 43% in study versus 44.4% in control group, P = 0.730; 40-42 years, 21% in study versus 23% in control group, P = 0.786). LIMITATIONS, REASONS FOR CAUTION: A retrospective cohort study design was the only way to study the impact of legislation on CDR. Owing to the retrospective nature of this analysis over a long period of time, our data are potentially influenced by improvements in techniques and therefore improved success rates in ART over time. WIDER IMPLICATIONS OF THE FINDINGS: This 'Belgian model' can now be considered for application worldwide in countries with the aim to reduce the main ART side effect (high MPR) and its associated costs without a negative effect on the main intended effect (high CDR). STUDY FUNDING/COMPETING INTEREST(S): The authors have no conflict of interest to declare. No funding was obtained for this study.

The history of Belgian assisted reproduction technology cycle registration and control: a case study in reducing the incidence of multiple pregnancy.

STUDY QUESTION: What is the effect of a legal limitation of the number of embryos that can be transferred in an assisted reproductive technology (ART) cycle on the multiple delivery rate? SUMMARY ANSWER: The Belgian national register shows that the introduction of reimbursement of ART laboratory costs in July 2003, and the imposition of a legal limitation of the number of embryos transferred in the same year, were associated with a >50% reduction of the multiple pregnancy rate from 27 to 11% between 2003 and the last assessment in 2010, without any reduction of the pregnancy rate per cycle. WHAT IS KNOWN ALREADY: Individual Belgian IVF centres have published their results since the implementation of the law, and these show a decrease in the multiple pregnancy rate on a centre by centre basis. However, the overall national picture remains unpublished. STUDY DESIGN, SIZE, DURATION: Cohort study from 1990 to 2010 of all ART cycles in Belgium (2685 cycles in 1990 evolving to 19 110 cycles in 2010), with a retrospective analysis from 1990 to 2000 and prospective online data collection since 2001. PARTICIPANTS/MATERIALS, SETTING, METHODS: Registration evolved from paper written reports per centre to a compulsory online registration of all ART cycles. From 2001 up to mid-2009, data were collected from Excel spread sheets or MS Access files into an MS Access database. Since mid-2009, data collection is done via a remote and secured web-based system (www.belrap.be) where centres can upload their data and get immediate feedback about missing data, errors and inconsistencies. MAIN RESULTS AND THE ROLE OF CHANCE: National Belgian registration data show that reimbursement of IVF laboratory costs in July 2003, coupled to a legal limitation in the number of embryos transferred in utero, were associated with a 50% reduction of the multiple pregnancy rate from 27 to 11% without reduction of the pregnancy rate per cycle, and with an increase in the number of fresh and frozen ART cycles due to improved access to treatment. LIMITATIONS, REASONS FOR CAUTION: There is potential underreporting of complications of ART treatment, pregnancy outcome and neonatal health. WIDER IMPLICATIONS OF THE FINDINGS: Over the 20 years of registration, the pregnancy rate has remained constant, despite the reduction in the number of embryos transferred, optimization of laboratory procedures and stimulation protocols, introduction of quality systems and implementation of the EU Tissue Directive over the period 2004-2010. STUDY FUNDING/COMPETING INTEREST(S): No external funding was sought for this study. None of the authors has any conflict of interest to declare.

Quality indicators for all dimensions of infertility care quality: consensus between professionals and patients.

STUDY QUESTION: What is the relative importance of the six dimensions of quality of care according to different stakeholders and can a quality indicator set address all six quality dimensions and incorporate the views from professionals working in different disciplines and from patients? SUMMARY ANSWER: Safety, effectiveness and patient centeredness were the most important quality dimensions. All six quality dimensions can be assessed with a set of 24 quality indicators, which is face valid and acceptable according to both professionals from different disciplines and patients. WHAT IS KNOWN ALREADY: To our knowledge, no study has weighted the relative importance of all quality dimensions to infertility care. Additionally, there are very few infertility care-specific quality indicators and no quality indicator set covers all six quality dimensions and incorporated the views of professionals and patients. STUDY DESIGN, SIZE AND DURATION: A three-round iterative Delphi survey including patients and professionals from four different fields, conducted in two European countries over the course of 2011 and 2012. PARTICIPANTS/MATERIALS, SETTINGS AND METHODS: Dutch and Belgian gynaecologists, embryologists, counsellors, nurses/midwifes and patients took part (n = 43 in round 1 and finally 30 in round 3). Respondents ranked the six quality dimensions twice for importance and their agreement was evaluated. Furthermore, in round 1, respondents gave suggestions, which were subsequently uniformly formulated as quality indicators. In rounds 2 and 3, respondents rated the quality indicators for preparedness to measure and for importance (relation to quality and prioritization for benchmarking). Providing feedback allowed selecting indicators based on consensus between stakeholder groups. Measurable indicators, important to all stakeholder groups, were selected for each quality dimension. MAIN RESULTS: All stakeholder groups and most individuals agreed that safety, effectiveness and patient centeredness were the most important quality dimensions. A total of 498 suggestions led to the development of 298 indicators. Professionals were sufficiently prepared to measure 204 of these indicators. Based on importance, 52 (7-15 per dimension; round 2) and finally 24 (4 per dimension; round 3) quality indicators were selected. LIMITATIONS, REASONS FOR CAUTION: The final quality indicator set does not cover the entire care process, but rather takes a 'sample' of each quality dimension. Although the quality indicators are face valid and acceptable, their psychometric characteristics need to be tested by further research. WIDER IMPLICATIONS OF THE FINDINGS: Quality management should focus on safety, effectiveness and patient centeredness of care. Clinics can use the quality indicator set to assess all quality dimensions of their care.

Semi-automated morphometric analysis of human embryos can reveal correlations between total embryo volume and clinical pregnancy.

STUDY QUESTION: Is there a link between morphometric characteristics measured by a computer-assisted scoring system and clinical pregnancy outcome? SUMMARY ANSWER: The results confirm that computer-assisted assessment of the total embryo volume is associated with clinical pregnancy outcome and can be used to complement current procedures of embryo selection. WHAT IS KNOWN ALREADY: Morphometric analysis of a large group of embryos has revealed the potential to optimize algorithms for image-analysis systems for the grading of embryos and predicting pregnancy outcomes. STUDY DESIGN, SIZE, DURATION: Oocytes and embryos were obtained from 458 patients who underwent single embryo transfer on Day 3 after IVF/ICSI, between September 2006 and December 2010 at the Leuven University Fertility Center, Belgium. In total, the data set contained 2796 embryos including 458 embryos that were transferred on Day 3. Ongoing pregnancy was defined as the presence of at least one intrauterine gestational sac at 20 weeks. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients included in this study were younger than 36 years, entering their first (n = 375) or second (n = 83) IVF/ICSI cycle and were only included once. Patients were excluded if the cycle included biopsy for PGD or if donor sperm/donor oocytes were used. Based on the 26 sequential images of the same embryo taken at one time point in different planes, the software calculates the total cytoplasmic volume for each time point, from which any reduction or change in the volume with time can be assessed (which helps interpret the degree of fragmentation) and the size of blastomeres. The diameter of the smallest and largest blastomere and the total volume of each embryo were extracted from the computer-assisted scoring system database and the coefficient of diversity was calculated for Days 1, 2 and 3. A logistic regression analysis was performed to determine the range of embryo volume associated with an increased chance of pregnancy. MAIN RESULTS AND THE ROLE OF CHANCE: On Day 3, blastomeres of 8-cell stage embryos were less divergent in size than those of 6-, 7-, 9-cell stage embryos. Although, the coefficients of diversity (ratio of the largest:smallest blastomeres) of implanted embryos tended to be lower than for non-implanted embryos, the difference was only significant for 6-cell stage embryos (P = 0.02). After logistic regression, an association between total embryo volume and pregnancy was observed which had a quadratic nature: both lower and higher volumes were associated with a lower probability of successful pregnancy. A significant association was identified between total embryo volume and pregnancy rate on both Days 2 (P = 0.003) and 3 (P = 0.0003). Diagnostic measures (sensitivity, specificity, positive predictive value, accuracy and c-statistics) of the defined volume range were relatively poor. However, results showed a good negative predictive value [76.86% (95% confidence interval 71.03-82.02) on Day 3]. LIMITATIONS, REASONS FOR CAUTION: A general disadvantage of studies evaluating the impact of a characteristic on the implantation potential of an embryo is the fact that the best embryo is chosen for transfer. No comparisons can therefore be made with the other embryos. Moreover, the decision process is currently based on a non-automated, standard scoring system, which means that a 'bias' in the selection process is always present. WIDER IMPLICATIONS OF THE FINDINGS: Our results are an important step towards the development of an automated computer-assisted scoring system for the morphological characteristics of human embryos to improve embryo selection for optimizing implantation potential. Total embryo volume appears to be one of the objective characteristics that should be included. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: Not applicable.

Dutch translation of the ICMART-WHO revised glossary on ART terminology.

BACKGROUND: A standardized set of definitions was needed in the field of medically assisted reproduction (MAR) to standardize and harmonize international data acquisition and to monitor the availability, efficacy, and safety of assisted reproductive technology (ART) worldwide. In order to provide accurate national data, the use of a terminology list which was composed and negotiated by the International Committee for Monitoring Assisted Reproductive Technology (ICMART) is essential, and a translation into Dutch was crucial for its implementation in Belgium and the Netherlands. METHOD: The authors of the Dutch article translated the English publication that appeared simultaneously in Human Reproduction and Fertility and Sterility in 2009. A consensus text was obtained after evaluation by experts in the field of MAR both in the Netherlands and in Belgium and then by the board of the respective organizations of obstetrics and gynecology. It was then sent to the World Health Organization (WHO) for approval of publication. RESULT: A translation into Dutch of the ICMART terminology of 2009 was obtained after consensus was reached on clinical and laboratory procedures, outcome variables, and birth. CONCLUSION: The availability and use of standardized terminology and its translation into Dutch will add to a more standardized communication between professionals responsible for the practice of ART and for those responsible for national, regional, and international registries.

BMI-related metabolic composition of the follicular fluid of women undergoing assisted reproductive treatment and the consequences for oocyte and embryo quality.

STUDY QUESTION: Is the metabolic composition of the follicular fluid of women undergoing assisted reproductive treatment (ART) related to serum composition and BMI and is it associated with oocyte and embryo quality? SUMMARY ANSWER: We showed that metabolic alterations in the serum are reflected in the follicular fluid and that some of these alterations may affect oocyte quality, irrespective of BMI. WHAT IS KNOWN ALREADY: Many studies have focused on the effect of metabolic disorders, such as obesity and type 2 diabetes, on assisted reproduction outcomes. There are, however, only few studies focusing on the importance of the correlation between serum and follicular fluid compositions and the composition of the follicular fluid as the oocyte's micro-environment, affecting its development and subsequent embryo quality. DESIGN, PARTICIPANTS AND SETTING: In this prospective cohort study, patient information, fertility treatment outcome data, follicular fluid and serum were obtained from women undergoing ART. Patients were categorized according to their BMI (kg/m(2)) as normal (n = 60), overweight (n = 26) or obese (n = 20). Serum and follicular fluid samples were analyzed for urea, total protein, albumin, cholesterol, high-density lipoprotein cholesterol, triglycerides, non-esterified fatty acids, apolipoprotein A1, apolipoprotein B, glucose, lactate, C-reactive protein, insulin-like growth factor -1 (IGF-1), IGF-binding protein 3 (only in follicular fluid), free carnitine and total carnitine. Metabolite concentrations in serum and follicular fluid samples were correlated and were associated with BMI and fertility treatment outcome. MAIN RESULTS: Most serum metabolite differences between patients were reflected in the follicular fluid (P < 0.05). Follicular fluid apolipoprotein A1 and follicular fluid total protein concentrations negatively affected oocyte quality parameters (P < 0.05). However, overall BMI-related associations were poor. BIAS, CONFOUNDING AND OTHER REASONS FOR CAUTION: In this study, we included every patient willing to participate. Within this cohort, women with a BMI transcending 35 kg/m(2) were scarce (n = 2), because extremely overweight women are mostly advised to lose weight before starting ART. Furthermore, the number of patients in each BMI group was different, possibly masking associations between the metabolic composition of serum and follicular fluid and oocyte quality parameters. GENERALIZABILITY TO OTHER POPULATIONS: There were significant associations indicating that metabolic changes in the serum are reflected in the follicular fluid, potentially affecting oocyte quality, irrespective of the patient's BMI. For ethical reasons, this study only focused on women already in need of artificial reproductive treatment. From a metabolic point of view, we consider this cohort as a representative sample of all women of reproductive age. STUDY FUNDING: This study was funded by the special research fund, university of Antwerp (BOF UA). None of the authors has any conflict of interest to declare.

Clinical effectiveness of elective single versus double embryo transfer: meta-analysis of individual patient data from randomised trials.

OBJECTIVE: To compare the effectiveness of elective single embryo transfer versus double embryo transfer on the outcomes of live birth, multiple live birth, miscarriage, preterm birth, term singleton birth, and low birth weight after fresh embryo transfer, and on the outcomes of cumulative live birth and multiple live birth after fresh and frozen embryo transfers. DESIGN: One stage meta-analysis of individual patient data. DATA SOURCES: A systematic review of English and non-English articles from Medline, Embase, and the Cochrane Central Register of Controlled Trials (up to 2008). Additional studies were identified by contact with clinical experts and searches of bibliographies of all relevant primary articles. Search terms included embryo transfer, randomised controlled trial, controlled clinical trial, single embryo transfer, and double embryo transfer. Review methods Comparisons of the clinical effectiveness of cleavage stage (day 2 or 3) elective single versus double embryo transfer after fresh or frozen in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI) treatments were included. Trials were included if the intervention differed only in terms of the intended number of embryos to be transferred. Trials that involved only blastocyst (day five) transfers were excluded. RESULTS: Individual patient data were received for every patient recruited to all eight eligible trials (n=1367). A total of 683 and 684 women randomised to the single and double embryo transfer arms, respectively, were included in the analysis. Baseline characteristics in the two groups were comparable. The overall live birth rate in a fresh IVF cycle was lower after single (181/683, 27%) than double embryo transfer (285/683, 42%) (adjusted odds ratio 0.50, 95% confidence interval 0.39 to 0.63), as was the multiple birth rate (3/181 (2%) v 84/285 (29%)) (0.04, 0.01 to 0.12). An additional frozen single embryo transfer, however, resulted in a cumulative live birth rate not significantly lower than the rate after one fresh double embryo transfer (132/350 (38%) v 149/353 (42%) (0.85, 0.62 to 1.15), with a minimal cumulative risk of multiple birth (1/132 (1%) v 47/149 (32%)). The odds of a term singleton birth (that is, over 37 weeks) after elective single embryo transfer was almost five times higher than the odds after double embryo transfer (4.93, 2.98 to 8.18). CONCLUSIONS: Elective single embryo transfer results in a higher chance of delivering a term singleton live birth compared with double embryo transfer. Although this strategy yields a lower pregnancy rate than a double embryo transfer in a fresh IVF cycle, this difference is almost completely overcome by an additional frozen single embryo transfer cycle. The multiple pregnancy rate after elective single embryo transfer is comparable with that observed in spontaneous pregnancies.

Cumulative live-birth delivery after IVF/ICSI since the progressive introduction of single-embryo transfer.

The only way to decrease the incidence of multiple pregnancies in the IVF/intracytoplasmic sperm injection (ICSI) population is to introduce single-embryo transfer (SET). This study investigated the impact of the progressive introduction of SET for the whole IVF/ICSI population from the patients' point of view by calculating the cumulative live-birth delivery rate. During a 5-year period (2001-2005), the outcome of 2164 cycles with oocyte aspiration in 1047 patients was analysed. A subanalysis was made to calculate the additional effect of frozen-thawed cycles. Survival analysis was performed with the Kaplan-Meier method and the endpoint was live-birth delivery. In this 5-year period, the cumulative live-birth delivery rate per patient was 51% after three IVF/ICSI cycles and 58% after six cycles. With a more permissive method of survival analysis, these results were 64% and 85%, respectively. The additional effect of the frozen-thawed cycles since reimbursement was only 5%. SET was progressively introduced in this period leading to a twin live-birth delivery rate of only 6.7%. It is concluded that a favourable outcome was observed for the cumulative live-birth delivery rate since the introduction of SET but with a disappointing additional effect of the frozen-thawed cycles.