Clinical outcomes of uninterrupted embryo culture with or without time-lapse-based embryo selection versus interrupted standard culture (SelecTIMO): a three-armed, multicentre, double-blind, randomised controlled trial.

BACKGROUND: Time-lapse monitoring is increasingly used in fertility laboratories to culture and select embryos for transfer. This method is offered to couples with the promise of improving pregnancy chances, even though there is currently insufficient evidence for superior clinical results. We aimed to evaluate whether a potential improvement by time-lapse monitoring is caused by the time-lapse-based embryo selection method itself or the uninterrupted culture environment that is part of the system. METHODS: In this three-armed, multicentre, double-blind, randomised controlled trial, couples undergoing in-vitro fertilisation or intracytoplasmic sperm injection were recruited from 15 fertility clinics in the Netherlands and randomly assigned using a web-based, computerised randomisation service to one of three groups. Couples and physicians were masked to treatment group, but embryologists and laboratory technicians could not be. The time-lapse early embryo viability assessment (EEVA; TLE) group received embryo selection based on the EEVA time-lapse selection method and uninterrupted culture. The time-lapse routine (TLR) group received routine embryo selection and uninterrupted culture. The control group received routine embryo selection and interrupted culture. The co-primary endpoints were the cumulative ongoing pregnancy rate within 12 months in all women and the ongoing pregnancy rate after fresh single embryo transfer in a good prognosis population. Analysis was by intention to treat. This trial is registered on the ICTRP Search Portal, NTR5423, and is closed to new participants. FINDINGS: 1731 couples were randomly assigned between June 15, 2017, and March 31, 2020 (577 to the TLE group, 579 to the TLR group, and 575 to the control group). The 12-month cumulative ongoing pregnancy rate did not differ significantly between the three groups: 50·8% (293 of 577) in the TLE group, 50·9% (295 of 579) in the TLR group, and 49·4% (284 of 575) in the control group (p=0·85). The ongoing pregnancy rates after fresh single embryo transfer in a good prognosis population were 38·2% (125 of 327) in the TLE group, 36·8% (119 of 323) in the TLR group, and 37·8% (123 of 325) in the control group (p=0·90). Ten serious adverse events were reported (five TLE, four TLR, and one in the control group), which were not related to study procedures. INTERPRETATION: Neither time-lapse-based embryo selection using the EEVA test nor uninterrupted culture conditions in a time-lapse incubator improved clinical outcomes compared with routine methods. Widespread application of time-lapse monitoring for fertility treatments with the promise of improved results should be questioned. FUNDING: Health Care Efficiency Research programme from Netherlands Organisation for Health Research and Development and Merck.

Women's preferences concerning IVF treatment: a discrete choice experiment with particular focus on embryo transfer policy.

STUDY QUESTION: What outcomes are important for women to decide on the day of embryo transfer (ET) in IVF? SUMMARY ANSWER: The highest cumulative live birth rate (cLBR) per treatment was the most important treatment outcome for women undergoing an IVF treatment, regardless of the number of transfers needed until pregnancy and impact on quality of life. WHAT IS KNOWN ALREADY: Cleavage stage (Day 3) and blastocyst stage (Day 5) ETs are common transfer policies in IVF. The choice for one or the other day of ET differs between clinics. From the literature, it remains unclear whether the day of transfer impacts the cLBR. Patient preferences for the day of ET have not been examined yet. STUDY DESIGN SIZE AND DURATION: A discrete choice experiment (DCE) was performed to investigate female patients' preferences and their values concerning various aspects of an IVF treatment, with a particular focus on ET policy. A multicenter DCE was conducted between May 2020 and June 2020 in which participants were asked to choose between different treatments. Each treatment was presented using hypothetical scenarios containing the following attributes: the probability of a healthy live birth per IVF treatment cycle, the number of embryos available for transfer (for fresh and frozen-thawed ET), the number of ETs until pregnancy and the impact of the treatment on the quality of life. PARTICIPANTS/MATERIALS SETTING METHODS: Women (n = 445) were asked to participate in the DCE at the start of an IVF treatment cycle in 10 Dutch fertility clinics. Participating women received an online questionnaire. The attributes' relative importance was analyzed using logistic regression analyses. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 164 women participated. The most important attribute chosen was the cLBR. The total number of embryos suitable for transfer also influenced women's treatment preferences. Neither the number of transfers needed until pregnancy, nor the impact on quality of life influenced the treatment preferences in the aggregated data. For women in the older age group (age ≥36 years) and the multipara subgroup, the impact on quality of life was more relevant. Naive patients (patients with no prior experience with IVF treatment) assigned less value to the number of ETs needed until pregnancy and assigned more value to the cLBR than the patients who had experienced IVF. LIMITATIONS REASONS FOR CAUTION: An important limitation of a DCE study is that not all attributes can be included, which might be relevant for making choices. Patients might make other choices in real life as the DCE scenarios presented here are hypothetical and might not exactly represent their personal situation. We tried to avoid potential bias by selecting the attributes that mattered most to the patients obtained through patient focus groups. The final selection of attributes and the assigned levels were established using the input of an expert panel of professionals and by performing a pilot study to test the validity of our questionnaire. Furthermore, because we only included women in our study, we cannot draw any conclusions on preferences for partners. WIDER IMPLICATIONS OF THE FINDINGS: The results of this study may help fertility patients, clinicians, researchers and policymakers to prioritize the most important attributes in the choice for the day of ET. The present study shows that cLBR per IVF treatment is the most important outcome for women. However, currently, there is insufficient information in the literature to conclude which day of transfer is more effective regarding the cLBR. Randomized controlled trials on the subject of Day 3 versus Day 5 ETs and cLBR are needed to allow evidence-based counseling. STUDY FUNDING/COMPETING INTERESTS: This work received no specific funding and there are no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Effect of parental and ART treatment characteristics on perinatal outcomes.

STUDY QUESTION: Do parental characteristics and treatment with ART affect perinatal outcomes in singleton pregnancies? SUMMARY ANSWER: Both parental and ART treatment characteristics affect perinatal outcomes in singleton pregnancies. WHAT IS KNOWN ALREADY: Previous studies have shown that singleton pregnancies resulting from ART are at risk of preterm birth. ART children are lighter at birth after correction for duration of gestation and at increased risk of congenital abnormalities compared to naturally conceived children. This association is confounded by parental characteristics that are also known to affect perinatal outcomes. It is unclear to which extent parental and ART treatment characteristics independently affect perinatal outcomes. STUDY DESIGN, SIZE, DURATION: All IVF clinics in the Netherlands (n = 13) were requested to provide data on all ART treatment cycles (IVF, ICSI and frozen-thawed embryo transfers (FET)), performed between 1 January 2000, and 1 January 2011, which resulted in a pregnancy. Using probabilistic data-linkage, these data (n = 36 683) were linked to the Dutch Perinatal Registry (Perined), which includes all children born in the Netherlands in the same time period (n = 2 548 977). PARTICIPANTS/MATERIALS, SETTING, METHODS: Analyses were limited to singleton pregnancies that resulted from IVF, ICSI or FET cycles. Multivariable models for linear and logistic regression were fitted including parental characteristics as well as ART treatment characteristics. Analyses were performed separately for fresh cycles and for fresh and FET cycles combined. We assessed the impact on the following perinatal outcomes: birth weight, preterm birth below 37 or 32 weeks of gestation, congenital malformations and perinatal mortality. MAIN RESULTS AND THE ROLE OF CHANCE: The perinatal outcomes of 31 184 out of the 36 683 ART treatment cycles leading to a pregnancy were retrieved through linkage with the Perined (85% linkage). Of those, 23 671 concerned singleton pregnancies resulting from IVF, ICSI or FET. Birth weight was independently associated with both parental and ART treatment characteristics. Characteristics associated with lower birth weight included maternal hypertensive disease, non-Dutch maternal ethnicity, nulliparity, increasing duration of subfertility, hCG for luteal phase support (compared to progesterone), shorter embryo culture duration, increasing number of oocytes retrieved and fresh embryo transfer. The parental characteristic with the greatest effect size on birth weight was maternal diabetes (adjusted difference 283 g, 95% CI 228-338). FET was the ART treatment characteristic with the greatest effect size on birth weight (adjusted difference 100 g, 95% CI 84-117) compared to fresh embryo transfer. Preterm birth was more common among mothers of South-Asian ethnicity. Preterm birth was less common among multiparous women and women with 'male factor' as treatment indication (compared to 'tubal factor'). LIMITATIONS, REASONS FOR CAUTION: Due to the retrospective nature of our study, we cannot prove causality. Further limitations of our study were the inability to adjust for mothers giving birth more than once in our dataset, missing values for several variables and limited information on parental lifestyle and general health. WIDER IMPLICATIONS OF THE FINDINGS: Multiple parental and ART treatment characteristics affect perinatal outcomes, with birth weight being influenced by the widest range of factors. This highlights the importance of assessing both parental and ART treatment characteristics in studies that focus on the health of ART-offspring, with the purpose of modifying these factors where possible. Our results further support the hypothesis that the embryo is sensitive to its early environment. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by Foreest Medical School, Alkmaar, the Netherlands (grants: FIO 1307 and FIO 1505). B.W.M. reports grants from NHMRC and consultancy for ObsEva, Merck KGaA, iGenomics and Guerbet. F.B. reports research support grants from Merck Serono and personal fees from Merck Serono. A.C. reports travel support from Ferring BV. and Theramex BV. and personal fees from UpToDate (Hyperthecosis), all outside the remit of the current work. The remaining authors report no conflict of interests. TRIAL REGISTRATION NUMBER: N/A.

Transfer of fresh or frozen embryos: a randomised controlled trial.

STUDY QUESTION: Is IVF with frozen-thawed blastocyst transfer (freeze-all strategy) more effective than IVF with fresh and frozen-thawed blastocyst transfer (conventional strategy)? SUMMARY ANSWER: The freeze-all strategy was inferior to the conventional strategy in terms of cumulative ongoing pregnancy rate per woman. WHAT IS KNOWN ALREADY: IVF without transfer of fresh embryos, thus with frozen-thawed embryo transfer only (freeze-all strategy), is increasingly being used in clinical practice because of a presumed benefit. It is still unknown whether this new IVF strategy increases IVF efficacy. STUDY DESIGN, SIZE, DURATION: A single-centre, open label, two arm, parallel group, randomised controlled superiority trial was conducted. The trial was conducted between January 2013 and July 2015 in the Netherlands. The intervention was one IVF cycle with frozen-thawed blastocyst transfer(s) versus one IVF cycle with fresh and frozen-thawed blastocyst transfer(s). The primary outcome was cumulative ongoing pregnancy resulting from one IVF cycle within 12 months after randomisation. Couples were allocated in a 1:1 ratio to the freeze-all strategy or the conventional strategy with an online randomisation programme just before the start of down-regulation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants were subfertile couples with any indication for IVF undergoing their first IVF cycle, with a female age between 18 and 43 years. Differences in cumulative ongoing pregnancy rates were expressed as relative risks (RR) with 95% CI. All outcomes were analysed following the intention-to-treat principle. MAIN RESULTS AND THE ROLE OF CHANCE: Two-hundred-and-five couples were randomly assigned to the freeze-all strategy (n = 102) or to the conventional strategy (n = 102). The cumulative ongoing pregnancy rate per woman was significantly lower in women allocated to the freeze-all strategy (19/102 (19%)) compared to women allocated to the conventional strategy (32/102 (31%); RR 0.59; 95% CI 0.36-0.98). LIMITATIONS, REASONS FOR CAUTION: As this was a single-centre study, we were unable to study differences in study protocols and clinic performance. This, and the limited sample size, should make one cautious in using the results as the basis for definitive policy. All patients undergoing IVF, including those with a poor prognosis, were included; therefore, the outcome could differ in women with a good prognosis of IVF treatment success. WIDER IMPLICATIONS OF THE FINDINGS: Our results indicate that there might be no benefit of a freeze-all strategy in terms of cumulative ongoing pregnancy rates. The efficacy of the freeze-all strategy in subgroups of patients, different stages of embryo development, and different freezing protocols needs to be further established and balanced against potential benefits and harms for mothers and children. STUDY FUNDING/COMPETING INTEREST(S): The Netherlands Organisation for Health Research and Development (ZonMW grant 171101007). S.M., F.M. and M.v.W. stated they are authors of the Cochrane review 'Fresh versus frozen embryo transfers in assisted reproduction'. TRIAL REGISTRATION NUMBER: Dutch Trial Register, NTR3187. TRIAL REGISTRATION DATE: 9 December 2011. DATE OF FIRST PATIENT'S ENROLMENT: 8 January 2013.

The composition of human preimplantation embryo culture media and their stability during storage and culture.

STUDY QUESTION: What is the composition and stability during storage and culture of fifteen commercially available human preimplantation embryo culture media? SUMMARY ANSWER: No two culture media had the same composition, and both storage and culture had an effect on the concentrations of multiple components. WHAT IS KNOWN ALREADY: The choice of embryo culture medium not only affects the success rate of an IVF treatment, but also affects the health of the future child. Exact formulations of embryo culture media are often not disclosed by manufacturers. It is unknown whether the composition of these media changes during storage or culture in the IVF laboratory. Without details on the exact concentrations, it is not possible to determine which components might be responsible for the differences in IVF success rates and health of the resulting children. STUDY DESIGN, SIZE, DURATION: Between October 2014 and October 2015, all complete human preimplantation embryo culture media, i.e. ready to use for IVF, that were commercially available at that time, were included (n = 15). Osmolality and the concentration of thirty seven components including basic elements, metabolites, immunoglobulins, albumin, proteins and 21 amino acids were tested immediately upon arrival into the IVF laboratory, after three days of culture without embryos (sham culture) starting from the day of arrival, just before the expiry date, and after three days of sham culture just before the expiry date. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ions, glucose, immunoglobulins, albumin and the total amount of proteins were quantified using a combination of ion selective electrodes and photometric analysis modules, and lactate, pyruvate and 21 amino acids were analysed by ultra performance liquid chromatography mass spectrometry. Osmolality was analysed by an advanced micro-osmometer. Statistical analysis was done using multivariate general linear models. MAIN RESULTS AND THE ROLE OF CHANCE: The composition varied between media, no two media had the same concentration of components. Storage led to significant changes in 17 of the 37 analyzed components (magnesium, chloride, phosphate, albumin, total amount of proteins, tyrosine, tryptophan, alanine, methionine, glycine, leucine, glutamine, asparagine, arginine, serine, proline, and threonine). Storage affected the osmolality in 3 of the 15 media, but for all media combined this effect was not significant (p = 0.08). Sham culture of the analyzed media had a significant effect on the concentrations of 13 of the 37 analyzed components (calcium, phosphate, albumin, total amount of proteins, tyrosine, alanine, methionine, glycine, leucine, asparagine, arginine, proline, and histidine). Sham culture significantly affected the osmolality of the analysed culture media. Two media contained 50% D-lactate, which a toxic dead-end metabolite. In a secondary analysis we detected human liver enzymes in more than half of the complete culture media. LIMITATIONS, REASONS FOR CAUTION: The analyzed culture media could contain components that are not among the 37 components that were analyzed in this study. The clinical relevance of the varying concentrations is yet to be determined. WIDER IMPLICATIONS OF THE FINDINGS: The presence of D-lactate could be avoided and the finding of human liver enzymes was surprising. The wide variation between culture media shows that the optimal composition is still unknown. This warrants further research as the importance of embryo culture media on the efficacy and safety in IVF is evident. Companies are urged to fully disclose the composition of their culture media, and provide clinical evidence supporting the composition or future changes thereof. STUDY FUNDING/COMPETING INTEREST(S): None.

Age-related gene expression profiles of immature human oocytes.

STUDY QUESTION: What is the difference between the gene expression profiles of single human germinal vesicle (GV) oocytes from women of different ages? SUMMARY ANSWER: There were no statistically significant differences in gene expression profiles of human GV oocytes from women of different ages (range: 25-43). WHAT IS KNOWN ALREADY: It is well established that reproductive capacity declines as women age, which is attributed to oocyte quality since this decline is counterbalanced in older women receiving young donor oocytes. Altered gene expression of human oocytes at different stages of development in relation to female age is one of the suggested mechanisms that could explain the decrease in oocyte quality. STUDY DESIGN, SIZE, DURATION: Between 2012 and 2014, 40 human GV oocytes of 40 women were obtained during follicular aspiration as part of routine ICSI treatment. Gene expression profiles of 38 GV oocytes were determined in four different age groups: 25-30, 31-35, 36-38 and 39-43 years of age. PARTICIPANTS/MATERIALS, SETTING, METHODS: GV oocytes were donated for research and frozen between 3.5 and 7.5 h after follicular aspiration. Subsequently, GV oocytes were thawed and prepared for gene expression profile analysis using Agilent microarrays containing ~42 000 Human Gene Expression probe-sets. Gene expression profiles were visualized by hierarchical clustering and the top 500 most differing genes were determined by multidimensional scaling (MDS). Transcripts were analysed in a class comparison between the four age groups and for indicators of biological age: antral follicle count (AFC) and the total dosage of FSH needed for ovarian stimulation. Individual transcripts were analysed using linear regression. A false discovery rate <0.05 was considered statistically significant. MAIN RESULTS AND THE ROLE OF CHANCE: Visualization of gene expression profiles of GV oocytes with hierarchal clustering and MDS demonstrated no clear grouping of samples based on female age, AFC or FSH dosage. The gene expression profile of GV oocytes classified in four age groups revealed no significantly differentially expressed genes between the four different age groups. There were also no significantly differentially expressed genes in the linear regression analysis for individual transcripts against age. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: Immature (GV) oocytes obtained from ovarian stimulation cycles were used. Findings may therefore differ for oocytes at other developmental stages and for in-vivo matured oocytes under physiological conditions. Due to our relatively large, but still limited study sample (40 GV oocytes), we cannot exclude that there might be smaller age-related gene-expression differences, i.e. due to a lack of power. WIDER IMPLICATIONS OF THE FINDINGS: We did not find an effect of female age on gene expression profiles of individual human GV oocytes. Other studies have suggested that gene-expression profiles are affected in mature oocytes, which might imply that female age affects oocyte maturation. Alternatively, other mechanisms in human oocytes might cause the age-related fertility decline. STUDY FUNDING/COMPETING INTEREST(S): This study received no external funding and there are no competing interests.

High-quality human preimplantation embryos actively influence endometrial stromal cell migration.

PURPOSE: The purpose of this paper is to study whether human preimplantation embryos regulate endometrial stromal cell (hESC) migration. METHODS: Primary hESCs were isolated from fertile patients undergoing hysterectomy for benign conditions (uterine scar niche n = 3, dysmenorrhea n = 2; no hormonal treatment). Migration and proliferation assays were performed by culturing decidualized or non-decidualized hESCs in the presence of embryo conditioned medium (ECM) from high-quality embryos (fragmentation ≤ 20%) or from low-quality embryos (fragmentation > 20%) or in non-conditioned medium from the same dishes (control). ECM samples from 425 individually cultured human embryos were used in this study. RESULTS: ECM from high-quality embryos, i.e., with a low percentage of fragmentation, actively stimulated decidualized hESC migration (p < 0.001). This effect was consistent throughout embryonic development from cleavage stage embryos with 2-7 cells (high quality vs. control; p = 0.036), 8-18 cells (high quality vs. control; p < 0.001) to morulae (high quality vs. control; p = 0.003). Additionally, linear regression analysis showed that hESC migration was influenced by embryo quality (fragmentation, ß - 0.299; p = 0.025) and not developmental stage (cell number, ß 0.177; p = 0.176) or maternal age (ß - 0.036; p = 0.78). Opposite to decidualized hESCs, the migration response of non-decidualized hESCs was inhibited by ECM from high-quality embryos (p = 0.019). ECM from low-quality embryos, i.e., with a high percentage of fragmentation, did not cause an altered migration response in decidualized hESCs (p = 0.860) or non-decidualized hESCs (p = 0.986). Furthermore, ECM of both high- and low-quality human embryos did not influence the number of proliferating cells (p = 0.375) and the cell cycle time (p = 0.297) of non-decidualized or decidualized hESCs. CONCLUSION: This study reveals a mechanism by which high-quality human preimplantation embryos actively interact with the endometrium to increase their chances of successful implantation.

Equipoise and the RCT.

Clinical decisions in reproductive medicine are often made in uncertainty. To reduce uncertainty and to improve clinical decision-making, RCTs are increasingly called upon. A key concept underpinning the ethics of RCTs is equipoise. Here, we aimed to dissect the basic reasoning behind the concept of equipoise and we proposed a line of thinking delineating under which conditions it is ethical to design and execute an RCT. This might prevent a priori negative trials, reduce research waste and aid in the design of meaningful ones. It is these trials that will provide insight on how to safely and effectively assist subfertile couples.

The addition of a low-quality embryo as part of a fresh day 3 double embryo transfer does not improve ongoing pregnancy rates.

STUDY QUESTION: Does the addition of a low-quality embryo in fresh Day 3 double embryo transfer (DET) affect the ongoing pregnancy rate (OPR) and multiple gestation rate in patients with only one or no high-quality embryos available? SUMMARY ANSWER: In patients with only one- or no high-quality embryo available, the addition of a low-quality embryo in fresh Day 3 DET does not improve the OPR but increases multiple gestation rates in fresh DET. WHAT IS KNOWN ALREADY: Pregnancy rates after DET are considered to be higher compared to single embryo transfer (SET) when analyzed per first embryo transfer only. However, these conclusions are based on RCTs in which mostly patients with two or more high-quality embryos were included, and can therefore not be applied to patients with only one or no high-quality embryo available. This is particularly relevant since it has been suggested that low-quality embryos could impair the implantation of simultaneously transferred embryos by paracrine signaling. Hence, we investigated in patients with only one or no high-quality embryo available whether the addition of a low-quality embryo in DET affects the OPR, multiple gestation rate and miscarriage rate. STUDY DESIGN SIZE DURATION: This was a retrospective cohort study of 5050 patients receiving 7252 fresh embryo transfers on Day 3 after fertilization in IVF/ICSI cycles from 2012 to 2015 in two academic hospitals. PARTICIPANTS/MATERIALS SETTING METHODS: We included all women that received fresh SET or DET with any combination of high-quality embryos (7, 8 or 9 blastomeres, with equal to or <20% fragmentation) or low-quality embryos (all other embryos). Outcomes were OPR (primary outcome, defined as a positive fetal heartbeat by transvaginal ultrasound at least 10 weeks after oocyte retrieval), miscarriage rate and multiple gestation rate. We used a generalized estimating equations model adjusting for maternal age, number of oocytes retrieved, center of treatment and the interaction between maternal age and number of oocytes retrieved. Other baseline characteristics, including infertility diagnosis, fertilization method and the number of consecutive fresh embryo transfers per patient, did not contribute significantly to the GEE model and were therefore excluded, and not adjusted for. MAIN RESULTS AND THE ROLE OF CHANCE: Compared to SET with one high-quality embryo, DET with two high-quality embryos resulted in a higher OPR (adjusted odds ratio (OR) 1.38, 95% CI 1.14-1.67), while DET with one high- and one low-quality embryo resulted in a lower OPR (adjusted OR 0.65, 95% CI 0.49-0.90). However, SET in patients with only one high-quality embryo available resulted in a lower OPR compared to SET in patients with two or more high-quality embryos available (adjusted OR 0.52, 95% CI 0.39-0.70). After adjusting for this confounding factor, we found that both DET with two high-quality embryos (adjusted OR 0.99, 95% CI 0.74-1.31) and DET with one high- and one low-quality embryo (adjusted OR 0.78, 95% CI 0.47-1.27) resulted in a not significantly different OPR compared to SET with one high-quality embryo. If only low-quality embryos were available, DET did not increase the OPR as compared to SET with one low-quality embryo (adjusted OR 0.84, 95% CI 0.55-1.28). Multiple gestation rates were higher in all DET groups compared to SET (DET with ≥1 high-quality embryo(s) compared to SET with one high-quality embryo; DET with two low-quality embryos compared to SET with one low-quality embryo; all comparisons P < 0.001). Miscarriage rates were not different in all DET groups compared to SET (DET with ≥1 high-quality embryo(s) compared to SET with one high-quality embryo; DET with two low-quality embryos compared to SET with one low-quality embryo; all comparisons P > 0.05). LIMITATIONS REASONS FOR CAUTION: Limitations to this study include the retrospective design and possible bias between study groups related to embryo transfer policies between 2012 and 2015. Consequently, we may have underestimated pregnancy chances in all DET groups. Furthermore, the OPR was calculated as a percentage of the number of fresh embryo transfers in each study group, and not the total number of started IVF/ICSI cycles. Therefore, the reported pregnancy outcomes may not truly reflect the pregnancy chances of couples at the start of treatment. A possible confounding effect of maternal age in our study is acknowledged but we could not compare clinical outcomes in different age groups separately owing to small sample sizes. Analysis of pregnancy outcomes in lower prognosis patients (higher maternal age, fewer oocytes retrieved) separately is an avenue for future research. WIDER IMPLICATIONS OF THE FINDINGS: The decision to perform DET rather than SET in order to increase the OPR per fresh embryo transfer seems not to be justified for those patients with only one or no high-quality embryo(s) available. However, owing to the limitations of this study, prospective RCTs are needed that specifically investigate pregnancy outcomes in patients with only one or no high-quality embryo(s) available in SET and DET. STUDY FUNDING/COMPETING INTERESTS: This study was funded by a grant from the joint Amsterdam Reproduction & Development Institute of the Academic Medical Center and VU University Medical Center (www.amsterdam-reproduction-and-development.org). The authors have no conflicts of interest to declare.

Factors affecting the gene expression of in vitro cultured human preimplantation embryos.

STUDY QUESTION: What is the relative effect of common environmental and biological factors on transcriptome changes during human preimplantation development? SUMMARY ANSWER: Developmental stage and maternal age had a larger effect on the global gene expression profile of human preimplantation embryos than the culture medium or oxygen concentration used in in vitro culture. WHAT IS KNOWN ALREADY: Studies on mouse and bovine embryos have shown that different conditions in the in vitro culture of embryos can lead to changes in transcriptome profiles. For humans, an effect of developmental stage on the transcriptome profile of embryos has been demonstrated, but studies on the effect of maternal age or culture conditions are lacking. STUDY DESIGN, SIZE, DURATION: Donated, good quality, day 4 cryopreserved human preimplantation embryos (N = 89) were randomized to be cultured in one of two culture media (G5 medium or HTF medium) and one of two oxygen concentrations (5% or 20%), with stratification for maternal age. Next to these variables, developmental stage after culture was taken into account in the analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Embryos that developed to morula or blastocyst stage during these 2 days whose amplified mRNA passed our quality control criteria for microarray hybridization were individually examined for genome-wide gene expression (N = 37). MAIN RESULTS AND THE ROLE OF CHANCE: Based on the number of differentially expressed genes (DEGs), developmental stage (3519 DEGs) and maternal age (1258 DEGs) had a larger effect on the global gene expression profile of human preimplantation embryos than either tested culture medium (596 DEGs) or oxygen concentration (492 DEGs) used during in vitro culture. Interactions between the factors were found, indicating that culture conditions might have a different effect depending on the developmental stage or the maternal age of the embryos. Affected pathways included metabolism, cell cycle processes and oxidative phosphorylation. LIMITATIONS, REASONS FOR CAUTION: Culture of embryos for only 2 days might have limited the effect on global gene expression by the investigated culture conditions. Earlier stages of development (Day 0 until Day 4) were not analyzed and these embryos might respond differently to the experimental conditions. The freezing and thawing procedures might have had an effect on gene expression. RT-PCR validation was not performed due to scarcity of the material. WIDER IMPLICATIONS OF THE FINDINGS: Our results show that when studying gene expression in single human preimplantation embryos under various experimental conditions, one should take into account the confounding effect of biological variables, such as developmental stage and maternal age. This makes these experiments different from gene expression experiments where these variables can be tightly controlled, for example when using cell lines. STUDY FUNDING/COMPETING INTERESTS: This study received no external funding and there were no competing interests.

Pathophysiological aspects of thyroid hormone disorders/thyroid peroxidase autoantibodies and reproduction.

BACKGROUND: Thyroid hormone disorders and thyroid peroxidase autoantibodies (TPO-Ab) in women are associated with subfertility and early pregnancy loss. Here, we aim to provide a comprehensive overview of the literature on the pathophysiology of these associations. METHODS: A review of the literature in the English language was carried out. Relevant studies were identified by searching Medline, EMBASE and the Cochrane Controlled Trials Register from 1975 until March 2014. RESULTS: From a total of 6108 primary selected articles from the literature search, 105 articles were selected for critical appraisal. Observational data indicate that altered thyroid hormone levels are associated with disturbed folliculogenesis, spermatogenesis, lower fertilization rates and lower embryo quality. Triiodothyronine (T3) in combination with FSH enhances granulosa cell proliferation and inhibits granulosa cell apoptosis by the PI3K/Akt pathway. T3 is considered a biological amplifier of the stimulatory action of gonadotrophins on granulosa cell function. T3 increases the expression of matrix metalloproteinases (MMP), MMP-2, MMP-3, fetal fibronectin and integrin α5ß1T3 in early placental extravillous trophoblasts. Thyroid hormone transporters and receptors are expressed in the ovary, early embryo, endometrium, uterus and placenta. No other data explaining the associations could be retrieved from the literature. The presence of TPO-Ab is negatively associated with spermatogenesis, fertilization and embryo quality, but no data are available on the potential pathophysiological mechanisms. CONCLUSIONS: Thyroid hormone disorders and TPO-Ab are associated with disturbed folliculogenesis, spermatogenesis, fertilization and embryogenesis. The pathophysiology of these associations remains largely unknown, as evidence is limited and includes studies using small sample sizes, and often restricted to animal models. There are no studies on the pathophysiology underlying the association between TPO-Ab and reproduction. The available evidence, although limited, supports a role of thyroid hormone in fertility and early pregnancy. This justifies clinical intervention studies on the effects of thyroid hormone supplementation in women with subclinical hypothyroidism and in women prone to develop hypothyroidism due to the presence of TPO-Ab. In addition, more research is needed to identify the underlying mechanisms. This would be of particular interest in women undergoing IVF to pinpoint the effects of thyroid hormone on different parameters of reproduction.

Time-lapse in the IVF-lab: how should we assess potential benefit?

Time-lapse imaging of embryos has been widely introduced to fertility laboratories worldwide with the aim of identifying the best quality embryos to transfer that will ultimately improve IVF success rates. In this opinion paper, we explore the lack of evidence of benefit of this novel intervention, analyse the methodological flaws of current studies, offer ideal study designs that assess the various features of time-lapse imaging, and discuss forthcoming studies. In particular, we emphasize the ethical aspects of hastily adopting a costly technology without current high level evidence of improved live birth rates, safety and cost effectiveness.

Embryo culture media and IVF/ICSI success rates: a systematic review.

BACKGROUND The media that are used to culture human preimplantation embryos are considered to be an important factor for the success rates of IVF/ICSI. Here, we present a systematic review of randomized controlled trials (RCTs) on the effect of culture media on IVF/ICSI success rates. METHODS RCTs published between January 1985 and July 2012 were eligible for inclusion. The primary outcome was live birth. Secondary outcomes were health of babies born, ongoing pregnancies, clinical pregnancies, miscarriages, multiple pregnancies, implantation rate, cryopreservation rate, embryo quality and fertilization rate. For those media that were evaluated in more than one comparison, an unconventional meta-analysis was performed by pooling the data of the media they were compared to. RESULTS Twenty-two RCTs were included that evaluated 31 different comparisons. Conventional meta-analysis was not possible for any of the outcomes as nearly all trials compared different culture media. Only four trials reported on live birth, and one of them reported a significant difference. Nine trials reported on ongoing and/or clinical pregnancy rates, of which four showed a significant difference. Pooling the data did not reveal a superior culture medium. CONCLUSIONS It is yet unknown what culture medium leads to the best success rates in IVF/ICSI. Given the potential importance of culture media for treatment outcome, rigorously designed RCTs are needed for currently available, as well as newly introduced culture media.

The effect of recombinant LH on embryo quality: a randomized controlled trial in women with poor ovarian reserve.

BACKGROUND: Poor ovarian response is a common clinical problem, affecting up to 26% of IVF cycles. For these women, addition of recombinant luteinizing hormone (rLH) to ovarian hyperstimulation with recombinant FSH has a beneficial effect on ongoing pregnancy rates, but its effect on the yield of top-quality embryos is unknown. METHODS: We conducted a randomized controlled trial in women expected to respond poorly under ovarian hyperstimulation during their first IVF cycle [all women aged 35-41 and women with FSH > 12 IU/ml and antral follicle count (AFC) ≤ 5]. Women were randomly allocated to rFSH and rLH (2:1 ratio) or rFSH alone (control group) after down-regulation with a GnRH agonist. The primary outcome was the proportion of top-quality embryos per woman on the day of transfer. Secondary outcomes were the number of stimulation days, the number of follicles ≥17 mm, the number of oocytes, the fertilization rate, the number of embryos, the number of women with ≥1 top-quality embryo, the biochemical, clinical and ongoing pregnancy rates and the miscarriage rate. RESULTS: There were 116 women allocated to the rLH group and 128 allocated to the control group. The proportion of top-quality embryos per woman was 17% in the rLH group and 11% in the control group [mean difference 0.06; 95% confidence interval (CI) -0.01-0.14]. In the rLH and control groups respectively, 47 (41%) and 41 (32%) women had at least one top-quality embryo on the day of transfer (relative risk: 1.3, 95% CI 0.91-1.77). The ongoing pregnancy rate was 13 versus 12% (relative risk: 1.1; 95% CI 0.57-2.16) for the rLH group compared with the control group. CONCLUSIONS: This study found no significant difference in embryo quality after the addition of rLH to rFSH for ovarian stimulation in women with poor ovarian reserve. CLINICAL TRIALS IDENTIFIER: NTR1457.

Preimplantation genetic screening: a systematic review and meta-analysis of RCTs.

BACKGROUND: Preimplantation genetic screening (PGS) has increasingly been used in the past decade. Here we present a systematic review and meta-analysis of RCTs on the effect of PGS on the probability of live birth after IVF. METHODS: PubMed and trial registers were searched for RCTs on PGS. Trials were assessed following predetermined quality criteria. The primary outcome was live birth rate per woman, secondary outcomes were ongoing pregnancy rate, miscarriage rate, multiple pregnancy rate and pregnancy outcome. RESULTS: Nine RCTs comparing IVF with and without PGS were included in our meta-analysis. Fluorescence in situ hybridization was used in all trials and cleavage stage biopsy was used in all but one trial. PGS significantly lowered live birth rate after IVF for women of advanced maternal age (risk difference: -0.08; 95% confidence interval: -0. 13 to -0.03). For a live birth rate of 26% after IVF without PGS, the rate would be between 13 and 23% using PGS. Trials where PGS was offered to women with a good prognosis and to women with repeated implantation failure suggested similar outcomes. CONCLUSIONS: There is no evidence of a beneficial effect of PGS as currently applied on the live birth rate after IVF. On the contrary, for women of advanced maternal age PGS significantly lowers the live birth rate. Technical drawbacks and chromosomal mosaicism underlie this inefficacy of PGS. New approaches in the application of PGS should be evaluated carefully before their introduction into clinical practice.

What next for preimplantation genetic screening? More randomized controlled trials needed?

The recent debate on preimplantation genetic screening (PGS) has raised questions about its routine use in clinical practice. It has been suggested that the most effective way to resolve the debate about the usefulness of PGS is to perform more well-designed and well-executed randomized controlled trials (RCTs). However, in view of the lack of evidence for the effectiveness of PGS and the accumulating evidence for its harmfulness, it is our opinion that it is unethical to perform additional RCTs for the indication advanced maternal age using cleavage stage biopsy.

Preimplantation genetic screening for abnormal number of chromosomes (aneuploidies) in in vitro fertilisation or intracytoplasmic sperm injection.

BACKGROUND: In both in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI), selection of the most competent embryo(s) for transfer is generally based on morphological criteria. However, many women fail to achieve a pregnancy after transfer of good quality embryos. One of the presumed causes is that such morphologically normal embryos show an abnormal number of chromosomes (aneuploidies). In preimplantation genetic screening (PGS), embryos are analysed for aneuploidies and only embryos that are euploid for the chromosomes tested are transferred. This technique has been suggested and used to improve pregnancy rates for the following indications: (i) advanced maternal age, (ii) repeated IVF failure, (iii) repeated miscarriage and (iv) testicular sperm extraction (TESE)-ICSI. Although PGS is used more and more often, its effectiveness is still unclear. OBJECTIVES: To assess the effectiveness of PGS in terms of live births in women undergoing IVF or ICSI treatment. SEARCH STRATEGY: We searched the Cochrane Menstrual Disorders and Subfertility Group Trials Register, the Cochrane Central Register of Controlled Trials (CENTRAL) (Cochrane Library Issue 1, 2005), MEDLINE (1966 to present) and EMBASE (1980 to present) (searched March 2005) and reference lists of articles. We also contacted authors for providing additional data when necessary. SELECTION CRITERIA: Trials for all four suggested indications as mentioned above were sought. All relevant published randomised controlled trials were selected. They were eligible for inclusion if the comparison dealt with IVF/ICSI with PGS versus IVF/ICSI without PGS. DATA COLLECTION AND ANALYSIS: Relevant data were extracted independently by two authors. All trials were screened and analysed according to predetermined quality criteria. Validity was assessed in terms of method of randomisation, completeness of follow-up, intention-to-treat analysis and presence or absence of blinding. The primary outcome measure was live birth rate per woman. Secondary outcome measures were the proportion of women reaching embryo transfer, mean number of embryos transferred per transfer, clinical pregnancy rate, multiple pregnancy rate, miscarriage rate, ongoing pregnancy rate, proportion of women reaching embryo transfer after cryopreservation and proportion of women whose child has a congenital malformation. MAIN RESULTS: Two randomised controlled trials met our predetermined eligibility criteria. These trials used PGS for advanced maternal age. The primary outcome of live birth rate per woman was not significantly different in the PGS and control groups, though data were only available from one study. The live birth rate was 11% (21 out of 199) in the PGS group, versus 15% (29 out of 190) in the control group (OR 0.65; 95% CI 0.36 to 1.19). For a control group rate of 15%, these data suggest a live birth rate using PGS of between 4% and 17%. Ongoing pregnancy rate was provided in both studies. This was not significantly different with a combined odds ratio of 0.64 (95% CI 0.37 to 1.09). For a control group rate of 20%, this suggests an ongoing pregnancy rate using PGS of between 8% and 21%. AUTHORS' CONCLUSIONS: To date there is insufficient data to determine whether PGS is an effective intervention in IVF/ICSI for improving live birth rates. Available data on PGS for advanced maternal age showed no difference in live birth rate and ongoing pregnancy rate. However, only two randomised trials were found, of which one included only 39 patients. For both studies comments on their methodological quality can be made. Therefore more properly conducted randomised controlled trials are needed. Until such trials have been performed PGS should not be used in routine patient care.

[Preimplantation genetic screening for numerical chromosomal abnormalities in embryos from women of 35 years of age and older; first results in The Netherlands]. / Preïmplantatiegenetische screening op numerieke chromosoomafwijkingen bij embryo's van vrouwen van 35 jaar en ouder; de eerste resultaten in Nederland.

OBJECTIVE: To assess the results of preimplantation genetic screening (PGS) for numerical chromosomal abnormalities in embryos from women of 35 years of age and older. DESIGN: Prospective, descriptive. METHOD: Women who were at least 35 years received standard IVF/ICSI treatment including ovarian hyperstimulation, after which matured oocytes were recovered and inseminated. Three days after insemination, one cell was biopsied from each of the available embryos. In these cells, the copy number of 5 (first 21 patients) or 8 chromosomes was determined using fluorescence in situ hybridisation (FISH). Only embryos with a normal or unknown FISH result were implanted in the uterus. Data were collected in an electronic database. RESULTS: PGS was done for 28 IVF- and 22 ICSI-treatments; the average age of the 50 women at the beginning of treatment was 38.5 years. There were 360 embryos generated; of the 288 biopsied embryos 156 (54%) contained an abnormal number of chromosomes. In 45 women, 1 or 2 embryos were transferred. This resulted in 8 ongoing pregnancies (8/50; 16%) and the birth of 9 children, all of whom were found to be healthy on a paediatric examination at 3 to 10 months of age. In 4 cases there was no embryo transfer because all the embryos were chromosomally abnormal. CONCLUSION: In the first 50 patients in The Netherlands, PGS resulted in an ongoing pregnancy rate of 16% per woman. All children showed normal growth and development.