The purpose of this study was to compare blastocyst development on the same cohort of oocytes using a single-step medium with or without three antioxidants (α-lipoic acid, acetyl L-carnitine and acetyl L-cysteine). A multicentre, prospective randomized, double blinded, sibling oocyte trial. Couples were enrolled in the study after fertilization check (day 1). A minimum of 6 normally fertilized oocytes were required for participation. Embryos were randomly allocated (1:1) into two study arms comparing embryo development on a time-lapse system (under 5% O2) using a single-step culture medium with (G-TLX) or without (G-TL) antioxidants. Percentage of good quality blastocysts (GQB) per fertilized oocyte on day 5 was the primary end-point in the study. One hundred and twenty six patients participated in the study resulting in 1167 zygotes that were randomized into the two study arms. There were similar numbers of top quality embryos on day 3 in the G-TL (20.2%) and G-TLX (18.8%) groups, p=0.58. Percentage of day 5 good quality blastocysts was 29.8% (SD 28.2%) and 30.2% (SD 28.5%) in the G-TL and G-TLX groups, respectively. The mean difference 0.45 (95% CI -5.39; 6.29) between the two media systems for the primary end-point was non-significant (p = 0.88). However, the ongoing clinical pregnancy rate was 41.5 % vs 54.8% in G-TL (n=41) and G-TLX (n=49), respectively, p=0.38, although, the study was not powered for significance of clinical outcome data. Addition of the combination of antioxidants (α-lipoic acid, acetyl L-carnitine and acetyl L-cysteine) to sequential media has previously been shown to improve embryo quality and subsequent viability in the mouse model. Although we could not measure any effect on the embryo development or blastocyst morphology grading by the addition of the antioxidants to a single-step culture medium, there was a tendency towards a better pregnancy rate in the medium containing the antioxidants, indicating possible positive effects of this combination of antioxidants on embryo viability when cultured in-vitro.

The use of time-lapse monitoring in ART is spreading rapidly and is already considered an important part of treatment in many centers internationally, but studies are lacking on patients experience of using this technology. We consider it important not to forget patient-oriented aspects of new technologies. The study question was therefore; how do patients value the chance to view time-lapse sequences of their embryos at the time of embryo transfer? We performed a prospective, observational questionnaire study at three of IVF Sweden`s centers, including 242 patients. We report the results from self-evaluated scores of patient´s experience which were reported anonymously. Participants in the study were patients who were offered to view time-lapse sequences of embryos at the time of embryo transfer. A 5-grade Likert-scale instrument was used for assessing patient’s experience. A majority of the patients found viewing the sequences to be a relevant and important aspect of their treatments. Patients felt increased participation in, and increased satisfaction with the treatment. There was a clear interest in obtaining a copy of the time-lapse sequence but only if the treatment resulted in a pregnancy. If time-lapse technology is available in a clinic it could be an additive value to share the sequences with the patients as deepened information about their treatment. If a copy of the sequences is made available to the patients we recommend to delay the time for that at least until there is a verified ongoing pregnancy.

STUDY QUESTIONWhat is the value of embryo selection by metabolomic profiling of culture medium with near-infrared (NIR) spectroscopy as an adjunct to morphology, compared with embryo selection by morphology alone, based on an individual patient data meta-analysis (IPD MA)?SUMMARY ANSWERThe IPD MA indicates that the live birth rate after embryo selection by NIR spectroscopy and morphology is not significantly different compared with the live birth rate after embryo selection by morphology alone.WHAT IS KNOWN ALREADYRetrospective proof of principle studies has consistently shown that high NIR viability scores are correlated with a high implantation potential of embryos. However, randomized controlled trials (RCTs) have generally shown no benefit of the NIR technology over embryo morphology, although there have been some conflicting results between pregnancy outcomes on different days of embryo transfer.STUDY DESIGN, SIZE, DURATIONThis IPD MA included all existing RCTs (n = 4) in which embryo selection by morphology was compared with embryo selection by morphology and the use of NIR spectroscopy of spent embryo culture medium by the Viametrics-E(™).PARTICIPANTS/MATERIALS, SETTING, METHODSSearches of PubMed, the Cochrane Library and the WHO International Clinical Trials Registry were conducted and the sole manufacturer of the Viametrics-E(™) was consulted to identify clinics where an RCT comparing embryo selection by morphology to embryo selection by morphology and the use of the Viametrics-E(™) (NIR viability score) was performed. A total of 20 citations were potentially eligible for inclusion, two of which met the inclusion criteria. The manufacturer of the Viametrics-E(™) provided two additional clinical sites of use. In total, four RCTs were identified as eligible for inclusion. The IPD MA was based on a fixed effect model due to the lack of heterogeneity between included studies. Differences between study groups were tested and reported using logistic regression models adjusted for significant confounders. The pooled analysis of the primary outcome led to a total sample size of 924 patients: 484 patients in the control group (embryo selection by morphology alone) and 440 patients in the treatment group (embryo selection by morphology plus NIR spectroscopy).MAIN RESULTS AND THE ROLE OF CHANCEThe live birth rates in the control group and the NIR group were 34.7% (168 of 484) and 33.2% (146 of 440), respectively. The pooled odds ratio (OR) was 0.98 [95% confidence interval (CI) 0.74-1.29], indicating no difference in live birth rates between the two study groups. The data of the four studies showed no significant heterogeneity (I(2) = 26.2% P = 0.26). The multivariate regression analysis including all confounders show that maternal age (OR 0.90, 95% CI 0.87-0.94) and the number of previous IVF cycles (OR 0.83, 95% CI 0.71-0.96) were significantly related to live birth. The study group (i.e. embryo selection by morphology or embryo selection by morphology plus NIR) was not related to live birth (OR 0.97, 95% CI 0.73-1.29).LIMITATIONS AND REASONS FOR CAUTIONThe availability of at least two similar best quality embryos as an inclusion criterion prior to transfer in the two largest RCTs might have caused a selection bias towards a better prognosis patient group.WIDER IMPLICATIONS OF THE FINDINGSThere is at present no evidence that NIR spectroscopy of spent embryo culture media in its current form can be used in daily practice to improve live birth rates.

STUDY QUESTION What pre-freeze and post-thaw morphological parameters can be used to predict live birth outcomes after frozen-thawed blastocyst transfer cycles? SUMMARY ANSWER Pre-freeze blastocoele expansion and trophectoderm (TE) grade and post-thaw degree of re-expansion are the most significant predictors of live birth in frozen-thawed blastocyst transfer cycles. WHAT IS KNOWN ALREADY Currently, blastocoele re-expansion after thawing is used to indicate blastocyst cryosurvival and reproductive potential. The predictive roles of other pre-freeze and post-thaw morphological parameters are neglected. STUDY DESIGN, SIZE, DURATION This was a retrospective study of all the patients who received a frozen-thawed single blastocyst transfer (n = 1089) at our clinic between March 2008 and October 2011. PARTICIPANTS/MATERIALS, SETTING, METHODS Pre-freeze morphological parameters analyzed for all blastocysts included grade of blastocoele expansion, inner cell mass and TE. A group of blastocysts (n = 243) were also graded for post-thaw parameters: degree of blastocoele re-expansion, viability and cell contour. Univariate and multivariate generalized estimating equations (GEEs) models were used to identify the confounders that statistically significantly affected live birth outcomes and to investigate the independent effect of significant pre-freeze and post-thaw morphological parameters. Stepwise logistic regression analysis was used to select the best independent morphological predictors of live birth. Pearson correlations and linear regression analyses were performed to determine the relationship between morphological parameters and possible covariates. MAIN RESULTS AND THE ROLE OF CHANCE Multivariate GEE models estimated that the odds of live birth increased by ∼36% for each grade of expansion (P = 0.0061) and decreased by 29% for blastocysts with grade B TE compared with grade A TE (P = 0.0099). Furthermore, the odds of live birth increased by ∼39% (P = 0.0042) for each 10% increase in degree of re-expansion. Blastocoele expansion and TE grade were selected as the most significant pre-freeze morphological predictors of live birth and degree of re-expansion was selected as the best post-thaw parameter for prediction of live birth. LIMITATIONS, REASONS FOR CAUTION Blastocysts with poorer grades of morphology were not cryopreserved or transferred, limiting the ability to generalize our findings for grades of morphology not included in this study. WIDER IMPLICATIONS OF THE FINDINGS Blastocysts with higher pre-freeze grades of expansion and TE, irrespective of day of cryopreservation, should be given priority when thawing. Subsequently, re-expanding blastocysts, assessed within 2-4 h, with >60% viability should be transferred. STUDY FUNDING/COMPETING INTEREST(S) No external funding was obtained for this study. There was no competing interest. TRIAL REGISTRATION NUMBER not applicable.

BACKGROUND:In order to select the best blastocyst for transfer, in humans, three morphological parameters have routinely been used, i.e. degree of blastocoele expansion and appearance of both the trophectoderm (TE) and the inner cell mass (ICM). Although it has been shown that blastocysts with highest scores for all three parameters achieve highest implantation rates, their independent ability to predict pregnancy outcome remains unclear.METHOD:This study is a retrospective analysis of 1117 fresh day 5 single blastocyst transfers and their live birth outcome related to each morphological parameter.RESULTS:All three parameters had a significant effect on live birth however, once adjusted for known significant confounders, it was shown that TE was the only statistically significant independent predictor of live birth outcome.CONCLUSIONS:This study has shown, for the first time, the predictive strength of TE grade over ICM for selecting the best blastocyst for embryo replacement. It may be that, even though ICM is important, a strong TE layer is essential at this stage of embryo development, allowing successful hatching and implantation.

ObjectiveIn Sweden, as a rule, single embryo transfer (SET) is recommended to avoid multiple pregnancy. Thus, a high implantation rate is required to maintain good clinical results in IVF.In 2006, blastocyst culture and vitrification was introduced in our clinic on a large scale. The objective was to increase the cumulative live birth rate (CLBR) per ovum pick-up (OPU). In order to evaluate the outcome of this introduction of blastocyst culture we now have analysed the CLBR for our patients.DesignA retrospective analysis.Materials and MethodsWe calculated the CLBR per OPU in a three-year period before the introduction, 2003-2005 and compared with the following period of 2006-2008. We included all patients who started their treatments and had OPU in these two periods including all frozen/thawed cycles. Several variables known to affect the birth rate after IVF were adjusted for in the analysis: female age, number of follicles, aspirated oocytes, previous IVF cycles, SET, total FSH dose, indication and method. In order to study live birth between the two periods adjusted for confounding variables multiple logistic regression models were used with the method of generalized estimating equations in order to adjust for the dependence within women.ResultsThe results were based on 2738 OPU (fresh + all frozen/thawed) in 2003-2005 and 2720 in 2006-2008. The CLBR per OPU for 2003-2005 and 2006-2008 was 29.8% respectively 34.8% (P<0.0001). The adjusted OR for CLBR per OPU between the two periods was 1.35 (1.18-1.54) in favour the period with blastocyst culture (P<0.0001). Adjustments were made for all variables given in methods.ConclusionDuring these two 3-year periods the only major change performed in our clinic was the introduction of blastocyst culture and vitrification. The patients' demographics during these two time periods were similar although the proportion of SET increased. The implementation of blastocyst culture along with vitrification of blastocysts significantly increased the CLBR per OPU. ObjectiveIn Sweden, as a rule, single embryo transfer (SET) is recommended to avoid multiple pregnancy. Thus, a high implantation rate is required to maintain good clinical results in IVF.In 2006, blastocyst culture and vitrification was introduced in our clinic on a large scale. The objective was to increase the cumulative live birth rate (CLBR) per ovum pick-up (OPU). In order to evaluate the outcome of this introduction of blastocyst culture we now have analysed the CLBR for our patients. In Sweden, as a rule, single embryo transfer (SET) is recommended to avoid multiple pregnancy. Thus, a high implantation rate is required to maintain good clinical results in IVF. In 2006, blastocyst culture and vitrification was introduced in our clinic on a large scale. The objective was to increase the cumulative live birth rate (CLBR) per ovum pick-up (OPU). In order to evaluate the outcome of this introduction of blastocyst culture we now have analysed the CLBR for our patients. DesignA retrospective analysis. A retrospective analysis. Materials and MethodsWe calculated the CLBR per OPU in a three-year period before the introduction, 2003-2005 and compared with the following period of 2006-2008. We included all patients who started their treatments and had OPU in these two periods including all frozen/thawed cycles. Several variables known to affect the birth rate after IVF were adjusted for in the analysis: female age, number of follicles, aspirated oocytes, previous IVF cycles, SET, total FSH dose, indication and method. In order to study live birth between the two periods adjusted for confounding variables multiple logistic regression models were used with the method of generalized estimating equations in order to adjust for the dependence within women. We calculated the CLBR per OPU in a three-year period before the introduction, 2003-2005 and compared with the following period of 2006-2008. We included all patients who started their treatments and had OPU in these two periods including all frozen/thawed cycles. Several variables known to affect the birth rate after IVF were adjusted for in the analysis: female age, number of follicles, aspirated oocytes, previous IVF cycles, SET, total FSH dose, indication and method. In order to study live birth between the two periods adjusted for confounding variables multiple logistic regression models were used with the method of generalized estimating equations in order to adjust for the dependence within women. ResultsThe results were based on 2738 OPU (fresh + all frozen/thawed) in 2003-2005 and 2720 in 2006-2008. The CLBR per OPU for 2003-2005 and 2006-2008 was 29.8% respectively 34.8% (P<0.0001). The adjusted OR for CLBR per OPU between the two periods was 1.35 (1.18-1.54) in favour the period with blastocyst culture (P<0.0001). Adjustments were made for all variables given in methods. The results were based on 2738 OPU (fresh + all frozen/thawed) in 2003-2005 and 2720 in 2006-2008. The CLBR per OPU for 2003-2005 and 2006-2008 was 29.8% respectively 34.8% (P<0.0001). The adjusted OR for CLBR per OPU between the two periods was 1.35 (1.18-1.54) in favour the period with blastocyst culture (P<0.0001). Adjustments were made for all variables given in methods. ConclusionDuring these two 3-year periods the only major change performed in our clinic was the introduction of blastocyst culture and vitrification. The patients' demographics during these two time periods were similar although the proportion of SET increased. The implementation of blastocyst culture along with vitrification of blastocysts significantly increased the CLBR per OPU. During these two 3-year periods the only major change performed in our clinic was the introduction of blastocyst culture and vitrification. The patients' demographics during these two time periods were similar although the proportion of SET increased. The implementation of blastocyst culture along with vitrification of blastocysts significantly increased the CLBR per OPU.

BACKGROUND:Near infrared (NIR) spectroscopy is a technology proposed to facilitate non-invasive screening for the most optimal human embryo for uterine transfer. It has been proposed that the NIR spectral profile of an embryo's spent culture medium can be used to generate a viability score that correlates to implantation potential. As the initial proof of principle studies were all retrospective, our aim was to investigate whether NIR spectroscopy on spent embryo culture medium in an on-site, prospective setting could improve the ongoing single embryo transfer (SET) pregnancy rate after Day 2 and 5 transfers.METHODS:We conducted a single-centre, double-blinded, randomized controlled trial in which the NIR group was compared with a control group. The primary outcome was the clinical pregnancy rate after 6-7 weeks of gestation per randomized patient. In the control group embryo selection was based only on traditional morphological evaluation while in the treatment group NIR spectroscopy was added to the morphological evaluation.RESULTS:The study was terminated early as the analysis of the Data Safety Monitoring Board showed a very low conditional power of superiority for the primary outcome. Of the 752 patients calculated to be included in the study, 164 and 163 patients were randomized into the NIR and control groups, respectively. No significant difference in the ongoing pregnancy rate per randomized patient was found between the NIR and the control group, 34.8 versus 35.6%, (P= 0.97). The proportional difference between the study groups mean was -0.8% (95% confidence interval -11.4 to 10.2).CONCLUSIONS:This study shows that adding NIR spectroscopy, in its present form, to embryo morphology does not improve the chance of a viable pregnancy when performing SET. The NIR technology appears to need further development before it can be used as an objective marker of embryo viability.CLINICAL TRIALS IDENTIFIER:ISRCTN23817363.

BACKGROUNDIt has been claimed that the risks to the child resulting from vitrification as compared with the slow-freezing technique, may be higher owing to the high concentrations of potentially toxic cryoprotectants. We therefore retrospectively compared the obstetric and neonatal outcomes in a cohort of children born after transfer of vitrified blastocysts, fresh blastocysts and slow-frozen early cleavage stage embryos.METHODSAll children born after transfer of vitrified blastocysts (n = 106), fresh blastocysts (n = 207) and slow-frozen early cleavage stage embryos (n = 206) during the period January 2006 to May 2008 at Fertility Center Scandinavia were included. Data on obstetric and neonatal outcomes were obtained from medical records from the antenatal and delivery clinics.RESULTSFor singletons, there were no significant differences between the groups in gestational age, mortality or birth defects. After adjustment for parity and BMI, birthweight was significantly higher in singletons born after transfer of vitrified blastocysts as compared with after transfer of fresh blastocysts (median 3560 versus 3510 g, P = 0.0311). More singletons born after transfer of fresh blastocysts were small for gestational age compared with singletons born after transfer of vitrified blastocysts (12.1 versus 3.0%, P = 0.0085). A higher rate of major post-partum haemorrhage was observed in the vitrified blastocyst group as compared with the other two groups (25.0 versus 6.0 and 7.5%).CONCLUSIONSNo adverse neonatal outcomes were observed in children born after transfer of vitrified, as compared with fresh blastocysts or after transfer of slow-frozen early cleavage stage embryos.

BACKGROUND In a randomized controlled study aiming to test the effectiveness of preimplantation genetic screening (PGS) in women of advanced maternal age, embryos diagnosed as chromosomally abnormal and those with no diagnosis were fixed for reanalysis. The aim of this study was to determine how well the chromosomal constitution of one biopsied blastomere reflects the status of the entire embryo. METHODS One hundred and seventy-three embryos diagnosed as chromosomally abnormal, 22 with no PGS result and four degenerated embryos originally diagnosed as normal were fixed and reanalysed by fluorescence in situ hybridization. RESULTS In total, 199 embryos were fixed, of which 166 were successfully reanalysed. One hundred and sixty embryos were found to be chromosomally abnormal; 48 of the reanalysed embryos with an initial diagnosis (149) had at least one cell with exactly the same chromosomal constitution shown in the first PGS analysis (34.2%). The reanalysis confirmed the initial overall chromosomally abnormal status of the embryo in 95.9% of the cases. Of all chromosomally abnormal embryos, 4.1% were diagnosed as false positive. The risk for false negative rate was at least 4.1%. CONCLUSIONS PGS seems to be a good method for selecting against chromosomally abnormal embryos but not for determining an embryo's exact chromosomal constitution.

OBJECTIVE: We have recently reported that non-invasive metabolomic profiling of embryo culture media using near infrared (NIR) spectroscopy correlates with the reproductive potential of embryos in IVF (Seli et al., 2007). This technology assesses modifications of the chemical composition of the embryo's surrounding medium using spectroscopy and generates a value termed the "ViaTest-E™ score". In the current study, we hypothesized that the use of a ViaTest-E score could further assist in determining the most viable embryo in addition to extended culture to blastocyst. DESIGN: NIR analysis of Day 5 embryo culture media samples and correlation with pregnancy outcome. MATERIALS AND METHODS: Single embryo Day 5 transfers (n=137) were performed at the Fertilitetscentrum, Gothenburg, Sweden and Shady Grove Fertility RSC, Rockville, Maryland, USA. Day 5 embryos were assessed for SET based on morphology. After SET a media sample from the transferred embryo and a blank were collected and cryostored. Media NIR analysis and a genetic algorithm were used to identify spectral regions, for eg. -SH, C=C, -CH, -OH, and –NH groups, that discriminated most between transferred embryos that did and did not result in pregnancy (fetal cardiac activity detected at 12 weeks of gestation). Relative ViaTest-E scores were calculated by quantification of these spectral regions, adjusting for parallel blank media controls. RESULTS: The overall pregnancy rate from the two clinical sites using SET was 69/137 (50.4%). When the transferred embryo's ViaTest-E scores were assessed, we found a large variability in metabolic profiles (range: -0.5 to 1.5). A significant (Pearsons: P<0.001) positive correlation was observed with increasing ViaTest-E score quintiles and their associated implantation rates (Table 1). CONCLUSIONS: The ViaTest-E score helps discriminate between day 5 embryos, confirming that the embryo's metabolism varies widely regardless of morphology. The added armory of metabolomic profiling by NIR spectroscopy, coupled with bioinformatics, to current morphological assessment protocols seems to allow a greater discrimination for selection of embryos for transfer, and has considerable promise to improve IVF outcomes.Table 1ViaTest-E scores of Day 5 embryos and implantation ratesViaTest-E scorePregnantNot PregnantImplantation Rate (%)<0.252271926.90.2522-0.445162122.20.4451-0.5415131448.10.5415-0.700421677.8>0.700422873.3 Open table in a new tab

BACKGROUND:Advanced maternal age (AMA) is an important parameter that negatively influences the clinical pregnancy rate in IVF, in particular owing to the increased embryo aneuploidy rate. It has thus been suggested that only transferring euploid embryos in this patient group would improve the pregnancy rate. The purpose of this study was to test whether employing preimplantation genetic screening (PGS) in AMA patients would increase the clinical pregnancy rate.METHODS:We conducted a two-center, randomized controlled trial (RCT) to analyze the outcome of embryo transfers in AMA patients (>or=38 years of age) after PGS using FISH analysis for chromosomes X, Y, 13, 16, 18, 21 and 22. The PGS group was compared with a control group. The primary outcome measure was clinical pregnancy rate after 6-7 weeks of gestation per randomized patient.RESULTS:The study was terminated early as an interim analysis showed a very low conditional power of superiority for the primary outcome. Of the 320 patients calculated to be included in the study, 56 and 53 patients were randomized into the PGS and control groups, respectively. The clinical pregnancy rate in the PGS group was 8.9% (95% CI, 2.9-19.6%) compared with 24.5% (95% CI, 13.8-38.3%) in the control group, giving a difference of 15.6% (95% CI, 1.8-29.4%, P = 0.039).CONCLUSIONS:Although the study was terminated early, this RCT study provides evidence against the use of PGS for AMA patients when performing IVF.TRIAL REGISTRATION NUMBER:ISRCTN38014610.

To determine if metabolomic profiling of OM biomarkers during embryonic development (D2 to D5) in-vitro, was associated with pregnancy in a single embryo transfer (SET) model. Prospective study. Embryos were cultured in individual drops of cleavage media (Cook) under oil. Embryo scoring and transfer was performed on Day 2 (N = 75) while remaining embryos were transferred to new cleavage media (CCM-30, Vitrolife) and later selected for transfer at Day 3 (N = 27) or Day 5 (N = 49) based on morphological criteria. Only SET was performed. Spent media was collected and stored in liquid N2. Pregnancy was determined by the presence of foetal cardiac activity (FCA) at (5–6) weeks. Near infrared spectroscopy (NIR) measurements of blinded, randomized samples were conducted using an InGaAs spectrometer. Sample volume was 7 μL. Control media samples were used to compensate for any drift in signal. Sample properties were quantified from the resulting mean centered NIR spectra by determining the most parsimonious combination of variables in selected wavelength domains using a genetic algorithm optimization. Selected wavelength regions were weighted by a coefficients calculated by inverse least-squares regression. Each samples' pregnancy viability was estimated by a leave-one-out cross validation method. Notch box plots were used to plot the resulting viability indices, and t-tests were applied to determine significant differences. A 151 media specimens were analyzed. Embryos from each media specimen were considered viable if pregnancy ensued following SET as determined by positive FCA. NIR spectral analysis reproducibly defined 5 OM biomarker regions associated with changes in ROH, C = C, -SH, -CH and -NH functional groups that were used to distinguish between FCA+ vs. FCA- samples based on bioinformatic analysis of the data. The FCA+ and FCA- groups were statistically different at 95% confidence level. Morphology scores did not correlate with neither pregnancy outcome nor metabolomic activity. Unique metabolomic profiles obtained at each day of embryo development (D2 through D5) were significantly correlated with FCA+ or FCA− embryos in a SET study design not observed by morphological scoring alone. Further prospective randomized trials are planned with the aim of choosing between morphologically similar embryos and to distinguish their implantation potential.