Comparing the cumulative live birth rate of cleavage-stage versus blastocyst-stage embryo transfers between IVF cycles: a study protocol for a multicentre randomised controlled superiority trial (the ToF trial).

INTRODUCTION: In vitro fertilisation (IVF) has evolved as an intervention of choice to help couples with infertility to conceive. In the last decade, a strategy change in the day of embryo transfer has been developed. Many IVF centres choose nowadays to transfer at later stages of embryo development, for example, transferring embryos at blastocyst stage instead of cleavage stage. However, it still is not known which embryo transfer policy in IVF is more efficient in terms of cumulative live birth rate (cLBR), following a fresh and the subsequent frozen-thawed transfers after one oocyte retrieval. Furthermore, studies reporting on obstetric and neonatal outcomes from both transfer policies are limited. METHODS AND ANALYSIS: We have set up a multicentre randomised superiority trial in the Netherlands, named the Three or Fivetrial. We plan to include 1200 women with an indication for IVF with at least four embryos available on day 2 after the oocyte retrieval. Women are randomly allocated to either (1) control group: embryo transfer on day 3 and cryopreservation of supernumerary good-quality embryos on day 3 or 4, or (2) intervention group: embryo transfer on day 5 and cryopreservation of supernumerary good-quality embryos on day 5 or 6. The primary outcome is the cLBR per oocyte retrieval. Secondary outcomes include LBR following fresh transfer, multiple pregnancy rate and time until pregnancy leading a live birth. We will also assess the obstetric and neonatal outcomes, costs and patients' treatment burden. ETHICS AND DISSEMINATION: The study protocol has been approved by the Central Committee on Research involving Human Subjects in the Netherlands in June 2018 (CCMO NL 64060.000.18). The results of this trial will be submitted for publication in international peer-reviewed and in open access journals. TRIAL REGISTRATION NUMBER: Netherlands Trial Register (NL 6857).

Embryo selection using time-lapse analysis (Early Embryo Viability Assessment) in conjunction with standard morphology: a prospective two-center pilot study.

STUDY QUESTION: Does prospective embryo selection using the results from the Eava Test (Early Embryo Viability Assessment) in combination with standard morphology increase the pregnancy rate of IVF and ICSI patients compared to embryo selection based on morphology only? SUMMARY ANSWER: Embryo selection using the Eeva Test plus standard morphology on Day 3 results in comparable pregnancy rates as conventional morphological embryo selection. WHAT IS KNOWN ALREADY: Time-lapse monitoring of embryo development may represent a superior way to culture and select embryos in vitro. The Eeva Test records the development of each embryo with a cell-tracking system and predicts the likelihood (High, Medium or Low) that an embryo will form a blastocyst based on an automated analysis of early cell division timings. STUDY DESIGN, SIZE, DURATION: This trial was designed as a prospective, observational, two-center pilot study with a propensity matched control group. The analysis involved 280 of 302 enrolled patients who were included in the Eeva Test group in 2013 and 560 control patients who were treated in the years 2011-2013. The majority of transfers (98%) were single embryo transfers. PARTICIPANTS/MATERIALS, SETTING, METHODS: Two academic hospitals (VUmc Amsterdam and UZ Gent) enrolled patients <41 years old, with <3 previous attempts and ≥5 normally fertilized eggs. Propensity matching was used to identify a propensity matched control group from a cohort of 1777 patients based on age, cycle number, oocyte number and number of fertilized oocytes. MAIN RESULTS AND THE ROLE OF CHANCE: There was no difference in patient baseline characteristics between the two groups. The ongoing pregnancy rate (OPR) of patients enrolled in the Eeva Test group (34.3%; 96/280) did not differ significantly from the OPR in the propensity matched control group (34.6%, 194/560; P = 0.92). However, significantly less top quality embryos (eight-cell embryos with ≤25% fragmentation) were transferred in the Eeva Test group compared to the propensity matched control group (70.4% vs. 82.3%; P < 0.001). The transfer of Eeva High and Medium embryos resulted in a significantly higher OPR of 36.8% (89/242) compared to 18.4% (7/38) for Eeva Low embryos (P = 0.02). LIMITATION, REASONS FOR CAUTION: This pilot study is limited by its nonrandomized design with a concurrent and historical control. WIDER IMPLICATIONS OF THE FINDINGS: Our pilot data did not reveal significant differences between time-lapse based and conventional embryo selection. Interestingly, the pregnancy rates were comparable in both groups even though the morphological quality of the transferred embryos was significantly lower in the Eeva Test group compared to the propensity matched control group. A sufficiently powered three-armed randomized controlled trial (RCT) with a solid design should be performed to generate decisive evidence in the future. STUDY FINDING/COMPETING INTERESTS: Progyny Inc., formerly Auxogyn provided the Eeva scopes, software and technical support for this study. The funding sources did neither influence data collection, management, analysis and interpretation of the data, nor the preparation of the manuscript. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov: NCT01671644.

Embryo Transfer with Controlled Injection Speed to Increase Pregnancy Rates: A Randomized Controlled Trial.

BACKGROUND/AIMS: Catheter injection speed affects depth and placement of the embryo into the uterine cavity and is shown to be highly variable in, and between, subjects in a manually performed embryo transfer. In an effort to standardize the injection speed during embryo transfer, we developed an automated transfer pump: the pump-regulated embryo transfer (PRET) device. In this randomized controlled trial, we aimed to investigate if standardization of the injection speed and pressure with this PRET results in a better controlled positioning of the transferred embryo(s). METHODS: Five hundred ninety-nine in-vitro fertilization/intracytoplasmic sperm injection/frozen-thawed embryo transfer cycles were randomly assigned to the PRET or manual transfer. Positioning of the embryo(s) into the uterine cavity was measured with ultrasound. RESULTS: The PRET device generates a significantly smaller variance of the positioning of the embryo(s) into the uterine cavity. This resulted in an ongoing pregnancy rate of 21% in the PRET versus 17% in the manual (p = 0.22) transfer group; frozen-thawed embryo transfers resulted in 17.5 versus 10.9% (p = 0.097), respectively. CONCLUSION: The PRET results in better controlled positioning of the embryo(s), and it also gives the opportunity to standardize embryo transfer. Whether the PRET may positively influence pregnancy rates, needs to be investigated in a multicenter trial.

In vitro development of donated frozen-thawed human embryos in a prototype static microfluidic device: a randomized controlled trial.

OBJECTIVE: To compare the development of human embryos in microfluidic devices with culture in standard microdrop dishes, both under static conditions. DESIGN: Prospective randomized controlled trial. SETTING: In vitro fertilization laboratory. PATIENT(S): One hundred eighteen donated frozen-thawed human day-4 embryos. INTERVENTION(S): Random allocation of embryos that fulfilled the inclusion criteria to single-embryo culture in a microfluidics device (n = 58) or standard microdrop dish (n = 60). MAIN OUTCOME MEASURE(S): Blastocyst formation rate and quality after 24, 28, 48, and 72 hours of culture. RESULT(S): The percentage of frozen-thawed day-4 embryos that developed to the blastocyst stage did not differ significantly in the standard microdrop dishes and microfluidic devices after 28 hours of culture (53.3% vs. 58.6%) or at any of the other time points. The proportion of embryos that would have been suitable for embryo transfer was comparable after 28 hours of culture in the control dishes and microfluidic devices (90.0% vs. 93.1%). Furthermore, blastocyst quality was similar in the two study groups. CONCLUSION(S): This study shows that a microfluidic device can successfully support human blastocyst development in vitro under static culture conditions. Future studies need to clarify whether earlier stage embryos will benefit from the culture in microfluidic devices more than the tested day-4 embryos because many important steps in the development of human embryos already take place before day 4. Further improvements of the microfluidic device will include parallel culture of single embryos, application of medium refreshment, and built-in sensors. DUTCH TRIAL REGISTRATION NUMBER: NTR3867.

The association of the blastomere volume index (BVI), the blastomere symmetry index (BSI) and the mean ovality (MO) with ongoing implantation after single embryo transfer.

PURPOSE: To generate novel, objective variables that resemble embryo quality and relate them to ongoing implantation, using multilevel imaging of single-transferred embryos. METHODS: Retrospective analysis of multilevel images of 659 day 3 single-transferred embryos. Each embryo was photographed on seven different levels, in order to measure the largest diameter of every blastomere within an embryo. The volume of each blastomere was calculated using the equation [Formula: see text]. The blastomere volume index (BVI) represented the ratio between the total blastomeric volume of an embryo and the mean cytoplasmic volume of an oocyte on day 0. The blastomere symmetry index (BSI) represented the ratio between the greatest blastomere volume and the smallest blastomere volume within an embryo. The mean ovality (MO) represented the presence of non-spherical blastomeres. Analyses were performed to compare the BVI, BSI and MO between patients with and without an ongoing implantation. RESULTS: The mean BVI was significantly higher for embryos in the ongoing implantation group compared to the no ongoing implantation group. The mean BSI was associated with ongoing implantation for unevenly cleaved embryos. The MO of blastomeres within an embryo was similar for embryos in the ongoing implantation group compared to the no ongoing implantation group. The association of the BVI and BSI with ongoing implantation was confounded, because only female age and cleavage rate were significantly associated with ongoing implantation in multiple logistic regression analyses. CONCLUSIONS: The BVI, BSI and MO are objective variables that resemble embryo quality, but they are not suitable to use as embryo selection tools.

The influence of the type of embryo culture medium on neonatal birthweight after single embryo transfer in IVF.

STUDY QUESTION: Does the type of medium used to culture fresh and frozen-thawed embryos influence neonatal birthweight after single embryo transfer (SET) in IVF? SUMMARY ANSWER: A comparison of two commercially available culture media showed no significant influence on mean birthweight and mean birthweight adjusted for gestational age, gender and parity (z-scores) of singletons born after a fresh or frozen-thawed SET. Furthermore, we show that embryo freezing and thawing cycles may lead to a significantly higher mean birthweight. WHAT IS KNOWN AND WHAT THIS PAPER ADDS: Animal studies have shown that culture media constituents are responsible for changes in birthweight of offspring. In human IVF, there is still little knowledge of the effect of medium type on birthweight. Until now, only a small number of commercially available culture media have been investigated (Vitrolife, Cook(®) Medical and IVF online medium). Our study adds new information: it has a larger population of singleton births compared with the previously published studies, it includes outcomes of other media types (HTF and Sage(®)), not previously analysed, and it includes data on frozen-thawed SETs. DESIGN: This study was a retrospective analysis of birthweights of singleton newborns after fresh (Day 3) or frozen-thawed (Day 5) SET cycles, using embryos cultured in either of two different types of commercially available culture media, between 2008 and 2011. PARTICIPANTS AND SETTING: Before January 2009, a single-step culture medium was used: human tubal fluid (HTF) with 4 mg/ml human serum albumin. From January 2009 onwards, a commercially available sequential medium was introduced: Sage(®), Quinn's advantage protein plus medium. Singletons born after a fresh SET (99 embryos cultured in HTF and 259 in Sage(®)) and singletons born after a frozen-thawed SET (32 embryos cultured in HTF only, 41 in HTF and Sage(®) and 86 in Sage(®) only) were analysed. Only patients using autologous gametes without the use of a gestational carrier were considered. Also excluded were (vanishing) twins, triplets, babies with congenital or chromosomal abnormalities and babies born before 22 weeks of gestation. MAIN RESULTS AND THE ROLE OF CHANCE: Analysis of 358 singletons born after a fresh SET and 159 singletons born after a frozen-thawed SET showed no significant difference between the HTF and Sage(®) groups in terms of birthweight. Gestational age, parity and gender of the baby were significantly related to birthweight in multiple linear regression analyses, and other possible confounding factors included maternal age, BMI and smoking, the number of blastomeres in the transferred embryo and the type of culture medium. Maternal age, BMI and smoking, gestational age at birth, gender of the baby and the percentage of firstborns did not differ significantly between the HTF and Sage(®) groups; however, among the fresh embryos, those cultured in Sage(®) had significantly more blastomeres at the time of embryo transfer compared with the embryos cultured in HTF. Birthweights adjusted for gestational age and gender or gestational age and parity (z-scores) were not significantly different between the HTF and Sage(®) groups for fresh or frozen-thawed SETs. Mean birthweight, as well as the mean birthweight among firstborns and the mean birthweights adjusted for gestational age and gender or parity (z-scores) were significantly higher in the cryopreservation group compared with the fresh embryo transfer group. BIAS, CONFOUNDING AND OTHER REASONS FOR CAUTION: Our study is limited by its retrospective design and only two commercially available types of culture media were tested. More research is necessary to investigate the potential influence of culture media on gene expression. GENERALIZABILITY TO OTHER POPULATIONS: Although our data do not indicate the major influences of the HTF and Sage(®) culture media on birthweight, our results cannot be extrapolated to other culture media types. Furthermore, there remains a potential influence of embryo culture environment on epigenetic variation not represented by birthweight differences but by more subtle features.

Day 3 embryo selection by metabolomic profiling of culture medium with near-infrared spectroscopy as an adjunct to morphology: a randomized controlled trial.

STUDY QUESTION: Is the selection of a single Day 3 embryo by metabolomic profiling of culture medium with near-infrared (NIR) spectroscopy as an adjunct to morphology able to improve live birth rates in IVF, compared with embryo selection by morphology alone? SUMMARY ANSWER: The live birth rate after embryo selection by NIR spectroscopy and morphology is not significantly different compared with the live birth rate after embryos were selected by morphology alone. WHAT IS KNOWN ALREADY: The elevated incidence of pregnancy and neonatal problems associated with a high-twinning rate after IVF can only be successfully reduced by the transfer of one embryo. Current embryo assessment methods are unable to accurately predict the reproductive potential of an individual embryo. Today, a number of techniques are said to be more accurate at selecting the best embryo. One of these new technologies is metabolomic profiling of spent embryo culture media with the use of NIR spectroscopy. STUDY DESIGN, SIZE AND DURATION: A double-blind, randomized controlled trial was conducted between 2009 and 2011, and included 417 couples undergoing IVF with a single embryo transfer. Randomization was performed centrally just before Ovum Pick-Up (OPU), using a computerized randomization program. Both patient and physician were unaware of the treatment allocation. To ensure blinding, the allocations were placed in consecutively numbered, opaque envelopes. Patients were randomized (1:1) into either the control group (embryo selection by morphology only) or the treatment group (embryo selection by morphology plus NIR spectroscopy of embryo culture medium). PARTICIPANTS/MATERIALS, SETTING AND METHODS: At OPU, 208 patients were randomized to the morphology only group and 209 patients were randomized to the morphology plus viability score group. On Day 3, 163 patients in the control group and 146 patients in the treatment group met the inclusion criteria. The study was conducted in an academic hospital with IVF laboratory and three non-academic hospitals. MAIN RESULTS AND THE ROLE OF CHANCE: Patient demographics and baseline characteristics were distributed equally over the two groups, except for embryo fragmentation, which was significantly higher in the treatment group. In the intention to treat analysis, the live birth rates were 31.7 and 26.8% for the control group and the treatment group, respectively (relative risk 0.84; 95% confidence interval 0.63-1.14, P=0.27). In the per protocol analysis, the live birth rates were 31.3 and 29.5% for the control group and the treatment group, respectively (relative risk 0.94; 95% confidence interval 0.67-1.32, P=0.73). For the treatment group, the embryological technician's independent choice (by morphology) of which embryo to transfer was recorded 138 times. In 75.4% (104 of 138) of the transfers, the embryo with the best morphology did not have the highest viability score. The live birth rate of these 104 transferred embryos was 30.8%. LIMITATIONS, REASONS FOR CAUTION: A possible limitation of our study is the pre-selection of all embryos by morphology and dividing the cohort of available embryos into two groups: good quality embryos and poor quality embryos. As a consequence, we have probably selected for a better prognosis patient group. WIDER IMPLICATIONS OF THE FINDINGS: To avoid the use of incompetent embryo selection tools at the expense of the patient, an evidence-based proof of clinical usefulness is essential before the implementation of new diagnostic tools in IVF laboratories. TRIAL REGISTRATION NUMBERS: Dutch Trial Registry, registry number NTR1178.

Non-invasive viability assessment of day-4 frozen-thawed human embryos using near infrared spectroscopy.

This study investigated if metabolomic profiling of culture media using near infrared (NIR) spectroscopy was related to live-birth rates after single-embryo transfer of frozen-thawed embryos. Analysis of culture media of frozen-thawed embryos was performed by NIR spectroscopy. A viability score was calculated using a predictive multivariate algorithm of fresh day-5 embryos with known pregnancy outcomes. This algorithm generated with fresh day-5 embryos could help to identify the live-birth group from the no live-birth group. Multivariable regression models that tested the predictive ability of the viability score for live birth showed an odds ratio in the crude analysis of 1.50 (P=0.008), after adjustment for embryo morphology, 1.44 (P=0.022), and after adjustment for all variables, 1.71 (P=0.005); based on a 0.1 step increase in viability scores. In conclusion, higher viability scores resulted in higher live-birth rates. An algorithm generated from fresh embryos might be used to predict viability of frozen-thawed embryos. Frozen-thawed embryos have different metabolic activity which is related to implantation potential. Therefore, this method might be useful to select the best embryo for transfer within a group of embryos with similar morphology.

Noninvasive metabolomic profiling as an adjunct to morphology for noninvasive embryo assessment in women undergoing single embryo transfer.

OBJECTIVE: To determine whether metabolomic profiling of spent embryo culture media correlates with reproductive potential of human embryos. DESIGN: Retrospective study. SETTING: Academic and a private assisted reproductive technology (ART) programs. PATIENT(S): Women undergoing single embryo transfer after IVF. INTERVENTION(S): Spent embryo culture media were collected after single embryo transfer on day 3 (n = 304) or day 2 (n = 181) and analyzed by near infrared spectroscopy. Near infrared spectral regions were correlated to reproductive potential using a genetic algorithm optimization. Models of these spectral regions were used to calculate viability indices, and were validated by blinded analysis of a subset (n = 60) of samples. Implantation rates were also compared between embryos of higher (>or=0.3) and lower (<0.3) viability indices, and within each morphology grade. MAIN OUTCOME MEASURE(S): Viability index and embryo viability. RESULT(S): Mean viability indices of embryos that resulted in positive fetal cardiac activity were significantly higher compared with embryos that did not for both day 2 and day 3 embryos. Blinded validation of the day 2 model proved to be significant. Increasing viability index values correlated with an increase in pregnancy. Viability indices were found to be independent of morphology for both day 2 and day 3 embryos. Implantation rates were significantly higher among embryos with viability indices >or=0.3. CONCLUSION(S): Metabolomic profiling of human embryo culture media using near infrared spectroscopy is independent of morphology and correlates with reproductive potential of embryos.