A novel method for gas mixing and distribution in multi-chamber embryo incubators.

BACKGROUND: High-quality control of the gas environment in incubators is crucial for in vitro embryo development, which requires high accuracy, fast recovery, and low gas consumption. OBJECTIVE: In this study, we propose a novel gas mixing and distribution system and method as an alternative solution for multi-chamber embryo incubators. METHODS: The system-based embryo incubator enables a controllable gas circulation process and a quantitative supply of CO2 and N2. To determine the optimal parameters for the mixing time and flow rate of the circulated gases, we conducted contrast experiments on the system-based incubator. To evaluate the performance of the gas system in the incubator, we conducted tests under four different initial conditions, simulating various practical application scenarios. Furthermore, we performed a mouse embryo assay to assess the system's effectiveness. RESULTS: The results show that the system achieved a gas concentration accuracy of ± 0.2% (volume fraction) after stabilization, a minimum recovery time of 5 minutes, an average consumption of 8.9 L/d for N2 and 0.83 L/d for CO2 during routine operation, and a blastocyst rate exceeding 90% observed after 96 hours of culture in the incubator. CONCLUSION: The system and method demonstrate a significant advantage in terms of low gas consumption compared to existing incubators, while still maintaining high accuracy and fast recovery.

A compact, high-throughput semi-automated embryo vitrification system based on hydrogel.

RESEARCH QUESTION: What is the efficiency and efficacy of the novel Biorocks semi-automated vitrification system, which is based on a hydrogel? DESIGN: This comparative experimental laboratory study used mouse model and human day 6 blastocysts. Mouse oocytes and embryos were quality assessed post-vitrification. RESULTS: The Biorocks system successfully automated the solution exchanges during the vitrification process, achieving a significantly improved throughput of up to 36 embryos/oocytes per hour. Using hydrogel for cryoprotective agent delivery, 12 vessels could be processed simultaneously, fitting comfortably within an assisted reproductive technology (ART) workstation. In tests involving the cryopreservation of oocytes and embryos, the system yielded outcomes equivalent to the manual Cryotop method. For example, the survival rate for mouse oocytes was 98% with the Biorocks vitrification system (n = 46) and 95% for the manual Cryotop method (n = 39), of which 46% and 41%, respectively, progressed to blastocysts on day 5 after IVF. CC-grade day 6 human blastocysts processed with the Biorocks system (n = 39) were associated with a 92% 2 h re-expansion rate, equivalent to the 90% with Cryotop (n = 30). The cooling/warming rates achieved by the Biorocks system were 31,900°C/minute and 24,700°C/minute, respectively. Oocyte quality was comparable or better post-vitrification for Biorocks than Cryotop. CONCLUSIONS: The Biorocks semi-automated vitrification system offers enhanced throughput without compromising the survival and developmental potential of oocytes and embryos. This innovative system may help to increase the efficiency and standardization of vitrification in ART clinics. Further investigations are needed to confirm its efficacy in a broader clinical context.

Metabolomic Analysis Evidences That Uterine Epithelial Cells Enhance Blastocyst Development in a Microfluidic Device.

Here we report the use of a microfluidic system to assess the differential metabolomics of murine embryos cultured with endometrial cells-conditioned media (CM). Groups of 10, 1-cell murine B6C3F1 × B6D2F1 embryos were cultured in the microfluidic device. To produce CM, mouse uterine epithelial cells were cultured in potassium simplex optimized medium (KSOM) for 24 h. Media samples were collected from devices after 5 days of culture with KSOM (control) and CM, analyzed by reverse phase liquid chromatography and untargeted positive ion mode mass spectrometry analysis. Blastocyst rates were significantly higher (p < 0.05) in CM (71.8%) compared to control media (54.6%). We observed significant upregulation of 341 compounds and downregulation of 214 compounds in spent media from CM devices when compared to control. Out of these, 353 compounds were identified showing a significant increased abundance of metabolites involved in key metabolic pathways (e.g., arginine, proline and pyrimidine metabolism) in the CM group, suggesting a beneficial effect of CM on embryo development. The metabolomic study carried out in a microfluidic environment confirms our hypothesis on the potential of uterine epithelial cells to enhance blastocyst development. Further investigations are required to highlight specific pathways involved in embryo development and implantation.

Comparison of Embryo and Clinical Outcomes in Different Types of Incubator Between Two Different Embryo Culture Systems.

This study aimed to compare the clinical outcomes of an oxidative stress-reducing embryo culture system (ORES) containing compounds that minimize intercellular oxidative stress, with those of a standard embryo culture system (StES). Furthermore, we investigated the efficiency of the ORES regarding the type of incubator used (time-lapse incubator [TLI] or non-time-lapse dry incubator [non-TLI]) and maternal age. In this retrospective study, we analyzed 3610 oocyte retrieval cycles (in 2537 patients) and 1726 single vitrified-warmed blastocyst transfer (SVBT) cycles (in 1726 patients) performed in a single center between April 2018 and July 2019. Transfers of single vitrified-warmed blastocysts, confirmed by fetal heartbeat, were used to assess clinical outcomes. The clinical outcomes of ORES and StES were compared in both TLI and non-TLI. Groups were stratified according to maternal age (≤39 years old, young age group; ≥40 years old, advanced age group). A significant difference in ongoing pregnancy rates was observed between the ORES and StES groups when non-TLI was used (34.9 vs. 27.0%, respectively; p < 0.05), unlike when TLI was used. Furthermore, ongoing pregnancy rates were significantly higher in the ORES group (24.8%) than in the StES group (14.9%) in the advanced age group, unlike in the young age group when non-TLI was used. In conclusion, compared to StEs, the ORES during all in vitro fertilization procedures improved ongoing pregnancy rates in the advanced age group using the non-TLI.

Degenerated oocyte in the cohort adversely affects IVF outcome.

The presence of Degenerated Oocyte (DEG) was mostly described after intracytoplasmic sperm injection (ICSI), with fewer reports on DEG at the time of ovum pick-up (OPU). This study aims to assess morphokinetics of embryos cultured in a time-lapse incubator and compare cohorts with and without DEG at OPU. In a retrospective cohort study from January 1, 2016 until September 31, 2017 a total of 399 IVF/ICSI cycles and 2980 embryos were evaluated. In 81 of 399 cycles at least one DEG oocyte was observed at the time of OPU. The remaining 318 cycles with no DEG oocyte were compared as a control group. In the DEG group, significantly more oocytes were collected per patient (12.9 ± 7.2 vs. 10.1 ± 6.1. P < 0.001). Fertilization rate, pregnancy and clinical pregnancy rates were comparable between the two groups, however, the morphokinetics and developmental scores of the embryos were significantly worse in the DEG group, (KID 3.4 ± 1.6 vs. 3.2 ± 1.6 P = 0.002 and ESHRE 1.5 ± 1.1 vs. 1.4 ± 1.0 P = 0.046). Significantly more patients achieved top-quality embryos in the NON DEG group (58.8% vs. 53.0%, P = 0.03), however, comparable delivery rate was achieved in both groups. In the DEG group, the frequency of DEG oocyte per cycle was negatively correlated with pregnancy rate. GnRH agonist protocol and the 17-20G needle used for OPU were significant predictors for the presence of DEG oocyte at OPU. In conclusions DEG oocyte may negatively affect IVF outcome, however, younger patients, and significantly more oocytes collected in the DEG group compensate for the IVF results.

Successful vitrification of dromedary camel embryos using a novel embryo vitrification kit.

Embryos (n = 87) collected 8 days after mating and 7 days after ovulation were vitrified using a camel-specific vitrification kit. Vitrification solutions (VS) containing 20% foetal calf serum, with or without 2% bovine serum albumin (BSA) were used to cryopreserve embryos, in three steps VS1 (5 min), VS2 (5 min) and VS3 (30-35 s) at room temperature (RT) before being loaded into open pulled straws and immediately frozen in liquid nitrogen. Embryos were subsequently thawed in warming solutions (WS) in three steps: WS1 at 37 °C (1 min), WS2 at RT (5 min) then into holding media at RT (5-60 min) prior to transfer, in pairs, into recipient camels 6 days after ovulation. There were 42 of 43 embryos viable after vitrification in media without BSA and these were subsequently transferred into 21 recipient females which resulted in ten pregnancies 60 days after transfer (48% pregnancy rate). There were 38 of 44 viable embryos vitrified in media containing BSA that were transferred in pairs into 19 recipient females which resulted in five pregnancies 60 days after transfer (26% pregnancy rate; P > 0.05). Of the total 15 foetuses that developed to 60 days of gestation after vitrification, 11 resulted from embryos of 200-499 µm diameter and four from embryos of 500-700 µm diameter (P > 0.05). There were encouraging results with use of this novel vitrification kit for the commercial application of cryopreservation of camel embryos with a diameter of 300-550 µm.

Sealed culture system for supporting mouse preimplantation embryo development in vitro.

This study investigated the possibility of a sealed culture system in polymerase chain reaction (PCR) tubes to maintain embryo development. The embryo density that could support the development of 2-cell stage mouse embryos to the hatching stage was determined. At an embryo density of 1:2 (100 embryos cultured in 200µL CZB medium that had been pretreated with a reference gas containing 5% O2), the developmental rate was higher and fewer embryos exhibited reactive oxygen species- or hypoxia-induced injury compared with other sealed culture groups. Expression of growth factors (insulin-like growth factor (IGF) 1, IGF2, epidermal growth factor and transforming growth factor-α) and their receptors was evaluated, with similar expression patterns seen for embryos in sealed culture (5% O2, embryo density of 1:2) compared with the control group (embryos cultured in microdrops and placed in a 37°C, 5% CO2 water-jacketed incubator; P>0.05). After transfer of blastocysts generated by the sealed culture into recipients, there were no obvious differences in the rate of normal live pups births between the sealed culture and control groups (P>0.05). Thus, the sealed embryo culture system in PCR tubes is feasible for use in situations which cannot use a traditional incubator, such as in space and during the transport of embryos.

Time-lapse technology for embryo culture and selection.

Culturing of human embryos in optimal conditions is crucial for a successful in vitro fertilisation (IVF) programme. In addition, the capacity to assess and rank embryos correctly for quality will allow for transfer of the potentially 'best' embryo first, thereby shortening the time to pregnancy, although not improving cumulative pregnancy and live birth rates. It will also encourage and facilitate the implementation of single embryo transfers, thereby increasing safety for mother and offspring. Time-lapse technology introduces the concept of stable culture conditions, in connection with the possibility of continuous viewing and documenting of the embryo throughout development. However, so far, even when embryo quality scoring is based on large datasets, or when using the time-lapse technology, the morphokinetic scores are still mainly based on subjective and intermittent annotations of morphology and timings. Also, the construction of powerful algorithms for widespread use is hampered by large variations in culture conditions between individual IVF laboratories. New methodology, involving machine learning, where every image from the time-lapse documentation is analysed by a computer programme, looking for patterns that link to outcome, may in the future provide a more accurate and non-biased embryo selection.

Unstable osmolality of microdrops cultured in non-humidified incubators.

PURPOSE: To investigate the stability of osmolality in non-humidified and humidified incubators for assisted reproductive technologies (ART). METHODS: Drops of three single-step culture media (media A, B, and C) were incubated for 5 or 6 days covered with four different mineral oils (oils A, B, C, and D) in non-humidified incubator A, non-humidified incubator B, or humidified incubator C to investigate the effects of incubator environment (humidification), drop volume, culture media, and mineral oil on the stability of osmolality in microdrops. RESULTS: A significant and linear increase was shown in the osmolality of 50-µL and 200-µL microdrops covered with mineral oil during 5 days incubation in non-humidified benchtop incubators. The maximum increase was 20 mOsm/kg, and the extent of the increase was affected by microdrop volume and possibly by the type of mineral oil used to cover the drops. In contrast, the osmolality of 50-µL and 200-µL microdrops did not change during 5 days incubation in a humidified benchtop incubator. CONCLUSIONS: Mineral oil alone may not adequately prevent gradual changes in the osmolality of low-volume microdrops during extended in vitro culture of human embryos in non-humidified incubators. As a result, the osmolality may increase to high enough levels to stress some human embryos and adversely affect clinical outcomes. We therefore recommend that the stability of osmolality should be given more consideration to ensure optimal culture conditions for ART.

Rabbit Whole Embryo Culture.

The rabbit is a mainstay of regulatory developmental toxicity testing; however, due to the historic absence of experimental tools for this species, there is a dearth of information about its fundamental embryology and the mechanisms underlying developmental toxicity. Relatively recently, there have been advances in the methods of rabbit whole embryo culture (WEC), and this has prompted an increase in understanding of rabbit embryogenesis. Described herein are the methods used to remove early somite-stage embryos (gestation day 9) and sustain their growth for 48 h. Although there are similarities to the well-described rodent WEC, there are also important differences. Akin to rodent WEC, the major phases of organogenesis can be investigated, including neural tube development, cardiac looping, segmentation, and the development of the anlagen of the optic and otic regions, craniofacial development, somites, and early limb bud development. Unlike the rodent, rabbit WEC requires the use of an apparatus that allows for the continuous gassing of embryos, and one may observe the expansion and closure of the visceral yolk sac around the embryo. After completion of the culture period, embryos are examined across several growth and developmental parameters including a quantitative morphological scoring system. Embryonic growth and development in the absence of maternal influences allows for the study of the direct action of agents or their metabolites on the embryo. The use of both rodent and rabbit WEC together is a powerful strategy with which to investigate species-specific vulnerabilities to specific agents.

Cumulative live birth rates and perinatal outcomes with the use of time-lapse imaging incubators for embryo culture: a retrospective cohort study of 1882 ART cycles.

OBJECTIVE: Comparison of live birth rates and the perinatal outcomes after fresh and frozen embryo transfer between time-lapse imaging (TLI) and standard culture (SC) incubators. DESIGN: Retrospective cohort study. SETTING: A single tertiary level IVF unit. POPULATION: Women undergoing IVF between January 2014 and October 2015. METHODS: Comparison was done between 1064 IVF cycles using TLI (TLI cycles) and 818 IVF cycles using SC (SC cycles). MAIN OUTCOME MEASURES: Cumulative live birth rate per oocyte retrieval and perinatal outcomes including birthweight, gestational age, preterm birth (PTB) (<37 weeks), early preterm birth (PTB; <32 weeks), low birthweight (LBW; <2500 g), very LBW (<1500 g) and macrosomia (>4500 g). RESULTS: The fresh embryo transfer live birth rate was noted to be higher for TLI cycles [TLI 36.8 versus SC 33.9%, adjusted odds ratio (aOR) 1.28, 95% CI 1.05-1.57], but the frozen embryo transfer live birth rates were not significantly different. The mean birthweight was higher in the TLI group after both fresh [adjusted mean difference (aMD) 174.78 g, 95% CI 64.80-284.77] and frozen embryo transfers (aMD 175.91 g, 95% CI 16.98-334.84). After a fresh embryo transfer, there was a lower risk of early PTB and very LBW in the TLI group. Among frozen embryo transfers, there was a lower risk of early PTB and LBW in the TLI group. CONCLUSIONS: TLI incubators are associated with improved perinatal outcomes and higher mean birthweight after fresh and frozen embryo transfer. TWEETABLE ABSTRACT: Time-lapse imaging incubators in IVF improve perinatal outcomes after both fresh and frozen embryo transfers.

[Method validation of a critical parameter in human embryos culture: culture media pH]. / Validation de la mesure d'un paramètre critique en culture d'embryons humains : le pH.

In vitro human embryos culture depends largely on the atmospheric conditions within the incubators of the laboratory. The pH of culture media, an indirect reflection of the CO2 content inside these incubators, is a critical parameter. Collaboration between the biochemistry and reproductive biology departments enabled the automated measurement of the pH in the culture medium on a blood gas analyzer. This method has been validated and evaluated. It is applicable in all laboratories whatever the medium and the conditions of culture. It allows strict monitoring of this parameter for the optimization of the culture conditions necessary to improve the results of in vitro fertilization attempts.

Considerations Regarding Embryo Culture Conditions: From Media to Epigenetics.

There are numerous reports on embryo culture media and conditions in the laboratory, as the subject is multifaceted and complex, reflecting the variation in practice. In this scoping review, we attempt to approach the topic of culture media and conditions from the practitioners' perspective aiming to highlight, in a comprehensive fashion, important aspects regarding the options available, introduce points of debate and controversy, while maintaining the viewpoint of the practicing embryologist's concerns.

Microinjection of Exogenous Nucleic Acids into Eggs: Ciona Species.

Microinjection is a common technique used to deliver nucleic acids into eggs and embryos in Ciona species. There are three Ciona species that are commonly used for research-Ciona intestinalis type A (C. robusta), C. intestinalis type B (C. intestinalis), and C. savignyi. Here, we present the microinjection methods using eggs and embryos of C. intestinalis type A and C. savignyi; however, our methods would also be applicable to eggs and embryos of C. intestinalis type B. Microinjection is a classical and widely used delivery method, which involves the use of a glass micropipette, a hollow glass needle with a microscopic tip, to inject nucleic acids into eggs and embryos under a stereo microscope. The required amount of nucleic acids is much smaller for microinjection than for electroporation, another delivery method. Proteins, and other chemicals, such as fluorescent dye, can be introduced with nucleic acids using a microinjection.

Practical Guide for Ascidian Microinjection: Phallusia mammillata.

Phallusia mammillata has recently emerged as a new ascidian model. Its unique characteristics, including the optical transparency of eggs and embryos and efficient translation of exogenously introduced mRNA in eggs, make the Phallusia system suitable for fluorescent protein (FP)-based imaging approaches. In addition, genomic and transcriptomic resources are readily available for this ascidian species, facilitating functional gene studies. Microinjection is probably the most versatile technique for introducing exogenous molecules such as plasmids, mRNAs, and proteins into ascidian eggs/embryos. However, it is not practiced widely within the community; presumably, because the system is rather laborious to set up and it requires practice. Here, we describe in as much detail as possible two microinjection methods that we use daily in the laboratory: one based on an inverted microscope and the other on a stereomicroscope. Along the stepwise description of system setup and injection procedure, we provide practical tips in the hope that this chapter might be a useful guide for introducing or improving a microinjection setup.

Microinjection of Exogenous DNA into Eggs of Halocynthia roretzi.

Exogenous gene expression assays during development, including reporters under the control of 5' upstream enhancer regions of genes, constitute a powerful technique for understanding the mechanisms of tissue-specific gene expression regulation and determining the characteristics, behaviors, and functions of cells that express these genes. The simple marine chordate Halocynthia roretzi has been used for these transgenic analyses for a long time and is an excellent model system for such studies, especially in comparative analyses with other ascidians. In this study, I describe simple methods for microinjecting H. roretzi eggs with exogenous DNA, such as a promoter construct consisting of a 5' upstream region and a reporter gene, which are prerequisites for transgenic analyses. I also describe basic knowledge regarding this ascidian species, providing reasons why it is an ideal subject for developmental biology studies.

Randomized controlled trial comparing embryo culture in two incubator systems: G185 K-System versus EmbryoScope.

OBJECTIVE: To study whether the closed culture system, as compared with a benchtop incubator with similar culture conditions, has a positive impact on intracytoplasmic sperm injection (ICSI) outcomes. DESIGN: Randomized controlled trial. SETTING: University hospital. PATIENT(S): A total of 386 patients undergoing ICSI cycles with at least six mature oocytes were randomized. INTERVENTION(S): Of these patients, 195 were assigned to the group with culture in a time-lapse imaging (TLI) system (EmbryoScope) and 191 to the group with culture in the G185 K-System (G185). MAIN OUTCOME MEASURE(S): Rate of implantation (primary endpoint) and embryo morphology grade. RESULT(S): No significant differences were found in the implantation rates. The proportion of high-grade embryos on day 2 was significantly higher in the TLI group compared with the G185 group (40.4% vs. 35.2%). The impact of the incubator on embryo morphology remained significant in multivariate analysis, which took into account the woman's age, the rank of attempt, and the smoking status (TLI vs. G185: odds ratio = 1.27; 95% confidence interval, [1.04-1.55]). No difference was found in the mean number of frozen embryos, even though the total proportion of frozen embryos was significantly higher in the TLI group than in the G185 group (29.5% vs. 24.8%). CONCLUSION(S): No difference in implantation rate was found between the two incubators for fresh cycles. It remains to be determined whether the observed differences in embryo morphology and the total number of embryos cryopreserved would translate into higher cumulative outcomes with subsequent frozen embryo transfers. CLINICAL TRIAL REGISTRATION NO: NCT02722252.

Comparison of the development of human embryos cultured in either an EmbryoScope or benchtop incubator.

PURPOSE: In this current study, our main goal was to establish that EmbryoScope incubation environment is comparable to standard incubation. METHODS: The development of sibling human zygotes was compared after culture in either a benchtop incubator (SI) or an EmbryoScope time-lapse incubator (ES). Between May 2015 to April 2016, a total of 581 normally fertilized 2PN, pronuclear-stage embryos, from 47 patients were allocated to culture in either a benchtop incubator (SI) or an EmbryoScope incubator (ES). RESULTS: The development of embryos to cleavage (up to day 3) and blastocyst stages (day 5/6) was compared between the two different incubators. The proportion of good quality embryos was higher in the ES group compared to the SI on day 2 (66.8 vs. 50.5%, P = 0.014) and on day 3 (75.1 vs. 56.0%, P = 0.006). Those differences were statistically significant. A higher proportion of embryos developed to good quality blastocysts when cultured in the EmbryoScope compared to the benchtop (49.4 vs. 42.0%, P = 0.24), but this was not significant. Finally, no significant differences were noted with the proportion of blastocysts chosen for cryopreservation on day 5/6 in the two incubators. CONCLUSIONS: The findings support the view that the EmbryoScope incubator supports at least equivalent in vitro development of human embryos compared to other standard incubation methods and may promote improved development during early cleavage stages.

Humid versus dry incubator: a prospective, randomized, controlled trial.

OBJECTIVE: To evaluate the efficacy of a dry versus humidified incubator on human embryo development ex vivo. DESIGN: Prospective, double-blind, randomized, controlled trial. SETTING: Private fertility centers. PATIENT(S): A total of 297 women undergoing in vitro fertilization randomized into two groups. INTERVENTION(S): From days 0 to day 5 or 6 of culture, intervention group embryos exposed to dry culture and control group embryos exposed to humidified culture. MAIN OUTCOME MEASURE(S): Subsequent ongoing pregnancy rate. RESULT(S): After transfer of embryos, there were statistically significantly lower rates of clinical and ongoing pregnancy in the dry culture arm than in the humidified culture arm (odds ratio [OR] 0.57; 95% confidence interval [CI], 0.36-0.91; versus OR 0.54; 95% CI, 0.34-0.85). On day 3 of culture, embryo quality and compaction were lower in the dry culture group (OR 0.38; 95% CI, 0.32-0.45) than in the group exposed to humidified culture (OR 0.23; 95% CI, 0.19-0.27). On day 5 of culture, embryos in dry culture had a lower rate of blastocyst formation (OR 0.39; 95% CI, 0.33-0.46), quality (OR 0.34; 95% CI, 0.29-0.40), and cryopreservation (OR 0.41; 95% CI, 0.35-0.48). CONCLUSION(S): In this study, human embryos cultivated ex vivo in a dry incubator had statistically significantly decreased implantation and clinical and ongoing pregnancy rates. Our findings indicate the need for larger multicenter, randomized, controlled trials. CLINICAL TRIAL REGISTRATION NUMBER: NCT01695096.

Pregnancy rates of day 4 and day 5 embryos after culture in an integrated time-lapse incubator.

BACKGROUND: The aim of this study was to compare pregnancy rates in patients undergoing IVF/ICSI with embryo transfer after 4 and 5 days of culture in a closed incubation system with integrated time-lapse imaging. METHODS: Out of n = 2207 in vitro fertilization (IVF)/ intracytoplasmic sperm injection (ICSI) cycles performed between January 2011 and April 2016 at a tertiary referral university hospital, a total of n = 599 IVF/ICSI cycles with prolonged embryo culture in an integrated time-lapse system (EmbryoScope© (Vitrolife)) until day 4 or 5 were retrospectively analyzed with regard to embryo morphology and pregnancy rates. RESULTS: A transfer on day 5 compared to a transfer on day 4 did not result in higher implantation and clinical pregnancy rates (IR 29.4% on day 4 versus 33.0% on day 5, p = 0.310; CPR 45.2% on day 4 versus 45.7% on day 5, p = 1.0). The percentage of ideal embryos transferred on day 4 was comparable to the rate of ideal embryos transferred on day 5 (41.6% versus 44.1%, p = 0.508). However, on day 4 a significantly higher number of embryos was transferred (1.92 on day 4 versus 1.84 on day 5, p = 0.023), which did not result in higher rates of multiple pregnancies. CONCLUSIONS: Pregnancy rates in IVF/ICSI cycles with integrated time-lapse incubation and transfer on day 4 and 5 are comparable. This finding provides the clinician, IVF laboratory and patient with more flexibility. TRIAL REGISTRATION: This study was retrospectively registered by the local ethics committee of the University of Heidelberg on December 19, 2016 (registration number S-649/2016).