The higher the score, the better the clinical outcome: retrospective evaluation of automatic embryo grading as a support tool for embryo selection in IVF laboratories.

STUDY QUESTION: Is the automatic embryo grading function of specific time-lapse systems clinically useful as a decision support tool for IVF laboratories? SUMMARY ANSWER: Blastocyst grading according to the automatic scoring system is directly associated with the likelihood of implantation and live birth, at least in treatments without preimplantation genetic testing for aneuploidy (PGT-A). WHAT IS KNOWN ALREADY: Several embryo selection algorithms have been described since the introduction of time-lapse technology in IVF laboratories, but no one algorithm has yet been sufficiently consolidated for universal use. Multicentric models based on automated grading systems offer promise for standardization of embryo selection. STUDY DESIGN, SIZE, DURATION: A retrospective cohort study was performed including 1678 patients who underwent IVF treatments between 2018 and 2020 and whose embryos (n = 12 468) were cultured in time-lapse systems. PARTICIPANTS/MATERIALS, SETTING, METHODS: After obtaining the required parameters (division time to 2, 3, 4 and 5 cells; time of blastocyst formation; inner cell mass quality; and trophectoderm quality), the automatic embryo score was calculated using the software included in the appropriate workstation. First, embryo score was compared with conventional morphological quality and the subsequent clinical outcomes of 1952 single blastocyst transfers. Second, we quantified the contribution of the automatic embryo score and conventional morphological grade to implantation and live birth outcome with multivariate logistic regression analysis in different patient populations. MAIN RESULTS AND THE ROLE OF CHANCE: A higher embryo score was associated with a better clinical outcome of IVF treatment. The mean of the automatic embryo score varied significantly (P < 0.001) among embryos with different morphological categories, between euploid and aneuploid embryos, between embryos resulting in positive versus negative pregnancy, between implanted and non-implanted embryos, and between embryos resulting in positive and negative live birth. Embryo score was related to the odds of implantation and live birth in the oocyte donation program (odds ratio (OR)=1.29; 95% CI [1.19-1.39]; P < 0.001 for implantation and OR = 1.26; 95% CI [1.16-1.36]; P < 0.001 for live birth) and in conventional treatments with autologous oocytes (OR = 1.38; 95% CI [1.24-1.54]; P < 0.001 for implantation and OR = 1.47; 95% CI [1.30-1.65]; P < 0.001 for live birth). There was no significant association of embryo score with implantation or live birth in treatments involving PGT-A. LIMITATIONS, REASONS FOR CAUTION: This study is limited by its retrospective nature. Further prospective randomized trials are required to confirm the clinical impact of these findings. The single-center design should be taken into account when considering the universal application of the model. WIDER IMPLICATIONS OF THE FINDINGS: Evidence of the clinical efficiency of automated embryo scoring for ranking embryos with different morphological grade and potential in order to achieve higher implantation and live birth rates may make it a decision support tool for embryologists when selecting blastocysts for embryo transfer. STUDY FUNDING/COMPETING INTEREST(S): This research has been funded by a grant from the Ministry of Science, Innovation and Universities FIS (PI21/00283) awarded to M.M. There are no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.

Analysis of the morphological dynamics of blastocysts after vitrification/warming: defining new predictive variables of implantation.

OBJECTIVE: To describe the morphological dynamics of vitrified/warmed blastocysts and to identify quantitative morphological variables related to implantation. Subsequently, by using the most predictive parameters, to develop a hierarchical model by subdividing vitrified/warmed blastocysts into categories with different implantation potentials. DESIGN: Observational, retrospective, cohort study. SETTING: University-affiliated private IVF center. PATIENT(S): The study included 429 vitrified/warmed blastocysts with known implantation data, which were evaluated by time-lapse imaging. Blastocysts were routinely placed in EmbryoScope (Vitrolife) immediately after warming until transfer. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Embryos were vitrified and warmed by the Cryotop method (KitazatoBiopharma). The studied variables included the initial and minimum thicknesses of zona pellucida (µm), the initial and maximum areas (µm2), the area of inner cell mass (µm2), expansion (whether the embryo reexpands or not after warming), and collapsing or contraction after warming. After defining the optimal ranges according to the consecutive quartiles with the highest probability of implantation, a logistic regression analysis was performed by combining the former variables and the blastocyst morphological classification criteria defined by the Spanish Association of Embryologists into A, B, C, or D categories. RESULT(S): Reexpansion of vitrified/warmed blastocysts correlated strongly with implantation (44.6% for reexpanded vs. 6.5% for the blastocysts that did not reexpand after warming). Throughout the logistic regression analysis, the model identified the maximum blastocyst area, odds ratio (OR) = 0.41 (95% confidence interval [CI], 0.22-0.77), followed by the initial area, OR = 0.62 (95% CI, 0.35-1.08) as the most predictive variables related to implanting embryos. Blastocyst morphology was not considered relevant in our model. The hierarchical tree model subdivided embryos into four categories, A-D, with lowering expected implantation potentials (from 47.3% for A to 14.2% for D). CONCLUSION(S): The analysis of warmed blastocysts by time-lapse imaging may provide objective quantitative markers for the blastocyst implantation potential. We propose a hierarchical model to classify vitrified/warmed blastocysts according to their implantation probability. The observed correlations and the proposed algorithm should be validated in a prospective trial to evaluate its efficacy.

Kinetics of the early development of uniparental human haploid embryos.

OBJECTIVE: To describe morphokinetically the early development of human haploid parthenotes and androgenotes and to compare them with euploid embryos. DESIGN: Experimental study of kinetics. SETTING: University-affiliated private fertility center. PATIENT(S): Experimental haploid parthenotes and androgenotes. INTERVENTION(S): Kinetic study of early development (up to eight cells) of 8 parthenotes, 10 androgenotes, and 20 euploid embryos. MAIN OUTCOME MEASURE(S): Timing of the first seven cleavages determined according to embryo origin, then calculation of the duration of the second and third cell cycles (cc2 and cc3) of whole embryos and individual cells. RESULT(S): Parthenotes and androgenotes were experimentally produced by artificial oocyte activation and intracytoplasmic sperm injection of enucleated oocytes, respectively. Uniparental embryos having 6 to 10 cells were assessed for haploidy, their kinetics analyzed, retrospectively compared with euploid embryos. All the first seven cleavages occurred later in parthenotes than in both androgenotes and correctly fertilized embryos. The whole embryos and single cells showed that cc2 was longer in parthenotes than in both androgenotes and correctly fertilized embryos; cc3 was shorter in androgenotes than in both parthenotes and correctly fertilized embryos. The duration of cc2 versus cc3 was longer in correctly fertilized embryos and parthenotes than in androgenotes. CONCLUSION(S): Parthenotes and androgenotes have different kinetics. The former have a longer cc2, and the latter a consistently shorter cc3 in comparison with correctly fertilized embryos.

Clinical validation of embryo culture and selection by morphokinetic analysis: a randomized, controlled trial of the EmbryoScope.

OBJECTIVE: To determine whether incubation in the integrated EmbryoScope time-lapse monitoring system (TMS) and selection supported by the use of a multivariable morphokinetic model improve reproductive outcomes in comparison with incubation in a standard incubator (SI) embryo culture and selection based exclusively on morphology. DESIGN: Prospective, randomized, double-blinded, controlled study. SETTING: University-affiliated private in vitro fertilization (IVF) clinic. PATIENT(S): Eight hundred forty-three infertile couples undergoing intracytoplasmic sperm injection (ICSI). INTERVENTION(S): No patient intervention; embryos cultured in SI with development evaluated only by morphology (control group) and embryos cultured in TMS with embryo selection was based on a multivariable model (study group). MAIN OUTCOME MEASURE(S): Rates of embryo implantation, pregnancy, ongoing pregnancy (OPR), and early pregnancy loss. RESULT(S): Analyzing per treated cycle, the ongoing pregnancy rate was statistically significantly increased 51.4% (95% CI, 46.7-56.0) for the TMS group compared with 41.7% (95% CI, 36.9-46.5) for the SI group. For pregnancy rate, differences were not statistically significant at 61.6% (95% CI, 56.9-66.0) versus 56.3% (95% CI, 51.4-61.0). The results per transfer were similar: statistically significant differences in ongoing pregnancy rate of 54.5% (95% CI, 49.6-59.2) versus 45.3% (95% CI, 40.3-50.4) and not statistically significant for pregnancy rate at 65.2% (95% CI, 60.6-69.8) versus 61.1% (95% CI, 56.2-66.1). Early pregnancy loss was statistically significantly decreased for the TMS group with 16.6% (95% CI, 12.6-21.4) versus 25.8% (95% CI, 20.6-31.9). The implantation rate was statistically significantly increased at 44.9% (95% CI, 41.4-48.4) versus 37.1% (95% CI, 33.6-40.7). CONCLUSION(S): The strategy of culturing and selecting embryos in the integrated EmbryoScope time-lapse monitoring system improves reproductive outcomes. CLINICAL TRIAL REGISTRATION NUMBER: NCT01549262.

Reduced oxygen tension improves embryo quality but not clinical pregnancy rates: a randomized clinical study into ovum donation cycles.

OBJECTIVE: To investigate the effect of low O2 tension during in vitro culture in terms of ongoing pregnancy rates in ovum donation cycles. DESIGN: Randomized trial. SETTING: Private university-affiliated IVF center, university-based hospital. PATIENT(S): A total of 1,125 cycles of ovum donation. INTERVENTION(S): Embryo culture in an atmosphere of 5.5% CO2, 6% O2, and 88.5% N2 versus a dual-gas system of 5.5% CO2 in air. MAIN OUTCOME MEASURE(S): Ongoing clinical pregnancy rates per intention-to-treat (ITT) patients. RESULT(S): The use of low O2 tension achieved a 41.3% ongoing pregnancy rate per ITT compared with a 40.8% rate obtained for 5% CO2 in air. The mean number of blastomeres and the percentage of top-quality embryos were significantly higher after lower O2 concentration during in vitro culture (7.1 ± 3.6 and 28.6% vs. 7.3 ± 8.4 and 32.1%, respectively). CONCLUSION(S): In the ovum donation cycles undergoing day-3 embryo transfers, the use of low O2 tension did not improve ongoing pregnancy rates per cycle and per transfer. However, it benefited embryo quality, demonstrating the potential negative impact of high O2 tension on the in vitro embryo development.

Hormonal and embryonic regulation of chemokine receptors CXCR1, CXCR4, CCR5 and CCR2B in the human endometrium and the human blastocyst.

Chemokines are implicated in the implantation process. The aim of this study was to investigate mRNA expression and protein levels of chemokine receptors CXCR1, CXCR4, CCR5 and CCR2B in human endometrium throughout the menstrual cycle, during HRT and in the human blastocyst. The regulation of chemokine receptors in the endometrial epithelium was also studied using an in-vitro model for the apposition phase of human implantation. We found up-regulation of endometrial CXCR1 mRNA (419-fold increase), CCR5 mRNA (612-fold increase) and CCR2B mRNA (657 fold-increase) during the luteal phase peaking in the pre-menstrual endometrium. CXCR4 mRNA levels presented a specific although modest (18-fold increase) up-regulation during the implantation window. These findings were corroborated at the protein level in natural and HRT cycles. Immunoreactive CCR5 and CCR2B receptors were detected in human blastocysts whereas CXCR4 and CXCR1 were not present. Chemokine receptors in cultured endometrial epithelial cells showed an up-regulation and polarization of CXCR1, CXCR4 and CCR5 receptors when a human blastocyst was present. The specific distribution and regulation of chemokine receptors in the endometrial epithelium and the human blastocyst suggest a possible implication of these receptors in the apposition and adhesion phases of human implantation.