To transfer or not to transfer: the dilemma of mosaic embryos - a narrative review.

A frequent finding after preimplantation genetic diagnostic testing for aneuploidies using next-generation sequencing is an embryo that is putatively mosaic. The prevalence of this outcome remains unclear and varies with technical and external factors. Mosaic embryos can be classified by the percentage of cells affected, type of chromosome involvement (whole or segmental), number of affected chromosomes or affected cell type (inner mass cell, trophectoderm or both). The origin of mosaicism seems to be intrinsic as a post-zygotic mitotic error, but some external factors can play a role. As experience has increased with the transfer of mosaic embryos, clinical practice has gradually become more flexible in recent years. Nevertheless, clinical results show lower implantation, pregnancy and clinical pregnancy rates and higher miscarriage rates with mosaic embryo transfer when compared with the transfer of euploid embryos. Prenatal diagnosis is highly recommended after the transfer of mosaic embryos. This narrative review is intended to serve as reference material for practitioners in reproductive medicine who must manage a mosaic embryo result after preimplantation genetic testing for aneuploidies.

Association between clinical and IVF laboratory parameters and miscarriage after single euploid embryo transfers.

BACKGROUND: The goal of this study was to investigate which factors, excluding embryo aneuploidies, are associated with miscarriage in patients who have undergone a single euploid blastocyst transfer. METHODS: Retrospective, observational and multicenter study with 2832 patients undergoing preimplantational genetic testing for aneuploidies (PGT-A) due to repeated implantation failure, recurrent pregnancy loss, advanced maternal age or severe male factor were transferred one single euploid embryo. RESULTS: One of the main findings was a significant relationship between body mass index (BMI) and miscarriage rates (13.4% in underweight women, 12.1% in normal weight, 14.5% in overweight, and 19.2% in obese women, odds ratio [OD] 1.04; 95% confidence interval [CI], 1.01-1.07 p = 0.006). Endometrial thickness (OD 0.65; 95%, 0.52-0.77 p = 0.04) and type of endometrial preparation (natural cycle or hormone replacement cycle) (OD 0.77; 95%, 0.52-0.77, p = 0.04) were also associated with miscarriage rates. CONCLUSIONS: BMI was strongly associated to miscarriage rates. We also observed a weaker association with endometrial thickness and with the type of endometrial preparation (natural cycle or hormone replacement cycle). None of the other studied variables (biopsy day, maternal and male age, duration of infertility, cycle length, previous miscarriages, previous live births, previous In Vitro Fertilization (IVF) cycles, endometrial pattern and/or diagnosis) were associated with miscarriage rates.

Magnetic-Activated Cell Sorting (MACS): A Useful Sperm-Selection Technique in Cases of High Levels of Sperm DNA Fragmentation.

Magnetic-activated cell sorting (MACS) can be used to separate apoptotic sperm with high proportions of fragmented DNA from the rest, thus improving the overall quality of the seminal sample. Therefore, the aim of this retrospective study was to investigate the efficiency of the MACS technique to increase reproductive outcomes in patients with high levels of sperm DNA fragmentation (SDF) undergoing intracytoplasmic sperm-injection (ICSI) cycles. In this study, we analyzed a total of 724 assisted-reproduction-technique (ART) cycles that were divided into two groups: the study group (n = 366) in which the MACS selection technique was performed after density-gradient centrifugation (DGC), and the control group (n = 358) in which only DGC was used for sperm selection. Reproductive outcomes were analyzed in both groups according to three different ART procedures: preimplantation genetic testing for aneuploidy (PGT-A), and autologous and oocyte-donation cycles. The MACS group showed significantly lower miscarriage rates in autologous ICSI cycles, higher pregnancy rates in oocyte-donation cycles, and a significant increase in live-birth rates in both autologous and oocyte-donation cycles. Overall, these results suggested that the MACS technique can be effectively used to eliminate sperm with high SDF levels, and therefore may help to improve reproductive outcomes in couples undergoing ART.

Should we forget about embryos till day 5?

PURPOSE OF REVIEW: To find the way of having more and better blastocyst is essential. How to culture embryos up to blastocyst stage remains critical. RECENT FINDINGS: Several studies show how a blastocyst score can predict the implantation potential. If that score is enough to choose the best blastocyst, as culture conditions would not be affected in these days, we would not need to check early cleavage embryos, even it could be better for the embryo development. SUMMARY: The item that should be discussed is if it is better to evaluate or not embryos at early cleavage stages. If we do not check embryos on days 2 and 3, we should change our way to work and how to culture those embryos. First step would be to perform all embryo transfers on day 5 or 6. If we let embryos grow to blastocyst without any morphology evaluation, we should adapt several steps in our laboratory, for example we should move to a single-step culture medium or we should not do assisted hatching on day 3 embryos.

Type of chromosome abnormality affects embryo morphology dynamics.

OBJECTIVE: To study the differences in the cleavage time between types of embryo chromosomal abnormalities and elaborate algorithm to exclude aneuploid embryos according to the likelihood to be euploid. DESIGN: Retrospective cohort study. SETTING: University affiliated private center. PATIENT(S): Preimplantational genetic screening patients (n = 112) including cases of advanced maternal age, repeated implantation failure, and recurrent miscarriage. A total of 485 embryos were analyzed. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): All biopsied embryos were cultured in an incubator with time-lapse technology, cleavage timing from insemination to day 3 and all kinetic parameters that have been described in previous studies by our group. RESULT(S): Logistic regression analysis were used to identify morphokinetic parameters and some were strongly associated with complex aneuploid embryos; t3 (odds ratio = 0.590, 95% confidence interval 0.359-0.971) and t5-t2 (odds ratio = 0.151, 95% confidence interval 0.082-0.278). CONCLUSION(S): Embryo morphokinetics are affected by chromosome aneuploidy and further analysis of the chromosome content reveals higher differences when the complexity in the chromosome disorders is increased. The use of time-lapse monitoring, although not able to detect an abnormal embryo, may be potentially useful to discard those embryos with high risk of complex chromosomal abnormalities.

Automatic time-lapse instrument is superior to single-point morphology observation for selecting viable embryos: retrospective study in oocyte donation.

OBJECTIVE: To correlate the different categories provided by a commercial diagnostic test with blastocyst formation, quality, implantation potential, and ongoing pregnancy (OPR) for the purpose of validating the automatic annotations and the classification algorithm. DESIGN: Observational, retrospective, multicenter cohort study. SETTING: University-affiliated private IVF center. PATIENT(S): A total of 3,002 embryos, including 521 transferred embryos with known implantation, from 626 IVF cycles that were incubated in a conventional incubator and monitored with an automatic time-lapse test. INTERVENTIONS(S): None. MAIN OUTCOME MEASURE(S): Embryo selection was based on morphology and the classification provided by a commercial diagnostic test. Implantation was the primary end point, and OPR, blastocyst formation (BR), and embryo morphology were secondary end points. RESULT(S): BR and number of optimal blastocysts were related to the classification test. This correlation was also observed when analyzing implantation rates (day 3 transfer: high 38.2%, medium 31.7% and low 26.1%; day 5 transfer: high 66.7%, medium 50%, low 31%). Patients where no high embryos were transferred (n = 75) had an OPR of 46.70%, and those patients where at least one high embryo was transferred (n = 109) significantly increased OPR to 67%. A logistic regression analysis studying other confounding factors (day of transfer, number of oocytes obtained, and embryo morphology classification) was included. In that model, if at least one of the embryos was labeled as high, OPR was 2.567 times higher than a cycle where no high embryos were transferred. CONCLUSION(S): Our study presents, to our knowledge, the largest set of transferred embryos after time-lapse analysis with the use of an automatic time-lapse test. The provided classification was related to reproductive outcome. Our results suggest that the automated embryo diagnostic test provided extra information to the embryologist to select the best embryos, independently from clinical features of the patient or day of transfer.

Is there a relationship between time-lapse parameters and embryo sex?

OBJECTIVE: To study if it is possible to identify embryo sex from embryo cleavage timings. DESIGN: Retrospective and observational study. SETTING: University-affiliated private fertility center. PATIENT(S): Women undergoing preimplantion genetic diagnosis. INTERVENTION[S): All biopsied embryos were cultured in an Embryoscope incubator with time-lapse technology. MAIN OUTCOME MEASURE(S): Cleavage timing from insemination to day 3 and all kinetic parameters that have been described in previous studies by our group. RESULT(S): The study included 421 embryos from our Compressive Chromosome Screening program, conducted from January 2012 to December 2012. Embryos were grouped according to their sex: male (176 embryos) and female (161 embryos). Chromosomal abnormal rate was similar for the two groups (male 62.5%, female 58.4%). When morphokinetic parameters were separated in different quartiles and grouped, we found statistical differences between male or female embryos. By logistic regression analysis we found that two specific kinetic variables were relevant: second synchrony (>2 hours) and timing of morula formation (80.8-90.9 hours). With the use of these parameters, we propose an algorithm with four different categories reflecting the range from 71% to 42% in the likelihood of an embryo being female. CONCLUSION(S): Embryo development was affected by embryo sex, and the sex ratio could be affected by the embryo selection method for transfer based on kinetic parameters.

Oocyte versus embryo vitrification for delayed embryo transfer: an observational study.

The aim of this observational prospective study was to compare multiple embryological and pregnancy outcomes for vitrified oocytes against the same outcomes using vitrified embryos in patients at risk for ovarian hyperstimulation syndrome. Ninety-six patients were included and allocated to vitrification of oocytes (Group 1) or embryos (Group 2). No statistical differences in baseline characteristics between groups were detected. Implantation rate was 30.6% versus 33.1%, and clinical pregnancy rate was 41.9% versus 7.1% in groups 1 and 2, respectively. A higher clinical spontaneous abortion rate occurred in group 2 (9.7% versus 21.9% for groups 1 and 2, respectively), but the same cumulative clinical pregnancy rate was observed after three embryo transfers (62.0% in group 1 and 69.6% in group 2). The ongoing pregnancy rate per patient was similar in both groups (56.0% and 54.3% in groups 1 and 2, respectively). Also, live birth rate per stimulation was similar (72.0% and 69.6% in groups 1 and 2, respectively). No differences were observed in outcomes according to vitrification timing. Oocyte vitrification achieved the same live birth rate as embryo vitrification.

Increasing the probability of selecting chromosomally normal embryos by time-lapse morphokinetics analysis.

OBJECTIVE: To study the differences in the cleavage time between chromosomally normal and abnormal embryos and to elaborate an algorithm to increase the probability of noninvasively selecting chromosomally normal embryos. DESIGN: Retrospective cohort study. SETTING: University-affiliated infertility center. PATIENT(S): Preimplantation genetic screening patients (n = 125; n = 77 with ET), including cases of repeated implantation failure or recurrent miscarriage. A total of 504 embryos were analyzed. INTERVENTION(S): Embryo culture within a time-lapse system. MAIN OUTCOME MEASURE(S): Kinetic variables included the time to 2 (t2), 3 (t3), 4 (t4), and 5 (t5) cells as well as the length of the second (cc2 = t3 - t2) and third (cc3 = t5 - t3) cell cycle, the synchrony in the division from 2 to 4 cells (s2 = t4 - t3), and the interval t5 - t2. Implantation and clinical pregnancy rates were also analyzed. RESULT(S): A logistic regression analysis identified t5 - t2 (odds ratio [OR] = 2.853; 95% confidence interval [CI], 1.763-4.616), followed by cc3 (OR = 2.095; 95% CI, 1.356-3.238) as the most relevant variables related to normal chromosomal content. On the basis of these results, an algorithm for embryo selection is proposed to classify embryos from A to D. Each category exhibited significant differences in the percentage of normal embryos (A, 35.9%; B, 26.4%; C, 12.1%; D, 9.8%). CONCLUSION(S): Chromosomally normal and abnormal embryos have different kinetic behavior. On the basis of these differences, the proposed algorithm serves as a tool to classify embryos and to increase the probability of noninvasively selecting normal embryos.

Type of culture media does not affect embryo kinetics: a time-lapse analysis of sibling oocytes.

STUDY QUESTION: Are the morphokinetics of growing embryos affected by the type of culture media utilized? SUMMARY ANSWER: Morphokinetic parameters used for embryo selection are not affected between the two different concept culture media analyzed. WHAT IS KNOWN ALREADY: Studies on the effect of culture media on human embryos have focused on evaluating different in-house and commercially available media as well as comparing outcomes among different commercial media. Nonetheless, the evaluation of embryo development in these studies was based on static observations and very little is known from a dynamic point of view. STUDY DESIGN, SIZE, DURATION: Prospective cohort study, October 2010 and April 2011. PARTICIPANTS/MATERIALS, SETTING, METHODS: University-affiliated infertility center. Patients undergoing egg donation (n = 75) in which embryos were cultured with two different types of media in a time-lapse system. Embryo development was analyzed with time-lapse imaging for single step media (Global®) and sequential media (Sage® Cleavage). Variables studied included the timing to two cells (t2), three cells (t3), four cells (t4) and five cells (t5) as well as the length of the second cell cycle (cc2 = t3 - t2) and the synchrony in the division from two to four cells (s2 = t4 - t3). Implantation and clinical pregnancy rates were also analyzed. MAIN RESULTS AND THE ROLE OF CHANCE: No statistically significant differences were observed between the two media for all the variables analyzed. When analyzing the percentage of embryos falling within the optimal ranges proposed for s2, cc2 and t5, we did not find significant differences between the two media. Pregnancy and implantation rates were similar for the three types of transfers: 48.0% (CI 95% 28.4-67.6) and 42.0% (CI 95% 22.5-61.4) with Global media; 58.8% (CI 95% 35.4-82.2) and 38.2% (CI 95% 15.0-61.4) with Cleavage media; and 58.1% (CI 95% 40.7-75.4) and 37.1% (CI 95% 22.1-52.1) with mixed transferred, respectively. Multiple implantations (twins) were also similar among the three groups, with 24.0% (CI 95% 9.3-45.1) for transfers with embryos cultured in Global media, 17.6% (CI 95% 3.7-43.3) for transfers with embryos cultured in Cleavage media and 22.5% (CI 95% 9.5-41.0) with mixed transfers. LIMITATIONS, REASONS FOR CAUTION: The study was not powered to test differences in pregnancy rates between the two culture media, as this was not the hypothesis tested. Results are based on observations with embryos from oocyte donors and need to be repeated with embryos from infertile patients of different ages. WIDER IMPLICATIONS OF THE FINDINGS: The absence of differences in morphokinetics between two different media concepts validates the algorithm for embryo selection in diverse culture conditions. STUDY FUNDING/COMPETING INTEREST(S): No specific funding was obtained for this study; it was solely funded by IVI. None of the authors have any economic affiliation with Unisense Fertilitech A/S but IVI is a minor shareholder in Unisense Fertilitech A/S.

The impact of in-vitro maturation of oocytes on aneuploidy rate.

Chromosome abnormalities in embryos obtained through in-vitro maturation (IVM) of oocytes from 11 oocyte donors were compared with embryos from women undergoing fluorescence in-situ hybridization (FISH) analysis for sex selection. Thirty-three oocytes had reached metaphase II stage at 28-30 h (65%) and 27 were successfully fertilized by intracytoplasmic sperm injection. Blastomere biopsy was performed in 20 embryos (74%). For five embryos, two blastomeres were analysed, three of which were mosaic. FISH study revealed aneuploidies of chromosomes 13, 15, 16, 18, 21, 22, X and Y in 12 embryos (60%) and euploidy in the remaining eight (40%). The percentage of aneuploidies in the control group was 33%. Differences between IVM and control embryos were not statistically significant. The high incidence of chromosome abnormalities in embryos resulting from the IVM protocol may account for the low implantation rates reported by others. Although a greater incidence of miscarriage or congenital abnormalities in babies born alive following IVM versus conventional IVF has not been observed in previous studies, preimplantation genetic aneuploidy screening or prenatal chromosome studies may be recommended to these patients on the basis of the present results.