OBJECTIVE: Does double blastocyst vitrification and warming affect pregnancy, miscarriage or live birth rates, or birth outcomes, from embryos that have undergone preimplantation genetic testing for aneuploidies (PGT-A) testing?
METHODS: This retrospective observational analysis of embryo transfers was performed at a single centre between January 2017 and August 2022. The double-vitrification group included frozen blastocysts that were vitrified after 5-7 days of culture, warmed, biopsied (either once or twice) and re-vitrified. The single vitrification (SV) group included fresh blastocysts that were biopsied at 5-7 days and then vitrified.
RESULTS: A comparison of the 84 double-vitrification blastocysts and 729 control single-vitrification blastocysts indicated that the double-vitrification embryos were frozen later in development and had expanded more than the single-vitrification embryos. Of the 813 embryo transfer procedures reported, 452 resulted in the successful delivery of healthy infants (56%). There were no significant differences between double-vitrification and single-vitrification embryos in the pregnancy, miscarriage or live birth rates achieved after single-embryo transfer (55% versus 56%). Logistic regression indicated that while reduced live birth rates were associated with increasing maternal age at oocyte collection, longer culture prior to freezing and lower embryo quality, double vitrification was not a significant predictor of live birth rate.
CONCLUSIONS: Blastocyst double vitrification was not shown to impact pregnancy, miscarriage or live birth rates. Although caution is necessary due to the study size, no effects of double vitrification on miscarriage rates, birthweight or gestation period were noted. These data offer reassurance given the absence of the influence of double vitrification on all outcomes after PGT-A.

OBJECTIVE: Evaluate which factors are involved in the increased rate of mosaicism in embryos.
METHODS: A systematic review and meta-analysis was performed. After an exhaustive search of the literature, a total of seven papers were included in the analysis. In addition, data collected from IVF cycles performed in our fertility clinic were also analysed. Day of biopsy, embryo quality, maternal and paternal age and seminal quality were the chosen factors to be studied.
RESULTS: The results of the meta-analysis show that neither embryo quality nor seminal quality were related to mosaic embryo rate (OR: 1.09; 95% CI: 0.94-1.28 and OR: 1.10; 95% CI: 0.87-1.37, respectively). A positive association was observed for the variable \"biopsy day\" with embryos biopsied at day 6 or 7 having the highest rate of mosaicism (OR: 1.06; 95% CI: 1.01-1.11). In opposite to what happens with aneuploidy rate, which increases with maternal age, embryo mosaicism is higher in younger women (<34 years) rather than in older ones (≥34 years) (OR: 0.95; 95% CI: 0.92-0.98). However, for the \"paternal age\" factor, no association with mosaicism was found (OR: 1.04; 95% CI: 0.90-1.21).
CONCLUSIONS: With the present study, we can conclude that the factors related to the presence of mosaicism in embryos are the embryo biopsy day and maternal age. The rest of the studied factors showed no significant relationship with mosaicism. These results are of great importance as knowing the possible causes leading to mosaicism helps to improve the clinical results of reproductive treatments.

OBJECTIVE: To investigate whether the rate of euploidy and pregnancy outcomes are affected by smooth endoplasmic reticulum clusters (SERc) and other metaphase II human oocyte dysmorphisms.
METHODS: Retrospective analysis of the morphologies of metaphase II (MII) human oocytes, which had developed into 590 biopsied blastocysts derived from 109 patients that received preimplantation genetic testing for aneuploidies (PGT-A) cycles between March 2013 and December 2017. The euploid rate of blastocysts that originated from morphologically abnormal or normal oocytes were analyzed. The chromosome status of the blastocysts was determined and analyzed by array comparative genomic hybridization (aCGH) or next generation sequencing (NGS) following trophectoderm biopsy.
RESULTS: According to the odds ratios obtained for each oocyte morphotype, no statistically significant relationship was found between oocyte dysmorphisms and euploid rate. Specifically, although SERc-positive oocytes had a higher rate of arrest at two pronuclei, or 2 PN (26.7% vs. 19.4%, p > 0.05), the blastocyst formation rate was not affected as compared with SERc-negative oocytes (40.0% vs. 38.6%, p > 0.05). Among nine euploid embryos derived from oocytes with SERc, three single euploid embryo transfers were performed, of which one resulted in blighted ovum, and two resulted in the births of two healthy, singleton term babies.
CONCLUSIONS: The results presented here suggest that oocyte dysmorphisms do not affect the euploidy rate of the blastocyst. The occurrence of SERc in the oocyte does not seem to impair the developing blastocyst nor does it interfere with good embryo formation rate and euploid rate. Thus, the embryos derived from SERc-positive oocytes could still be considered for embryo transfer if there are no other embryos available.

Do clinical and neonatal outcomes differ between mosaic embryo transfers (MET) and euploid embryo transfers (EET)?
This retrospective cohort study compared the implantation rate, live birth rate (LBR) and miscarriage rate between 513 euploid embryos and 118 mosaic embryos (72 whole chromosome mosaic [WCM], 40 segmental mosaic and six complex mosaic). Blastocysts were analysed using preimplantation genetic testing for aneuploidies with next-generation sequencing, followed by a single vitrified-warmed embryo transfer. Trophectoderm biopsies were classified as mosaic if they had 20-80% abnormal cells.
Overall, EET resulted in a significantly higher implantation rate (47.0%) and LBR (40.7%) than MET (implantation rate 39.0%, P = 0.005; LBR 28.8%, P = 0.008) and WCM embryos (implantation rate 37.5%, P = 0.01; LBR 22.2%, P = 0.007) after covariate adjustment. Segmental mosaic embryos had an implantation rate (47.5%) and LBR (45.0%) comparable to those of euploid embryos. Mosaic embryos with a high percentage of aneuploid cells (≥60%) showed a significantly lower LBR (10.5% versus 40.7%, P = 0.03) than euploid embryos after covariate adjustment, with three of the five implantations of mosaic embryos resulting in miscarriage. Neonatal outcomes did not differ significantly between the mosaic and euploid groups. Of the 34 women with a live birth after MET, 13 had a prenatal or postnatal genetic testing result, and no abnormalities were found.
Mosaic embryos were associated with a lower LBR, while segmental mosaic embryos had similar clinical outcomes to euploid embryos. Mosaic embryos with a high aneuploidy percentage (≥60%) should be assigned a low transfer priority. Neonatal outcomes did not differ significantly between the euploid and mosaic groups.

OBJECTIVE: To investigate whether preimplantation genetic testing for aneuploidy (PGT-A) improves the clinical outcome in patients with advanced maternal age (AMA), recurrent miscarriages (RM), and recurrent implantation failure (RIF).
METHODS: Retrospective cohort study from a single IVF center and a single genetics laboratory. One hundred seventy-six patients undergoing PGT-A were assigned to three groups: an AMA group, an RM group, and a RIF group. Two hundred seventy-nine patients that did not undergo PGT-A were used as controls and subgrouped similarly to the PGT-A cohort. For the PGT-A groups, trophectoderm biopsy was performed and array comparative genomic hybridization was used for PGT-A. Clinical outcomes were compared with the control groups.
RESULTS: In the RM group, we observed a significant decrease of early pregnancy loss rates in the PGT-A group (18.1% vs 75%) and a significant increase in live birth rate per transfer (50% vs 12.5%) and live birth rate per patient (36% vs 12.5%). In the RIF group, a statistically significant increase in the implantation rate per transfer (69.5% vs 33.3%) as well as the live birth rate per embryo transfer (47.8% vs 19%) was observed. In the AMA group, a statistically significant reduction in biochemical pregnancy loss was observed (3.7% vs 31.5%); however, live birth rates per embryo transfer and per patient were not significantly higher than the control group.
CONCLUSIONS: Our results agree with recently published studies, which suggest caution in the universal application of PGT-A in women with infertility. Instead, a more personalized approach by choosing the right candidates for PGT-A intervention should be followed.

Male infertility contributes as the main factor in 30-50% of infertility cases. Conventional methods for sperm preparation have induced questioning of sperm recovery rates. The microfluidic sperm sorting (MSS) technique selects highly motile sperm with lower levels of SDF (sperm DNA fragmentation) compared to conventional sperm sorting techniques. This study aimed to determine whether utilizing this technique will reveal better embryo quality and euploidy rates in couples with repeated implantation failure (RIF) and high SDF in a new PGT-A (preimplantation genetic testing for aneuploidies) cycle. This retrospective study included couples referred to PGT-A for previous repeated ART (assisted reproductive techniques) cycle failures and with high SDF. In their new cycles, couples who accepted the technique were assigned to the MSS group, and the rest were managed with DGC (density-gradient centrifugation). Two groups were compared in terms of fertilization and euploidy rates, clinical miscarriage and live birth rates, the total number of blastocysts, and top quality blastocysts. There was no difference between the groups regarding fertilization rates, euploidy rates, clinical miscarriage, and live birth rates. The total number of blastocysts and top quality blastocysts were significantly higher in the MSS group. The MSS technique provides a higher number of top-quality blastocysts than DGC; however, neither euploidy nor live birth rates improved. Studies focusing on confounding factors to embryonic genomic status in the presence of high SDF are needed.

OBJECTIVE: Day of cryopreservation, inner cell mass (ICM) grade, trophectoderm grade and blastocyst expansion grade have been associated with differences in live birth rate in frozen embryo transfer (FET) cycles. This study sought to examine the likelihood of live birth and whether the morphological grade of the blastocyst is more or equally useful in FET cycles among preimplantation genetic testing for aneuploidies (PGT-A) tested and untested blastocysts.
METHODS: This was a retrospective cohort study of 6271 vitrified-warmed, autologous, single-embryo transfer cycles among patients undergoing IVF from July 2013 to December 2017 at a single, university-affiliated infertility practice. The primary outcome was live birth, calculated by generalized estimating equations.
RESULTS: Among PGT-A tested embryos, inferior ICM grade was associated with a lower chance of live birth (ICM grade B versus A: adjusted risk ratio [aRR] 0.91, 95% confidence interval [CI] 0.84-0.99). Among untested blastocysts there was a lower live birth rate in blastocysts cryopreserved on day 6 versus day 5 (aRR 0.87, 95% CI 0.78-0.96), and those with inferior pre-vitrification trophectoderm grade (trophectoderm grade B versus A: aRR 0.86, 95% CI 0.79-0.94). Blastocysts with a higher pre-vitrification expansion grade (pre-vitrification expansion grade 5 versus 4: aRR 1.1, 95% CI 1.01-1.2) were associated, but ICM grade was not associated (ICM grade B versus A: aRR 0.93, 95% CI 0.86-1.02), with chance of live birth.
CONCLUSIONS: Among PGT-A untested blastocysts, assessing embryo quality by day of cryopreservation, trophectoderm grade and expansion grade may help to identify embryos with the highest likelihood of live birth. Identifying euploid embryos by PGT-A appears to homogenize the cohort, making blastocyst morphological grade and day of cryopreservation less important.

Chromosomal mosaicism is at high occurrence in early developmental-stage embryos, but much lower in those at prenatal stage. Recent studies provided evidence on the viability of mosaic embryos by reporting pregnancy outcomes. Expanded research is warranted to evaluate its clinical significance. This is a multi-center prospective cohort study on 137 mosaic, 476 euploid and 835 non-preimplantation genetic testing (non-PGT) embryos from three in vitro fertilization (IVF) providers of three countries in Asia, applying the same preimplantation genetic testing for aneuploidies (PGT-A) reporting criteria. Mosaic embryo transfers (METs) resulted in a significantly lower clinical pregnancy rate (40.1% versus 59.0% versus 48.4%), lower ongoing/live birth rate (27.1% versus 47.0% versus 35.1%) and higher miscarriage rate (33.3% versus 20.5% versus 27.4%) than euploid and non-PGT transfers, respectively. Pregnancy losses after METs were different between embryos carrying numerical and segmental chromosomal abnormalities (p = 0.04). Our meta-analysis concluded that METs gave rise to pregnancies but were associated with a reduced ongoing/live birth rate and a higher miscarriage rate. All 37 MET live births were confirmed viable, among which 8 completed prenatal genetic testing with normal results. Longitudinal investigation on one MET pregnancy evidenced the aneuploidy depletion hypothesis. This is the first multi-center prospective study reporting a full MET pregnancy outcome with complementary information from prenatal genetic testing as compared to euploid and non-PGT cohorts.

This retrospective study aimed to assess the relationship between standard markers of embryo morphology, maternal age and blastocyst ploidy determined by trophectoderm (TE) biopsy and Next-generation Sequencing (NGS). A total of 774 oocytes and embryos from 288 PGT-A cycles were scored for pronuclear, cleavage stage and blastocyst morphology. Pronuclear oocytes aligned between the nuclei and presenting equal number of nucleolus precursor bodies (NPBs) were designated Z1, oocytes showing equal number of NPBs, but not aligned, as Z2 while Z3 oocytes had an unequal number of NBPs between the nuclei or NPBs aligned in one nucleus and non-aligned in the other. Pronuclear oocytes with unequal-sized or non-aligned nuclei were designated Z4. Blastocysts were graded as BL1 (AA, AB or BA), BL2 (BB or CB) and BL3 (BC or CC) based on the combination of inner cell mass (ICM) and TE scores. Pronuclear and blastocyst morphology were correlated with aneuploidy in a < 40-year-old group (p < 0.01 and p < 0.05, respectively), but not in those ≥40 years. Interestingly, BL3 blastocysts classified as Z1 or Z3-Z4 on day-1 had different aneuploidy rates (BL3/Z1 = 46.7% vs. BL3/Z3-Z4 = 90.0%, p < 0.05). In summary, our data showed that pronuclear and blastocyst morphology are associated with blastocyst ploidy in younger patients. This may help embryo selection for embryo transfer and decision-making on which blastocysts should be biopsied in PGT-A cycles.

Preimplantation genetic testing for aneuploidies (PGT-A) and PGT for monogenic disorders (PGT-M) have currently been used widely, aiming to improve IVF outcomes. Although with many years of unsatisfactory results, PGT-A has been revived because new technologies have been adopted, such as platforms to examine all 24 types of chromosomes in blastocysts. This report compiles current knowledge regarding the available PGT platforms, including quantitative PCR, array CGH, and next-generation sequencing. The diagnostic capabilities of are compared and respective advantages/disadvantages outlined. We also address the limitations of current technologies, such as assignment of embryos with balanced translocation. We also discuss the emerging novel PGT technologies that likely will change our future practice, such as non-invasive PGT examining spent culture medium. Current literature suggest that most platforms can effectively reach concordant results regarding whole-chromosome ploidy status of all 24 types of chromosomes. However, different platforms have different resolutions and experimental complexities; leading to different turnaround time, throughput and differential capabilities of detecting mosaicism, segmental mutations, unbalanced translocations, concurrent PGT-A and PGT-M etc. Based on these information, IVF staff can more appropriately interpret PGT data and counsel patients, and select suitable platforms to meet personalized needs. The present report also concisely discusses some crucial clinical outcomes by PGT, which can clarify the role of applying PGT in daily IVF programs. Finally the up-to-date information about the novel use of current technologies and the newly emerging technologies will also help identify the focus areas for the design of new platforms for PGT in the future.