The Impact of Embryo Quality on Pregnancy Outcomes in Single Day 5 versus Day 6 Euploid Blastocyst Transfer: A Retrospective Cohort Study.

As the ultimate purpose of in vitro fertilization (IVF) is to maximize the likelihood of a successful pregnancy and the delivery of a healthy baby following each embryo transfer cycle, the culture and selection of the embryos is a crucial step. Today, optimization of the in vitro embryo culture process allows for improving the yield of blastocysts after ovarian stimulation. Moreover, the employment of the vitrification technique results in nearly 100 percent post-thaw survival rates for cryopreserved embryos ( 1 ). Single frozen blastocyst transfer is becoming more prevalent in assisted reproduction centers in order to reduce the risk of ovarian hyperstimulation while lowering the risk of multiple pregnancies and other maternal or neonatal problems as well as economizing the health care expenses ( 2 ). In addition, preimplantation genetic testing for aneuploidies (PGT-A) of available blastocysts has been proposed to select embryos with the highest potential. Indeed, as euploid embryos are most likely to implant, the pregnancy rate per embryo transfer is enhanced and the miscarriage rate is reduced, hence minimizing the time-to-pregnancy. Indications for PGT-A usually include advanced maternal age, recurrent implantation failure, severe male factor and recurrent miscarriage ( 3 ). However, not every euploid embryo is likely to successfully implant because embryonic factors other than embryo morphology and genetic characteristics such as the time of blastocyst formation also influence embryo implantation potential. The usual time for an embryo to reach the blastocyst stage is day 5 (D5) following fertilization but some may not attain a blastocyst morphology until D6 or later. Some evidence exists that D6 embryos might have weak development rate due to their slower growth rate than D5 embryos ( 4 ). As reported by Tong et al. ( 5 ), the percentage of euploid embryos is higher in D5 than in D6 blastocysts but the pregnancy rate was similar. Abdala et al. ( 6 ) observed higher rates of pregnancy and clinical pregnancy in D5 as compared to D6 euploid embryos but that outcome was more influenced by endometrial thickness, patient age, body mass index (BMI) and inner cell mass (ICM) grade C than by the embryonic age at biopsy. In contrast, studies by Li et al. ( 7 ) and Irani et al. ( 8 ) concluded that the transfer of D5 euploid embryo results in a higher live birth rate than D6. Few studies with large sample sizes comparing the outcome of euploid D5 and D6 transfer have been published so far. Furthermore, embryo quality and parameters related to the uterine cavity and hydrosalpinx are not fully discussed ( 7 - 10 ). The aim of this study was to compare the live birth rates of euploid blastocyst transfers based on the timing of blastulation (D5 vs. D6) and the quality of embryos.

Overall, 774 patients underwent D5 euploid transfer and 155 patients underwent D6 euploid transfer. The D5 group had an average age of 35.5 years [95% confidence intervals (CI): 35.21-35.79] compared to the D6 group's 35.27 years (95% CI: 34.64-35.89). Both groups had nearly identical BMIs, with 21.54 kg/m 2 for D5 (95% CI: 21.39-21.69) and 21.44 kg/m 2 for D6 (95% CI: 21.1-21.78). Regarding infertility types, the D5 group reported 13.05% primary (those never achieving pregnancy) and 86.95% secondary (those previously conceiving but now facing difficulties). The D6 group had similar percentages, at 12.90 and 87.10% respectively. Among those in the D5 group, 67.01% (518 out of 774) were undergoing their first embryo transfer cycle, while this figure was 61.29% (95 out of 155) in the D6 group (P=0.170). Other factors, like duration of infertility, history of caesarean section, endometrial thickness, and rates of difficult embryo transfer, also showed analogous patterns between the two groups. However, we did note a statistically significant difference in the underlying causes of infertility. Specifically, 47.55% (368 out of 774) of infertility cases in the D5 group were attributed to female factors, compared to 40.00% (62 out of 155) in the D6 group. Conversely, male factors were more prevalent in the D6 group, accounting for 8.39% (13 out of 155) of the cases, as opposed to D5's 3.49% (27 out of 774) (P=0.030, Table 1 ). Table 2 summarizes the clinical outcome of FET cycles stratified according to the timing of blastulation. The live birth rate was significantly lower in group D6 compared to group D5 (38.71 vs. 55.04%, P=0.001). Similarly, the ongoing pregnancy rate was lower in Group D6 (38.71 vs. 55.17%, P=0.001). The pregnancy and clinical pregnancy rates were significantly lower in Group D6 compared to Group D5 (61.94 vs. 73.13%, P=0.013; and 48.39 vs. 62.66%, P=0.003, respectively). No significant difference was found in biochemical pregnancy rates between the groups. Birth outcomes including gestational age at delivery and birth weight showed no statistical disparity. Patient characteristics Data are presented as (95% CI) or n (%). *; Obtained by independent sample t test and Pearson’s chi-square test, and statistically significant difference at 0.05. CI; Confidence intervals and BMI; Body mass index. Embryos of excellent quality biopsied on D5 yielded the highest live birth rate (59.87%), followed by those of good quality (52.27%) and fair quality (33.33%) ( Table 3 ). In contrast, the live birth rate for embryos of excellent quality biopsied on D6 was only 53.33% and 38.6 and 15.79% for good quality and fair quality embryos, respectively. The significant statistical difference observed in Table 3 indicates that at least one category within the embryo quality and timing of blastulation variables differs from the others. It indicates that different levels of embryo quality and timing of blastulation may potentially lead to better outcomes in comparison to the remaining categories. This finding highlights the importance of conducting a multivariate analysis to assess the specific effects and relationships of these variables on the outcomes of interest. Table 4 provides the adjusted risk ratios (RR) for multiple clinical outcomes, benchmarked against the reference group of D5 excellent quality embryos. The data suggest that the transfer of fair quality D5 embryos is significantly associated with decreased live birth rates (RR=0.58, 95% CI: 0.42-0.79) and pregnancy rates (RR=0.71, 95% CI: 0.58-0.87). Transfers of excellent or good quality D6 embryos yielded lower live birth rates (RR=0.89 and RR=0.68 with 95% CI: 0.69-1.13 and 0.49-0.95, respectively) and pregnancy rates (RR=0.94 and 0.87 with 95% CI: 0.80-1.10 and 0.73-1.05, respectively). Notably, the transfer of fair quality D6 embryos is associated with substantially lower rates of live birth (RR=0.27, 95% CI: 0.14-0.54), pregnancy (RR=0.43, 95% CI: 0.28-0.67), clinical pregnancy (RR=0.27, 95% CI: 0.13-0.57), and ongoing pregnancy (RR=0.27, 95% CI: 0.13-0.57). Clinical outcomes stratified according to the timing of blastulation Data are presented as (95% CI) or n (%). *; Obtained by independent sample t test and Pearson’s chi-square test, and statistically significant difference at 0.05 and CI; Confidence intervals Association between clinical outcome, quality of embryos and timing of blastulation Data are presented as (95% CI) or n (%). *; Obtained by Pearson’s chi-square test and statistically significant difference at 0.05 and CI; Confidence intervals Adjusted risk ratios for clinical outcomes, relative to the day 5 excellent quality embryo reference group Data are presented as (95% CI) or n (%). RR; Risk ratios and CI; Confidence intervals.

Selecting the embryo with the greatest potential for implantation remains a challenge for embryologists and clinicians. The utilization of PGT-A in conjunction with morphological grading represents useful criteria for the selection process. According to our data, both live birth and ongoing pregnancy rates are notably lower following the transfer of D6 euploid embryos compared to their D5 counterparts. Additionally, the quality of the embryos played a crucial role in determining the clinical outcome. No consensus actually exists in the literature regarding the clinical potential of euploid D5 and D6 vitrified blastocysts. Our results are in line with previous studies that reported improved live birth rates following single D5 euploid blastocyst transfers compared to D6 transfers ( 7 - 10 ). In contrast, Abdala et al. ( 6 ) and Tong et al. ( 5 ), recently reported no significant differences in reproductive outcomes between the two groups. Discrepancies between the conclusions of these studies are likely related to differences in studied populations, methodologies or other unaccounted factors. The lower live birth and ongoing pregnancy rates for D6 euploid blastocysts observed in our study might suggest that D5 euploid blastocysts present a better potential for development than the D6 ones but it could also be related to asynchrony between the embryo and endometrium. Reduced potential of blastocyst development may result from various factors, including delayed timing of the initial cleavage division, subsequent cleavage divisions, or abnormal early cleavage events. These events could potentially influence the intricate process of embryonic genome activation, which involves the transition of gene transcription from the maternal to the embryonic genome, ultimately leading to the differentiation of the ICM and TE ( 14 , 15 ). Furthermore, it has been observed that D5 blastocysts possess a significantly greater quantity of mitochondrial DNA (mtDNA) than D6 blastocysts. The quantity of mtDNA could be a crucial factor affecting the developmental rate of blastocysts, as mitochondria play a vital role in supplying energy for cellular processes, including embryonic development ( 16 ). Compared to previous studies, this study has some strengths, including the large sample size and the use of contemporary technologies to assess embryo viability. Firstly, the study included a relatively large sample size of cycles [929], which is higher than other previous studies such as Tong et al. ( 5 ) [537 cycles] and Rao et al. ( 9 ) [126 cycles]. A large sample size increases the statistical power of the study and makes the results more reliable and generalizable to the population. Secondly, this study utilized next-generation sequencing (NGS) to perform PGT-A, which is considered a more reliable and accurate technology compared to the traditional array comparative genomic hybridization (aCGH) used by Irani et al. ( 8 ). The use of NGS for PGT-A allows detection of copy number variations (CNVs) and structural rearrangements that may be missed by aCGH. Therefore, the use of NGS in this study increases the accuracy of embryo selection, leading to better clinical outcomes. Despite the consistent findings in our study and previous research, it is important to acknowledge that there may be variations in embryo grading systems and laboratory practices across different studies and clinical settings. As a result, direct comparisons between studies should be made with caution. Furthermore, it should be noted that factors such as maternal age, infertility etiology, and endometrial receptivity might also influence the outcomes of euploid blastocyst transfers. Additional studies with larger sample sizes and more homogeneous populations of patients could help elucidate further the complex relationship between these factors and clinical outcomes. The study also demonstrated the importance of embryo quality in determining clinical outcomes. We observed that excellent quality embryos had the highest rates of live birth and pregnancy, regardless of the timing of blastulation. This finding aligned with previous research that has emphasized the role of embryo quality in predicting successful pregnancies ( 17 , 18 ). Interestingly, our study revealed that D6 embryos with excellent quality had a higher live birth rate than D5 embryos with fair quality. This suggests that the quality of the embryo may be more involved in the success of the transfer than the timing of blastulation. However, the study faced some potential limitations, such as the retrospective analysis of the data and the relatively small sample size of D6 embryos, which could impact the statistical power of our findings. Further research is needed to confirm this observation and explore the underlying mechanisms that contribute to the observed differences in outcomes between D5 and D6 euploid blastocyst transfers.

Timing of blastulation and embryo quality are important factors in determining the success of single euploid blastocyst transfers. The transfer of D5 embryos, particularly those with excellent quality, was associated with higher live births and ongoing pregnancy rates compared to D6 embryos. These findings provide valuable information to help clinicians in the selection of embryos for transfer.

A retrospective cohort study was conducted at the Assisted Reproductive Center of Tam Anh General Hospital enrolling all patients who underwent embryo transfer between January 2019 and February 2022. Criteria for selection included: i. Single D5 or D6 euploid transfer, ii. Endometrial thickness on the day of progesterone supplementation of at least 7 mm, and iii. Age between 20 and 45 years old. Exclusion criteria were the following: i. Abnormalities of the uterus or hydrosalpinx on hysterosalpingography, ii. Submucosal fibroid, iii. Oocyte donation, iv. Adenomyosis, and v. Sperm from retrieval procedures. Patient-related variables were extracted from the hospital information system. For outcomes after embryo transfer, if the patient was not followed up at the same hospital, they were interviewed by phone and their outcomes were recorded in the hospital’s data system. Before initiating any research activities, approval was obtained from the Tam Anh General Hospital Ethics Committee (IRB.TAHN.024) on October 20th, 2022. Stimulation was started on day 2 of a spontaneous menstrual cycle with the administration of recombinant follicle stimulating hormone (FSH, Follitrope, LG Chem, South Korea) or human menopausal gonadotropin (Menopur, Ferring, United Kingdom) at a dose of 150-300 IU daily. The initial and continuous gonadotropin dosages were adjusted according to patient age, baseline FSH level, body mass index (BMI), antral follicle count (AFC) and response to follicular growth. Pituitary suppression was started on day 6 of ovarian stimulation with daily administration of a GnRH antagonist (ganirelix, Orgalutran®, MSD, Australia; or cetrorelix, Cetrotide®, Merck, Netherlands) at a dose of 0.25 mg. Once the dominant follicle’s diameter reached 20 mm, or when three follicles reached 18 mm, the last stage of oocyte maturation was induced by administration of 0.2 mg of triptorelin (Diphereline, Ipsen Pharma Biotech, France) plus 2000 IU of hormone chorionic gonadotropin (hCG, IVF-C, LG Life Sciences, Korea). After 36 hours, oocyte retrieval was performed by transvaginal aspiration. Inseminated oocytes were cultured using continuous single culture medium (Continuous Single Culture Complete-Irvine). On day 3, assisted hatching and medium renewing took place in the afternoon. Embryos were cultured further up to the blastocyst stage (D5/6), underwent biopsy of trophectoderm (TE) cells and cryopreservation. The morphology of the embryos was evaluated immediately before TE biopsy using the Gardner and Schoolcraft system and divided into three groups: excellent (≥3AA/3AB), good (3-6BA/BB, 1 -2 AA), and fair (3-6BC/CB/CC, 1 -2 AB/BA). The grades given to ICM were as follows: A. Tightly packed and many cells, B. Loosely grouped and several cells, and C. Very few cells. The grades given to TE were as follows: A. Many cells form a cohesive epithelium, B. Few cells form a loose epithelium, and C. Very few cells struggle to form a very loose epithelium ( 11 ). On D5 following fertilization, if the ICM and TE of a blastocyst could be clearly seen, and the TE had a sufficient number of blastomeres to be taken (at least 5 cells), the biopsy procedure was performed by an embryologist on D5. If not, the blastocyst would be cultured until day 6. A biopsy pipette was used to aspirate a sample of 6-10 TE cells, which was then laser-removed. Following the biopsy, embryos were vitrified with Cryotech medium. PGT-A workflow for blastocysts was done using Ion Reproseq™ (Thermo Fisher Scientific, USA). Amplification and library preparation were performed with the Ion SingleSeq kit (Thermo Fisher Scientific, USA) for the Ion GeneStudio S5™ System (Thermo Fisher Scientific, USA). Data generated by the Ion S5 system was subjected to Torrent Suite v14.0 (Thermo Fisher Scientific, USA). Then the available reads were analyzed using Ion Reporter software v10.5 (Thermo Fisher Scientific, USA). Embryos identified by 23 pairs of chromosomes were considered euploid. Euploid blastocysts, selected based on their morphological grade, were warmed and cultured at 37oC (6% CO 2 and 5% O 2 ) for approximately 2 hours prior to transfer. Only cycles with fully intact blastocysts after thawing were included. On the second day of a menstrual cycle, patients started the endometrial preparation with estradiol 4-6 mg/ day (Progynova, Bayer, Singapore) and the dose was increased if the endometrial thickness was insufficient. Secretory phase transformation was performed by using vaginal progesterone 600-800 mg/day (Utrogestan, Besins, Thailand or Cyclogest, Actavis U.K. Limited, United Kingdom) and oral dydrogesterone 30 mg/day (Duphaston, Abbott Biologicals B.V., Netherlands) after 14 days of estradiol usage if the endometrial thickness was at least 7 mm. After approximately 120 hours (plus or minus 3 hours) from the initiation of progesterone supplementation, the blastocyst was transferred to the patient’s uterus ( 12 ). The embryo transfer was performed under abdominal ultrasound-guided, with procedures according to the 2017 ASRM guidelines ( 13 ). If the catheter was hampered to pass through the cervical canal into the uterine cavity, resulting in prolonged time and the need for a catheter with a malleable mandrel or cervical clamp pozzi, embryo transfer was recorded as difficult. The primary outcome of this study was the live birth rate. The study also assessed several secondary outcomes, including pregnancy (measured by beta-hCG levels of at least 5 IU/L on the 12th day after embryo transfer), biochemical pregnancy (characterized by a serum beta-hCG level of greater than 5 IU/L on the 12th day after embryo transfer, followed by a subsequent negative test result and no visualization of a gestational sac by ultrasound), clinical pregnancy (determined by ultrasound visualization of a gestational sac on the 21 st day after embryo transfer), ongoing pregnancy (pregnancies continuing beyond 12 weeks). Moreover, information on birth weight and gestational age at birth was also collected The statistical analysis used frequency and percentage for categorical variables together with the mean and 95% confidence intervals for continuous variables. The statistical significance was measured by using independent sample t-test for continuous variables and Pearson’s chi-square test for categorical variables. The retrospective cohort study design was allowed to generate relative risk (RR) and 95% confidence interval using the logarithm of regression for the binominal outcomes. Demographic factors such as age, BMI, duration of infertility, history of caesarean section, and presence of endometrial fluid, first embryo transfer cycle were included in the model for adjustment. The risk ratio was estimated using the generalized linear model (GLM) in this study. The reference model was the best fit model for D5 embryos with excellent quality, and it was compared to the finest models of different embryo types. We chose D5 excellent-quality embryos as our reference model based on both our center's data and existing literature, consistently indicating the highest live birth rates. Furthermore, these embryos not only constitute a significant portion of our samples but also consistently yield stable rates of clinical pregnancies and live births. The significance level of 5% was applied to determine the statistical significance of the results. All data analysis and management were conducted using Stata version 17.0 (TX Stata Corp LLC, USA, 2021).