Smooth Endoplasmic Reticulum Clusters in Oocytes From Patients Who Received Intracytoplasmic Sperm Injections Negatively Affect Blastocyst Quality and Speed of Blastocyst Development

Findings regarding the relationship between smooth endoplasmic reticulum clusters (SERCs) in oocytes and blastocyst development have been conflicting. In this study, the effects of SERCs on blastocyst quality and the speed of blastocyst development were evaluated. Patients who received intracytoplasmic sperm injections (ICSI) at our reproductive center from 2016 to 2020 were retrospectively analyzed. SERC (+) oocytes (n = 217) and SERC (–) oocytes (n = 822), as well as SERC (+) cycles (n = 146) and SERC (–) cycles (n = 1,951) were compared. There was no significant difference in embryological, clinical, and neonatal outcomes between the SERC (+) and SERC (–) cycles. The fertilization rate (73.9%), good quality blastocyst rate (26.7%) and the speed of blastocyst development (44.4%) were significantly lower (P < 0.05) in SERC (+) oocytes than in unaffected counterparts (86.2%, 44.1% and 63.4%, respectively). Furthermore, the proportion of blastocysts with trophectoderm (TE) grade C was significantly higher in the SERC (+) oocyte group than in the SERC (–) oocyte group (73.3 vs. 55.9%, P < 0.05). After adjusting for age, years of infertility, endometriosis, stimulation protocols (GnRHa), and male infertility, multiple logistic regression analysis revealed that the presence of SERCs in the oocytes significantly affected the speed of blastocyst development (odds ratio, 2.812; 95% CI, 1.257–6.292; P = 0.012). These findings suggest that the presence of SERCs in oocytes may negatively affect blastocyst quality and the speed of blastocyst development.

Impact of oxygen tension according to embryo stage of development: a prospective randomized study

Human embryo culture under 2–8% O2 is recommended by ESHRE revised guidelines for good practices in IVF labs. Nevertheless, notably due to the higher costs of embryo culture under hypoxia, some laboratories perform embryo culture under atmospheric O2 tension (around 20%). Furthermore, recent meta-analyses concluded with low evidence to a superiority of hypoxia on IVF/ICSI outcomes. Interestingly, a study on mice embryos suggested that oxidative stress (OS) might only have an adverse impact on embryos at cleavage stage. Hence, we aimed to demonstrate for the first time in human embryos that OS has a negative impact only at cleavage stage and that sequential culture conditions (5% O2 from Day 0 to Day 2/3, then «conventional» conditions at 20% O2 until blastocyst stage) might be a valuable option for human embryo culture. 773 IVF/ICSI cycles were included in this randomized clinical trial from January 2016 to April 2018. At Day 0 (D0), patients were randomized using a 1:2 allocation ratio between group A (20% O2; n = 265) and group B (5% O2; n = 508). Extended culture (EC) was performed when ≥ 5 Day 2-good-quality-embryos were available (n = 88 in group A (20% O2)). In subgroup B, 195 EC cycles were randomized again at Day 2 (using 1:1 ratio) into groups B’ (5% O2 until Day 6 (n = 101)) or C (switch to 20% O2 from Day 2 to Day 6 (n = 94). Fertilization rate, cleavage-stage quality Day 2-top-quality-embryo (D2-TQE), blastocyst quality (Day 5-top-quality-blastocyst (D5-TQB) and implantation rate (IR) were compared between groups A and B (= cleavage-stage analysis), or A(20% O2), B’(5% O2) and C(5%-to-20% O2). Overall, characteristics were similar between groups A and B. Significantly higher rates of early-cleaved embryos, top-quality and good-quality embryos on Day 2 were obtained in group B compared to group A (P < 0.05). This association between oxygen tension and embryo quality at D2 was confirmed using an adjusted model (P < 0.05). Regarding blastocyst quality, culture under 20% O2 from Day 0 to Day 6 (group A) resulted in significantly lower Day 5-TQB number and rates (P < 0.05) compared to both groups B’ and C. Furthermore, blastocyst quality was statistically equivalent between groups B’ and C (P = 0.45). At Day 6, TQB numbers and rates were also significantly higher in groups B’ and C compared to group A (P < 0.05). These results were confirmed analyzing adjusted mean differences for number of Day 5 and Day 6 top quality embryos obtained in group A when compared to those respectively in groups B’ and C (P < 0.05). No difference in clinical outcomes following blastocyst transfers was observed. These results would encourage to systematically culture embryos under hypoxia at least during early development stages, since OS might be detrimental exclusively before embryonic genome activation.

Transfer of hatched blastocyst can result in skewed sex ratio: a retrospective cohort study

Background The correlation between blastocyst quality and birthweight, neonatal outcomes is still controversial. There is a significantly higher male: female ratio among good quality blastocysts (advanced trophoderm morphology) but in the expansion degree, the significance for sex ratio is unclear. Methods A total of 617 and 6803 live singleton births resulting from the transfer of fresh and frozen-thawed single blastocysts in the Reproductive Medicine Center of Peking University Third Hospital from 2009 to 2020 were included. Live singleton births from fresh and frozen-thawed single blastocyst transfer were stratified by inner cell mass/trophoderm morphology and degree of blastocoel expansion. Multivariate linear regression was used to analyze the correlation between expansion, inner cell mass/trophoderm morphology, and birthweight, Z score, gestational weeks. Logistic regression was used to analyze the relationship between expansion, ICM/TE morphology and sex, neonatal outcomes. Results There was no significant correlation between birthweight, neonatal outcomes and blastocyst quality in fresh and frozen-thawed single blastocyst transfer cycles. However, the proportion of male infants in the hatched blastocyst (stage-6) group (67.9% vs. 54.2%; p < 0.001) [OR: 1.76 95% CI (1.34–2.32)] and hatching blastocyst (stage-5) group (61.7% vs. 54.2%; p = 0.001) [OR: 1.36 95 C.I (1.14ཞ1.62)] was significantly higher than that in the expanded blastocyst (stage-4) group. Conclusions The transfer of poor-quality blastocysts is unlikely to affect birthweight and neonatal health; however, transfer of stage-6 blastocysts can result in extremely skewed sex ratio.

Effect of nucleocytoplasmic ratio on the in vitro porcine embryo development after in vitro fertilization or parthenogenetic activation

Summary This study was conducted to examine whether the nuclear to cytoplasmic (N/C) ratio had any influence on the timing of embryo compaction and blastocoel formation, as well as formation rate and quality of blastocyst. First, we produced embryos with increased N/C ratio by removal of approximately one-third of the cytoplasm and with decreased N/C ratio by doubling the oocyte cytoplasm with an enucleated oocyte. The initiation of compaction and cavitation in reduced cytoplasm group was significantly earlier (P < 0.05) compared with the control and doubled cytoplasm groups. The rate of blastocysts in the reduced cytoplasm and doubled cytoplasm groups was significantly lower (P < 0.05) compared with the control group. Blastocyst quality in terms of total cell number in the reduced cytoplasm group was significantly lower (P < 0.05) compared with the doubled cytoplasm group, but not different from the control group. Next, we produced embryos with various N/C ratios by oocyte fusion combined with cytochalasin D treatment. The onset of compaction and cavitation in the 2N/2C group (decreased N/C ratio) was significantly delayed (P < 0.05) or had the tendency to be delayed (P = 0.064), respectively, compared with the control group (2N/1C). A significantly higher rate of blastocyst was observed in the 4N/2C group compared with the 1N/1C group (P < 0.05) but not different from the remaining groups. These results demonstrated that an increase in N/C ratio caused an earlier occurrence of morula compaction and blastocyst formation in both in vitro fertilization (IVF) and parthenogenetically activated pig embryos.

Effect of Blastocyst Quality on Human Sex Ratio at Birth in Single Blastocyst Frozen Thawed Embryo Transfer Cycle

ObjectiveThis study aimed to determine whether blastocyst quality affect on sex ratio at birth through in single blastocyst frozen thawed embryo transfer cycle.Materials and MethodsWe ran this retrospective study on 4205 singleton infants born following single blastocyst frozen thawed embryo transfer from January 2016 to October 2020 at a single institution. We compared the sex ratios of these infants with respect to blastocyst quality, embryo growth rate and morphology.ResultsThe main outcomes of this study were that the sex ratio at birth of single blastocyst frozen thawed embryo transfer babies was 56.67% which was higher than the normal level (51.22%). The sex ratio of mothers older than 40 years was significantly lower than that of mothers younger than 40 years (0.39vs1.3, P<0.05). Transplanting good quality blastocysts significantly increased the proportion of boys (1.35vs0.94, P<0.05). Transplanting grade 5 and 6 blastocysts significantly increased the proportion of male babies born compared with grade 3 and 4 blastocysts (1.91vs1.28, P<0.05). There were no significant differences in the sex ratio with respect to the inner cell mass (ICM) score. In addition, the higher the trophectoderm (TE) score, the higher the sex ratio (2.79vs1.18vs0.91, P<0.05).ConclusionsOur study indicated that blastocyst quality, especially trophoblastic cell score, had a significant effect on sex ratio.

Does Blastocyst Morphology Affect Live Birth Rate After Transfer of Single Euploid Embryo?

Background The aim of this study was to investigate whether the morphologic parameters of blastocyst influence the live birth rate (LBR) of euploid embryos transferred in subsequent single frozen-thawed embryo transfer (FET) cycles? Methods Women who received first preimplantation genetic testing for aneuploidy (PGT-A) and following underwent frozen-thawed single euploid blastocyst transfer cycles from June 2017 to May 2020 were divided into three age groups (< 30, 30–34 and ≥ 35 years). The primary outcome measure was LBR. Outcomes were compared between different blastocyst quality, inner cell mass (ICM) grade, trophectoderm (TE) grade and day of TE biopsy within the same age group. Results In the youngest group (< 30 years, n = 100), LBR were compared between cycles with various blastocyst quality (66.67% for good quality, 65.52% for average quality and 36.36% for poor quality; P = 0.013), ICM grade (61.11% for grade A and 51.22% for grade B; P = 0.466), TE grade (68.75% for grade A,65.00% for grade B and 36.30% for grade C; P = 0.012) and day of TE biopsy (65.38% for Day 5 and 39.58% for Day 6; P = 0.010). Similarly, in the 30–34 years group(n = 121) and the oldest group (≥ 35years, n = 58), LBR were also comparable between these subgroups, but no significant differences were seen between blastocyst morphologic grading and LBR (P > 0.05). Moreover, good quality (adjusted odds ratio [aOR] 3.30; 95% confidence interval [CI], 1.09 ~ 9.99; P = 0.035) and average quality (aOR 3.71; 95%CI, 1.25 ~ 11.01; P = 0.018) embryos were still yielded a significantly higher LBR than poor-quality embryos, TE grade B embryos were also associated with a statistically significantly higher LBR compared with TE grade C embryos (aOR 3.69;95%CI, 1.37 ~ 9.95; P = 0.010) after adjusting for the potential confounding factors. Conclusion Blastocyst quality and trophectoderm grading is a useful predictor of LBR in single frozen-thawed euploid embryo transfer cycles among women < 30 years old. However, these differences were not found in women older than 30 years.

P–304 The endometrial preparation protocol does not affect the live-birth-rate after vitrified-warmed euploid single blastocyst transfers: an analysis of 1884 procedures

Study question Is the live-birth-rate (LBR) different when comparing artificial (AC) and modified-natural (M-NC) cycle for endometrial preparation to vitrified-warmed euploid blastocyst transfer? Summary answer The LBR after vitrified-warmed euploid blastocyst transfer seem independent of the endometrial preparation administered. What is known already Only the transfer of a competent embryo on a receptive endometrium might result in successful implantation. Three main protocols for endometrial preparation to vitrified-warmed embryo transfer exist: NC, M-NC, and AC. None among them, though, has been shown more appropriate than the others to date, especially since, only in a few studies, the analysis was restricted to single euploid blastocyst transfers to limit the impact of embryonic issues on implantation. In conclusion, no clear consensus exists and the choice is still largely based on menstrual/ovarian cycle characteristics and patient’s needs. Study design, size, duration All first vitrified-warmed single euploid blastocyst transfers performed between April–2013 and March–2020 were included in the analysis. Endometrial preparation was conducted with either an AC (N = 1211) or a M-NC (N = 673). The protocol was chosen based on patients’ logistical reasons. The primary outcome was the LBR per transfer. Sub-analyses based on blastocyst quality and day of development were conducted. Birthweight, gestational age, gestational and perinatal issues were secondary outcomes. Participants/materials, setting, methods AC: oral estradiol-valerate 3-times/day from day2–3 of the cycle until the endometrial thickness reached ≥7mm, then 600 mg/day of micronized progesterone. The transfer was conducted on day6 of progesterone administration. M-NC: an intramuscular dose of 10,000IU hCG was administrated when the leading follicle was &gt;17 mm and the endometrium was thicker than 7mm and trilaminar, plus 400 mg/day of micronized-progesterone as luteal phase support starting 36–40hr post-hCG. The transfer was conducted on day7 after trigger. Main results and the role of chance The two groups were similar for maternal age at retrieval (38.0±3.3yr) and transfer (38.3±3.3yr), reproductive history, embryological outcomes of the IVF cycle, body-mass-index, basal hormonal levels, and blastocyst features (Gardner’s classification: AA = 73%, AB/BA=11%, BB/AC/CA=8%, CC/BC/CB=8%; day5=48%, day6=47%, day7=5%). The LBR was 46.7% (N = 565/1211) and 49.9% (N = 336/673) after AC and M-NC, respectively, resulting in an odds-ratio 1.14, 95%CI:0.94–1.37. The absence of significant differences was confirmed also when adjusted for blastocyst quality and day of full-development (1.16, 95%CI:0.96–1.41). Among the 565 and 336 deliveries, the birthweight was similar (3290.3±470.7 versus 3251.7±521.5 g, Mann-Whitney-U-test=0.5), the gestational age was similar (38.5±1.7 versus 38.4±1.9 weeks, Mann-Whitney-U-test=0.5). Also, the rates of newborns who were normal (81% versus 82%), large (8% versus 9%), and small (11% versus 9%) for gestational age were similar (Chi-squared-test=0.5). The rates of patients experiencing gestational (6% versus 7%) and/or perinatal issues (3% versus 3%) were also similar (Fisher’s-exact-tests=0.4). Limitations, reasons for caution This is a retrospective study conducted in poor prognosis patients indicated to preimplantation genetic testing for aneuploidies. Future randomized controlled trials and cost-effectiveness analysis are desirable, as well as studies in different patient populations. Lastly, each gestational/perinatal issue shall be analyzed per se (e.g. different placentation disorders). Wider implications of the findings: The absence of clinical and perinatal differences between the two protocols for endometrial preparation supports the adoption, whenever needed, of AC. This approach, in fact, allows a higher flexibility in patients’ and daily workload management. Trial registration number None

P–237 The good, the fast and the slow: which is better? Live birth rate following single blastocyst frozen embryo transfer

Study question Are embryo quality and day of vitrification (Day 5, 6 or 7) associated with live birth rates (LBR) following single blastocyst transfer (SBT) in frozen embryo transfer cycle (FET)? Summary answer Both blastocyst quality and day of vitrification are significantly associated with LBRs, with very low LBR when poor quality embryos are frozen on day 6. What is known already Evidence suggests that chromosomal status (ploidy) is strongly associated with blastocyst morphology and good quality embryos are more likely to be euploid. Furthermore, previous studies have shown a relationship between the time that embryos need to reach blastocyst stage and their euploidy rate with slowly developing blastocysts showing higher rate of aneuploidy. Nonetheless, despite all this evidence little is known about the actual effect of the combination of blastocyst quality and day of its vitrification. The scope of this study was to quantify the actual effect of the embryo quality and day of vitrification on live birth rates following FET. Study design, size, duration Retrospective analysis of 1546 FET cycles with SBT conducted between 2017 and 2019 in the university-affiliated private clinic. The embryos used for FET were obtained from IVF/ICSI: with PGT (FET-PGT) or without PGT (FET0) or from donated oocytes (FET-DON). Participants/materials, setting, methods FET with natural, natural-modified and completely medicated cycles to prepare endometrial lining were included. Blastocysts were classified according to Spanish Association for the Study of Reproductive Biology (ASEBIR) classification, ranging from A (the highest) to D (the lowest). The impact on LBR of different subgroups, formed within FET-PGT, FET0, FET-DON groups due to different day of vitrification and blastocyst quality, was assessed, using logistic regression after adjusting for age, day of vitrification and embryo quality. Main results and the role of chance We included 1546 FET cycles. Of those, 543 (35%) corresponded to FET-PGT; 648 (42%) to FET0 and 355 (23%) to FET-DON cycles. Overall, 1051 (68%) embryos were frozen on day 5(D5), 472 (30.5%) on day 6(D6) and 23 (1.5%) on day 7(D7). As far as embryo quality was concerned, 215 (13.9%) grade A; 957 (61.9%) B; 371(24%) C and 3(0.2%) D blastocysts were transferred. LBRs were significantly different between different embryos frozen on D5 44.3%; on D6 28.8% and on D7 8.7%, p &lt; 0.001. When blastocyst quality was considered, LBR were 48.4% for grade A; 42.5% for B; 25.1% for C and 0% for D, p &lt; 0.001. After applying logistic regression analysis, the odds ratio (OR) for transferring D6-blastocyst was 1.08, 95% CI[0.45; 2.62] and blastocyst with grade B and C; 0.71, 95% CI[0.51; 1.00]; 0.57,95% CI[0.36; 0.88] respectively. However, after transferring D6-blastocyst graded as C, the OR was 0.33, 95% CI[0.12; 0.90]. Our predictive model showed that the impact of the embryo quality on LBR was sustained across three groups. Transfer of D5/D6 grade A blastocyst resulted in the highest, while D6-C in the lowest LBR in all the groups. In the latter case vitrification on D6 impaired additionally the outcome. Limitations, reasons for caution The study should be interpreted with caution due to its retrospective character and the assessment of blastocyst quality on the day of vitrification and not on the day its transfer. Wider implications of the findings: Our robust findings could be considered a useful tool for counselling couples who seek advice regarding their expected success rates in the setting of FET with SBT. The very low livebirth rates in low quality (C) slow developing (D6) embryos should be communicated to patients prior to planning a FET. Trial registration number Not applicable

P–132 Centralized versus local embryologists scoring of blastocyst quality obtained in a large European multicenter clinical trial

Study question Does blastocyst quality scoring by central assessment deviate from local assessment and potentially lead to the selection of a different single blastocyst for transfer? Summary answer Central and local assessment provided the same quality classification (poor / good / top) in 69% of all blastocysts and 63% of all transferred blastocysts. What is known already Blastocyst quality is scored most frequently by three morphological parameters, namely expansion and hatching (EH) status, inner cell mass (ICM) grading and trophectoderm (TE) grading. The score is used to define the quality classification (poor / good / top) which determines which embryo is to be transferred or cryopreserved. Blastocyst scoring and grading can be highly subjective, which does influence the choice for transfer and cryopreservation. Time-lapse imaging technology captures additional input about embryo development as well as enables centralized data storage and sharing for independent central assessments. Study design, size, duration Pooled embryo analysis from a prospective, randomized, multicenter trial (RAINBOW) of 619 women undergoing ovarian stimulation with an individualized dose of follitropin delta in a long GnRH agonist protocol between May 2018 and January 2020. Blastocysts were centrally assessed using time-lapse images by two independent assessors and one adjudicator . Selection of the blastocyst for transfer by local assessment was based on morphological scoring and not on morphokinetic time-lapse parameters. Participants/materials, setting, methods Oocytes were fertilized by ICSI and cultured in the Embryoscopeâ (Vitrolife) up to day 5 for transfer or day 5/6 for cryopreservation. Embryos were assessed as either non-blastocyst or blastocyst. Blastocysts were graded centrally and locally at 116 hrs of development, based on EH status (1–6), ICM (A-D) and TE grading (A-D). Central assessors were blinded to local assessment and embryo transfer selection. Main results and the role of chance In total 4282 embryos were assessed centrally, of which 2046 day 5 embryos (48%) were adjudicated due to a scoring difference of at least one parameter between the two central assessors. In total 38% of day 5 embryos were judged as non-blastocysts and 62% as blastocysts of which 61% (i.e. 38% of all embryos) were determined to be of good or top quality. Identical results in terms of quality classification (poor / good / top) were obtained for 69% of blastocysts between local and central assessment and in 78%, between the two central assessors. Moreover, central and local scoring were identical in 62% for EH status, 53% for ICM grading and 57% for TE grading. For all transferred blastocysts (n = 508), central and local quality assessment was aligned for 63%. The ongoing pregnancy rate following single blastocyst transfer (SBT) was 41% (202/489), and similar to when considering only the transfers for which the central assessment had the same or a higher classification than the local assessment (166/411=40%). In 16% of all SBT, central quality assessment gave a lower score for the transferred blastocyst than the central assessment. This discrepancy could potentially have led to transfer of a different blastocyst. Limitations, reasons for caution This trial included assessments made by embryologists from 20 IVF centres. Some centres has limited experience with time-lapse technology for morphological blastocyst scoring. Scoring could therefore have been affected by differences in focal planes, magnification and contrast compared to inverted microscopy, with potential influence on blastocyst scores and quality classification. Wider implications of the findings: Local and central blastocyst quality classification based on morphology aligns well but remains subjective. Embryo assessment may benefit from using tools like artificial intelligence-based algorithms for a more objective analysis. Trial registration number NCT03564509