Blastocyst age,expansion, trophectoderm morphology,and number cryopreserved are variables predicting clinical implantation in single blastocyst frozen embryo transfers in freeze-only-IVF

PurposeTo determine which blastocyst assessment variables predict clinical implantations in single blastocyst frozen embryo transfers (FET) of freeze-only-IVF cycles, following improved vitrified-warmed blastocyst survival and developmental competence preservation.MethodIn this retrospective cohort study performed at a single private IVF center, the pregnancy outcomes of 1795 single blastocyst FET cycles were analyzed, from freeze-only-IVF retrievals performed between January 2017 and January 2020. Stepwise forward logistic regressions with clinical implantation (i.e., normal gestational sac and cardiac activity) as dependent variable were performed to identify the significant predictors. All blastocysts were vitrified using Cryotop technology, with before transfer (post-warming) blastocyst morphology scores used in all analyses.Result(s)The 1795 blastocysts transferred were vitrifıed on embryo days 4 (1057), 5 (716), and 6 (22). The overall clinical implantation rate was 50.9%; however, using blastocyst age and blastocyst morphological score the clinical implantation rates increased from 49.0% (day-4 1 and 2) and 25.2% (day-5 1 and 2) to 71.2% (day-4 4AA) and 64.3% (day-5 4AA), respectively. Whereas full (≥3) blastocysts with scores of AA and BA had similar clinical implantation rates (66.2 vs. 66.7%), the rate of full blastocysts with scores of AB was lower (58.9%). In stepwise forward logistic regressions, female age, blastocyst age, blastocyst expansion score, blastocyst trophectoderm score, and number of blastocysts vitrified were significant predictors of clinical implantation.Conclusion(s)Using blastocyst age and before transfer blastocyst expansion and trophectoderm morphology scores to select blastocysts, clinical implantation rates greater than 70% could be achieved for top-scoring blastocysts.     

Live birth rate from euploid blastocysts is not associated with infertility etiology or oocyte source following frozen-thawed embryo transfer (FET): analysis of 4148 cycles reported to SART CORS

PurposeTo investigate whether live birth rates from euploid blastocyst frozen-thawed embryo transfer (FET) cycles are associated with infertility diagnosis or oocyte source.DesignRetrospective analysis of FET cycles reported to SART CORS in 2014.MethodsData from fresh IVF cycles with preimplantation genetic testing for aneuploidy (PGT-A), linked to the first FET cycles, were collected from the 2014 SART CORS database for autologous and donor oocyte cycles. Inclusion criteria were patients undergoing FET with euploid embryos (n = 4148). Demographic data including age, BMI, prior fertility, and etiology of infertility were collected from the retrieval cycle and analyzed. Patients with uterine anomalies, preimplantation genetic testing-mutation (PGT-M) for genetic diseases, gender selection, HLA determination, or systemic and immunologic disorders were excluded. The primary outcome measure was live birth (LB) rate. Potential confounders such as age, prior fertility, and maximum baseline FSH values were analyzed with regression models as indicated.ResultsThough age, maximum baseline FSH, and infertility diagnosis were significantly different, LB was similar between patients undergoing autologous or donor oocyte FET cycles. Etiology of infertility was not significantly associated with LB in autologous cycles (p = 0.95). Potential confounders such as maternal age, prior fertility, and maximum baseline FSH were not associated with outcomes; however, maternal BMI was inversely related to LB in autologous cycles, with an odds ratio of 0.97 (95% CI: 0.96–0.98 (rho = − 0.08, p < 0.01)).ConclusionsAfter controlling for confounding variables, a euploid embryo derived from a donor or autologous oocyte results in similar LB in women with different infertility diagnoses.     

Day 5 expanded blastocysts transferred on same day have comparable outcome to those left for more extended culture and transferred on day 6

Objective

To study the outcome of blastocysts showing expansion on day 5 and transferred on day 5 or 6, in comparison with those unexpanded and transferred on day 6.

Study Design

Prospective cohort of 221 women prepared for BET classified into three groups according to timing of blastocyst expansion and day of embryo transfer. Group I; with expanded blastocysts on day 5 having day 5 transfer, group II; with expanded blastocysts on day 5 having day 6 transfer and group III ; with delayed expansion undergoing day 6 BET.

Results

Implantation rates, pregnancy rates, ongoing pregnancy rates, and live birth rates in the first 2 groups were almost double the rates in the third group. The figures for implantation rates were 40 % in the first two groups vs. 19 % in the third group (P < 0.05). Pregnancy rates were 60.9 % and 64 % vs. 31.8 % (P < 0.05) and ongoing pregnancy/ live-birth rates were 52.3 % & 56 % vs. 27.3 %.

Conclusion

The current study reports better implantation and pregnancy rates with earlier expanding blastocysts regardless of the time of transfer.     

The clinical outcomes of day 3 4-cell embryos after extended in vitro culture

ObjectiveTo evaluate the development potential and clinical significance of day 3 4-cell embryos after extended in vitro culture.MethodsThis study was a retrospective cohort study for patients with infertility treatment between January 2011 and July 2013. Patients undergoing blastocyst culture in controlled ovarian hyperstimulation cycles using surplus embryos were analyzed in the study. A total of 764 women undergoing blastocyst culture with 1,522 surplus 4-cell embryos on day 3 were analyzed. An additional 2,391 patients with embryos undergoing blastocyst culture during the same period with embryos having more blastomeres were chosen as control.ResultsAfter extended culture, 253 embryos from 183 cycles in the study group which developed to blastocysts were frozen, and 118 embryos were warmed in subsequent frozen embryo transfer cycles. Implantation rates, clinical pregnancy rates (PRs) and ongoing PRs were 33.3 %, 38.4 % and 31.4 %, respectively, which were similar to those of the control group. The singleton birth weights of newborns using these blastocysts showed no significant difference to that seen in the control group.ConclusionSurplus 4-cell embryos on day 3 displayed lower blastulation rates. However, once a blastocyst is obtained, it has equivalent clinical outcomes. Embryos that are developmentally lagging on day 3 can be observed in extended culture to increase the cumulative chances of a successful pregnancy.     

Human chorionic gonadotropin levels are equally predictive for pregnancy outcome after fresh and vitrified-warmed blastocyst transfer

Purpose

The aim of the present study was to evaluate if the live birth predictive values of β-hCG levels differ in fresh and vitrified-warmed blastocyst transfer cycles.

Methods

In the retrospectively designed study, 775 cycles with positive β-hCG values 13 days after fresh blastocyst transfer (fresh ET; n = 568) or vitrified-warmed blastocyst transfer (FET; n = 207) were selected for analysis. Average β-hCG levels stratified according to pregnancy outcome (biochemical pregnancy, spontaneous abortion, ectopic pregnancy, and singleton or twin birth) were compared between fresh ET and FET cycles. To determine the optimal sensitivity and specificity of β-hCG levels for live birth prediction, a ROC curve was constructed. Fisher’s exact test was used to compare the positive predictive values (PPV).

Results

Average β-hCG levels stratified according to pregnancy outcome were not statistically different between fresh ET and FET cycles. In fresh ET and in FET group, the β-hCG levels were significantly higher in pregnancies resulting in live birth compared to non-viable pregnancies (1,035 vs. 462 IU/L, p < 0.001 and 968 vs. 411 IU/L, p < 0.001). Optimal cut-off level for live birth prediction was 495 IU/L (sensitivity 83.0 %, specificity 71.8 %) after ET and 527 IU/L (sensitivity 80.0 % and specificity 76.6 %) after FET. The PPV for live birth rate in the groups after ET and FET were 90.6 % and 84.9 % respectively, without statistically significant difference (p > 0.05).

Conclusion

Beta-hCG levels after fresh and vitrified-warmed blastocyst transfer are equally predictive for pregnancy outcome. Clinicians can be encouraged to interpret β-hCG results in the same manner.     

Early blastulation of day 4 embryo correlates with the increased euploid rate of preimplantation genetic screening cycles

Objective

It is known that embryos with faster growing potential, especially in blastocyst development, correlate with the increased euploid rate. Our study investigated the preimplantation genetic screening cycle to analyze the correlation between early blastulation (EB) on day 4 embryo and the euploid rate.

囊胚内细胞团和滋养外胚层基因表达研究

目的研究人胚胎植入前囊胚内细胞团和滋养外胚层细胞基因的表达。方法收集辅助生殖来源人受精后6 d Gardner评分5AA囊胚,显微镜下微吸管机械分离内细胞团和滋养外胚层细胞,单细胞测序检测,选取筛选的差异表达基因并进行无监督层次聚类分析和Gene Ontology(GO)功能分类分析。结果对3枚受精后6 d的5AA囊胚内细胞团14个单细胞和滋养外胚层19个单细胞测序,结果显示内细胞团1 283个基因表达上调,其G O生物学功能主要是参与DNA依赖的转录、生物种间相互作用、抗原加工和表达、免疫反应、信号转导、氧化还原、细胞分化、抗凋亡、神经系统发育、细胞黏附等,参与丝裂原活化蛋白激酶(MAPKs)信号通路、肌动蛋白细胞骨架、黏附、轴引导、Jak-STAT、Wnt信号通路。滋养外胚层1 073个基因表达上调,其GO生物学功能主要是参与转录、DNA依赖的转录、细胞周期、蛋白分解代谢、蛋白氨基酸磷酸化、蛋白质运输、细胞分裂、有丝分裂、泛素依赖性蛋白分解、细胞内蛋白质转运等;参与泛素介导蛋白水解、鞘脂代谢、缬氨酸,亮氨酸和异亮氨酸降解、果糖和甘露糖代谢、氨基磷酸酯代谢、类固醇的生物合成、抗原处理和表达等信号通路。结论从空间维度揭示囊胚内细胞团和滋养外胚层的基因表达,显示两者相互协调,精细调节囊胚发育和胚胎植入过程,进一步数据分析有望寻找到调控胚胎植入的内源性关键特异分子。     

Ghrelin影响小鼠胚泡着床的体外实验研究

目的:探讨Ghrelin在小鼠妊娠围植入期子宫表达的变化及其对胚胎着床的影响。方法:用免疫组化方法检测Ghrelin蛋白在小鼠妊娠围植入期子宫表达的变化;采用体外胚泡培养方法,观察重组Ghrelin蛋白对胚泡的孵化、黏附与扩展率的影响。结果:在小鼠妊娠的第1至2天,Ghrelin蛋白主要于腔上皮和腺上皮表达,随妊娠进展,其表达水平逐渐下降。体外胚泡培养48h时,与正常对照组相比[孵化率、黏附率和扩展率分别为(61.25±5.27)%、(71.45±6.56)%和(43.26±5.87)%],Ghrelin重组蛋白可明显抑制胚泡的孵化率[(20.36±4.78%),P<0.01]、黏附率[(53.23±4.98)%,P<0.01)]及扩展率[(18.34±7.12)%,P<0.05]。结论:Ghrelin可抑制胚泡孵化、黏附与扩展及抑制胚泡植入。     

The influences of weather on patients with different ovarian responses in the treatment of assisted reproductive technology

Purpose: The purpose of this study was to evaluate the influences of tropical weather on patients with different ovarian responses in the treatment of assisted reproductive technology.Methods: Six-hundred fourty-seven women underwent their first treatment cycles were retrospectively analyzed. Patients received embryo transfer either 3 days or 5 days after oocyte retrieval, depending on the number and quality of embryos on day-2.Results: Significant correlations were demonstrated in the top quality embryo rates of day-3 and day-5 embryo transfers with temperature, humidity, and atmosphere pressure. The cumulative light hours negatively correlated with the implantation and pregnancy rates of day-3 embryo transfer (−.282 and −.282, respectively), while they positively correlated with those of day-5 embryo transfer (.225 and .224, respectively).Conclusions: These results clearly suggest that weather may exert influences on the outcome of assisted reproductive technology. Patients with different ovarian responses or blastocyst culture and transfer may modify weather influences.     

Effect of the size of zona pellucida opening by laser assisted hatching on clinical outcome of frozen cleaved embryos that were cultured to blastocyst after thawing in women with multiple implantation failures of embryo transfer: a retrospective study

Purpose To evaluate the effect of the size of zona pellucida opening by laser assisted hatching for frozen cleaved embryo that were thawed after both fresh and frozen cleaved embryo transfer cycles failed and were cultured to blastocyst after thawing in patients with multiple implantation failures. Materials and methods Of 101 consecutive procedures (October 2003 to June 2006), 30 patients declined to perform assisted hatching and were selected as control group, 40 patients had 40 μm opening of the zona (October 2003 to January 2005), 31 patients had 50% of the zona opening (February 2005 to June 2006). Results The pregnancy, implantation and delivery rates were significantly higher in 50% opening group (74%, 52% and 65%) compared to control group (17%, 10% and 13%; P < 0.01) and 40 μm opening group (43%, 27% and 38%; P < 0.04). Conclusions The size of the zona pellucida opening may affect the outcome of frozen cleaved embryo transfer. Capsule The size of the zona pellucida opening by laser assisted hatching may affect the outcome of frozen cleaved embryo transfer in multiple implantation failures.     

形态正常发育滞后胚胎冻融后的临床妊娠结局

目的:探讨形态正常但发育滞后胚胎的临床妊娠结局。方法:2006年10月至2007年10月在山东大学附属省立医院生殖中心接受体外授精-胚胎移植(IVF-ET)或卵胞浆内单精子显微注射(ICSI)的患者于第3天将形态正常(卵裂球均匀或轻度不均匀,无核碎片≤25%)但发育缓慢的胚胎(受精后64~66h胚胎卵裂球数3~5个)慢速冷冻,复苏后行胚胎移植,移植时按照第2天发育阶段的胚胎对待,随访其妊娠情况。结果:胚胎移植104例患者,冷冻复苏胚胎348个,复苏成活303个(成活率87.17%),继续培养后121个胚胎(39.93%)继续发育。移植后获得生化妊娠35例,生化妊娠率33.65%,临床妊娠30例,临床妊娠率28.84%。过夜培养后至少有一个胚胎继续发育61例,临床妊娠率37.70%,过夜培养无胚胎继续发育43例,临床妊娠率16.27%,两组差异有统计学意义(P<0.05)。结论:形态正常但发育滞后的胚胎有冷冻保存价值,可以在冷冻胚胎融解周期作为第2天胚胎融解,过夜培养后优先选择有发育生长的胚胎移植。     

The importance of the cleavage stage morphology evaluation for blastocyst transfer in patients with good prognosis

Purpose

To evaluate: (i) the influence of morphology at cleavage stage on blastocyst formation and implantation, and (ii) whether the transfer of low-quality embryos on day-three would be a better approach than the transfer at blastocyst stage.

Methods

This study included 8,444 embryos obtained from 1,125 patients undergoing ICSI cycles between January/2011 and September/2013. The influence of the quality of the embryo on days-two and -three on blastocyst formation and implantation success was evaluated. Moreover, the implantation potential of low-quality embryos, at cleavage stage, transferred on day-three was compared with the implantation potential of low-quality embryos, at cleavage stage, transferred on day-five.

Results

Low-quality embryos on day-two had an approximate 20 % decreased chance of achieving the blastocyst stage, and blastocysts derived from low-quality embryos on day-two had a nearly 40 % decrease in the implantation chance. Low-quality embryos on day-three had a 30 % decreased chance of achieving the blastocyst stage, and blastocysts derived from low-quality embryos on day-three had an almost 40 % decreased implantation chance. The implantation rate didn’t differ when low-quality embryos on the cleavage stage were transferred on day-three or left in culture and transferred on day-five.

Conclusions

The transfer of low-quality embryos on day-three is a better approach than transfer at the blastocyst stage. In addition, the embryo morphology evaluation at the cleavage stage is still needed for the selection of the embryo with the best implantation potential in extended embryo culture programmes.     

Quantitative and qualitative trophectoderm grading allows for prediction of live birth and gender

Purpose

Prolonged in vitro culture is thought to affect pre- and postnatal development of the embryo. This prospective study was set up to determine whether quality/size of inner cell mass (ICM) (from which the fetus ultimately develops) and trophectoderm (TE) (from which the placenta ultimately develops) is reflected in birth and placental weight, healthy live-birth rate, and gender after fresh and frozen single blastocyst transfer.

Methods

In 225 patients, qualitative scoring of blastocysts was done according to the criteria expansion, ICM, and TE appearance. In parallel, all three parameters were quantified semi-automatically.

Results

TE quality and cell number were the only parameters that predicted treatment outcome. In detail, pregnancies that continued on to a live birth could be distinguished from those pregnancies that aborted on the basis of TE grade and cell number. Male blastocysts had a 2.53 higher chance of showing TE of quality A compared to female ones. There was no correlation between the appearance of both cell lineages and birth or placental weight, respectively.

Conclusions

The presented correlation of TE with outcome indicates that TE scoring could replace ICM scoring in terms of priority. This would automatically require a rethinking process in terms of blastocyst selection and cryopreservation strategy.

     

Twin pregnancies and perinatal outcomes: a comparison between fresh and frozen embryo transfer: a two-centre study

Research questionWhat is the optimal timing for transfer in natural cycle vitrified–warmed embryo transfers (NC-VET)?DesignThis retrospective cohort study uses data from a large university-affiliated IVF clinic. The study included 341 NC-VET cycles with autologous oocytes and non-preimplantation genetic testing, vitrified embryos from January 2013 to September 2017. Each cycle was classified by timing of embryo transfer in relation to LH surge ≥20 IU/l. Group 1: LH ≥20 IU/l one day and blastocyst was transferred 6 days later; Group 2: LH ≥20 IU/l two consecutive days and blastocyst was transferred 6 days after the initial surge; Group 3: LH ≥20 IU/l two consecutive days and blastocyst was transferred 7 days after the initial surge. The primary outcome was ongoing pregnancy rate (OPR). The secondary objective was to compare OPR in relation to serum oestradiol dynamics and progesterone concentration (according to threshold 1.0 ng/ml) 6 days prior to embryo transfer.ResultsOPR were similar for all three groups (66.8%, 65.0%, 62.9% for Groups 1, 2 and 3, respectively). When stratified according to oestradiol and progesterone, no significant differences were noted in OPR.ConclusionsThe results suggest that the timing of blastocyst transfer in a natural cycle after LH surge is flexible within 24 h. Outcomes are equally good with day of embryo transfer 6 or 7 days after LH surge date. Oestradiol dynamics and progesterone concentration 6 days prior to NC-VET did not have a significant impact on OPR.     

A quantitative approach to blastocyst quality evaluation: morphometric analysis and related IVF outcomes

Purpose

To quantify blastocyst morphologic parameters with a feasible and standardized tool, investigating their predictive value on implantation outcome.

Method

The study retrospectively analyzes 124 blastocysts from 75 patients. Quantitative measurements of blastocyst expansion, inner cell mass and trophoectoderm were taken using digital image analysis software.

Result(s)

Blastocysts areas were found to be ranging from 11626.2 up to 35076.4 μm². The area of an early blastocyst is A ≤ 18500 μm² with a mean diameter d = 140 ± 9 μm, and the area of an expanded blastocyst is A ≥ 24000 with d = 190 ± 9 μm. While blastocyst mean area was not related to implantation rate, more expanded blastocysts displayed a significantly higher implantation rate. Trophoectoderm cell number is a predictor of positive outcome: since a higher of cells (25.6 ± 11.3 vs 16.3 ± 12.8) `forming a tightly knit epithelium is prognostic of implantation potential. Conversely, inner cell mass size is significantly related to implantation only in expanded blastocysts (3122.7 ± 739.0 vs. 2978.1 ± 366.0 μm²).

Conclusion(s)

Evaluation of blastocyst morphology with a digital image system could be a valuable tool to standardize blastocyst grading based on quantitative parameters. Therefore, digital analysis may be helpful in identifying the best blastocyst to transfer.