Advancing Age May Decrease Mitochondrial Activity in Cumulus Cells.

Background: The goal of this study was to compare mitochondrial activity in cumulus cells (CCs) between young and advancing-aged women, the factors that affect mitochondrial activity, and their association with blastocyst quality. Materials and methods: This prospective study included 80 infertile women who underwent ICSI between May and October 2023. Participants were divided into two groups: older and younger than 38. The oocyte mitochondrial activity from CCs was evaluated using MitoTracker, and the mean fluorescence intensity (MFI) was also evaluated. Results: The univariate and multivariate analyses revealed a significant difference in the MFI between the woman ≥ 38 age group and the lower age group (162.68 ± 79.87 vs. 228.39 ± 121.38; p-value = 0.005; 95%CI 19.97, 111.45). The factors that affected the MFI were women ≥ 38 years of age (p-value = 0.005; 95%CI -111.45, -19.91), total gonadotropin dosages (p-value = 0.006; 95%CI -0.08, 0.01), and gonadotropin-releasing hormone agonist (GnRHa) triggering (p-value = 0.006; 95%CI 36.46, 210.06). However, only women aged ≥38 years remained statistically significant after a multivariable regression analysis (p-value = 0.014; 95%CI -121.00, -14.30). In addition, only male age (mean age ± SD = 38.26 ± 5.13) was associated with high blastocyst quality in univariate and mixed multivariate analyses (OR 0.91; 95%CI 0.56, 3.04). The chemical pregnancy rate was not significantly different between the two age groups (34.5% vs. 56.7%; p-value = 0.162; 95%CI 0.2, 1.30). Conclusion: Advancing age decreased mitochondrial activity in CCs but did not affect blastocyst quality. By contrast, male age may be a predictor of high-grade blastocyst quality.

Preimplantation genetic testing for aneuploidy: predictive embryonic factors.

PURPOSE: In a preimplantation genetic testing for aneuploidy (PGT-A) cycle, does the blastocyst quality before biopsy, or the day of biopsy, or the embryo hatching status have an impact on either euploidy or the rate of embryo survival after freezing? METHODS: This was a retrospective study including 6130 biopsied blastocysts coming from 1849 PGT-A cycles performed in our center (2016-2022). Embryos were categorized according to the inner cell mass and trophectoderm quality, using Gardner's scoring (excellent: AA; good: AB, BA, BB; poor: AC, CA, BC, CB, CC); the day of biopsy (5 or 6); and their hatching status (fully hatched blastocysts [FHB] or non-fully hatched blastocysts [nFHB]). The independent relationship between each group and both euploidy and survival rate was assessed. RESULTS: Excellent-quality embryos were more euploid than both good- and poor-quality embryos (52.69%, 39.69%, and 26.21%; p < 0.001), and day 5-biopsied embryos were more euploid than day 6-biopsied embryos (39.98% and 34.80%; p < 0.001). Survival rates of excellent-quality (92.26%) and good-quality (92.47%) embryos were higher than survival rates in the poor-quality group (84.61%) (p = 0.011 and p = 0.002). Day 5-biopsied embryos survived better than day 6-biopsied embryos (93.71% vs. 83.69%; p < 0.001) and FHB had poorer survival than nFHB (78.61% vs. 93.52%; p < 0.001). CONCLUSIONS: Excellent-quality and day 5-biopsied embryos are more prone to be euploid than good and poor or day 6-biopsied embryos, respectively. Poor-quality, day 6-biopsied embryos, and FHB have significantly lower survival after biopsy and vitrification.

Energy metabolism disorders during in vitro maturation of bovine cumulus-oocyte complexes interfere with blastocyst quality and metabolism.

Glucose and fatty acids (FA) metabolism disturbances during oocyte in vitro maturation (IVM) affect their metabolism and surrounding cumulus cells, but only inhibition of glucose metabolism decreases embryo culture efficiency. Therefore, the present experiment aimed to reveal if glucose or FA metabolism inhibition leads to the disruption of embryo developmental potential, and to characterize the metabolic landscape of embryos reaching the blastocyst stage. Inhibitors of glucose (IO + DHEA) or FA (ETOMOXIR) metabolism were applied during IVM, and the control group was matured under standard conditions. Blastocysts obtained from experimental and control groups were analyzed with regard to lipidome and metabolome (mass spectrometry), transcriptome (RNA-Seq) and fluorescence lipid droplets staining (BODIPY). We showed that inhibition of glucose and fatty acid metabolism leads to cellular stress response compromising the quality of preimplantation embryos. The inhibition of energy metabolism affects membrane fluidity as well as downregulates fatty acids biosynthesis and gene expression of trophectoderm cell line markers. Therefore, we conclude that oocyte maturation environment exerts a substantial effect on preimplantation development programming at cellular and molecular levels.

Antimüllerian hormone (AMH) and age as predictors of preimplantation genetic testing for aneuploidies (PGT-A) cycle outcomes and blastocyst quality on day 5 in women undergoing in vitro fertilization (IVF).

PURPOSE: The objective of this study was to investigate whether women with diminished ovarian reserve who planned for PGT-A exhibit a lower number of blastocysts for biopsy, ploidy outcomes, and blastocyst quality on day 5, regardless of age. METHODS: A retrospective analysis was performed between March 2017 and July 2020 at ART Fertility Clinics Abu Dhabi, including couples that were triggered for final oocyte maturation in an ovarian stimulated cycle planned for PGT-A. Patients were stratified into four AMH groups: < 0.65 ng/ml, 0.65-1.29 ng/ml, 1.3-6.25 ng/ml, and > 6.25 ng/ml; four age categories: ≤ 30, 31-35, 36-40, and > 40 years. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 1410 couples with a mean maternal age of 35.2 ± 6.4 years and AMH of 2.7 ± 2.6 ng/ml were included. In a multivariate logistic regression analysis, controlling for age, the chance of having at least one blastocyst biopsied/stimulated cycle (1156/1410), the chance of having at least one euploid blastocyst/stimulated cycle (880/1410), and the chance of having one euploid blastocyst once biopsy was performed (880/1156) were affected in all patients with AMH < 0.65 ng/ml [AdjOR 0.18[0.11-0.31] p = 0.008)], [AdjOR 0.18 [0.11-0.29] p < 0.001], and [AdjOR 0.34 [0.19-0.61] p = 0.015] as well as in patients with AMH 0.65-1.29 ng/ml (AdjOR 0.52 [0.32-0.84] p < 0.001), (AdjOR 0.49 [0.33-0.72] p < 0.001), and (AdjOR 0.57 [0.36-0.90] p < 0.001), respectively. In a multivariate linear regression analysis, AMH values did not affect blastocyst quality (- 0.72 [- 1.03 to - 0.41] p < 0.001). CONCLUSION: Irrespective of age, patients with diminished ovarian reserve (AMH < 1.3 ng/ml) have a lower chance of having at least one blastocyst biopsied and lower chance of having at least one euploid blastocyst per ovarian stimulated cycle. Blastocyst quality was not affected by AMH values.

Effect of blastocyst quality on human sex ratio at birth in a single blastocyst frozen thawed embryo transfer cycle.

RESEARCH QUESTION: To determine whether blastocyst quality affects the sex ratio at birth through a single blastocyst frozen - thawed embryo transfer (SBFET) cycle. DESIGN: In this retrospective analysis, we examined 3,041 singleton infants born following SBFET between 2017 and 2020 at a single institution. We compared the sex ratios of these infants with respect to the blastocyst quality, embryo growth rate, and morphology. RESULTS: The main outcomes of this study were that the sex ratio (M/F) at birth of SBFET was 1.24. Mothers >40 years old had a considerably lower sex ratio than mothers <40 years old (0.39 vs. 1.23-1.28, p < .05). Transplanting high-quality blastocysts significantly increased the proportion of boys born (1.29 vs. 0.88, p < .05). There were no significant differences in the sex ratio with respect to the inner cell mass (ICM) score and expansion degree. Additionally, a high trophoblastic cell (TE) score resulted in a significantly higher sex ratio than the TE score with C (1.62 vs. 1.15 vs. 0.85, p < .001). Multivariable logistic regression analysis was performed to determine which variables were significant factors affecting sex ratio, and the outcomes were consistent with previous findings. CONCLUSIONS: Our study indicated that high-quality, especially good TE score, had a higher chance of resulting in a male infant than a female infant.

Development and evaluation of a live birth prediction model for evaluating human blastocysts from a retrospective study.

Background: In infertility treatment, blastocyst morphological grading is commonly used in clinical practice for blastocyst evaluation and selection, but has shown limited predictive power on live birth outcomes of blastocysts. To improve live birth prediction, a number of artificial intelligence (AI) models have been established. Most existing AI models for blastocyst evaluation only used images for live birth prediction, and the area under the receiver operating characteristic (ROC) curve (AUC) achieved by these models has plateaued at ~0.65. Methods: This study proposed a multimodal blastocyst evaluation method using both blastocyst images and patient couple's clinical features (e.g., maternal age, hormone profiles, endometrium thickness, and semen quality) to predict live birth outcomes of human blastocysts. To utilize the multimodal data, we developed a new AI model consisting of a convolutional neural network (CNN) to process blastocyst images and a multilayer perceptron to process patient couple's clinical features. The data set used in this study consists of 17,580 blastocysts with known live birth outcomes, blastocyst images, and patient couple's clinical features. Results: This study achieved an AUC of 0.77 for live birth prediction, which significantly outperforms related works in the literature. Sixteen out of 103 clinical features were identified to be predictors of live birth outcomes and helped improve live birth prediction. Among these features, maternal age, the day of blastocyst transfer, antral follicle count, retrieved oocyte number, and endometrium thickness measured before transfer are the top five features contributing to live birth prediction. Heatmaps showed that the CNN in the AI model mainly focuses on image regions of inner cell mass and trophectoderm (TE) for live birth prediction, and the contribution of TE-related features was greater in the CNN trained with the inclusion of patient couple's clinical features compared with the CNN trained with blastocyst images alone. Conclusions: The results suggest that the inclusion of patient couple's clinical features along with blastocyst images increases live birth prediction accuracy. Funding: Natural Sciences and Engineering Research Council of Canada and the Canada Research Chairs Program.

More than 50 million couples worldwide experience infertility. The most common treatment is in vitro fertilization (IVF). Fertility specialists collect eggs and sperm from the prospective parents. They combine the egg and sperm in a laboratory and allow the fertilized eggs to develop for five days into a multi-celled blastocyst. Then, the specialists select the healthiest blastocysts and return them to the patient's uterus. Since 1978, more than 8 million children have been conceived through IVF. Yet, only about 30% of IVF attempts result in a successful birth. As a result, fertility patients often undergo multiple rounds of IVF, which can be expensive and emotionally draining. Several factors determine IVF success, one of which is the health of the blastocysts selected for transfer to the uterus. Specialists select the blastocysts using several criteria. But these human assessments are subjective and inconsistent in predicting which ones are most likely to result in a successful birth. Recent studies suggest artificial intelligence technology may help select blastocysts. Liu et al. show that using artificial intelligence to assess blastocysts and fertility patient characteristics leads to more accurate predictions about which blastocysts are likely to result in a successful birth. In the experiments, the researchers trained an artificial intelligence computer program using pictures of 17,580 blastocysts with known birth outcomes and the parents' clinical characteristics. The model identified 16 parental factors associated with birth outcomes. The top 5 most predictive parental factors were maternal age, the day of blastocyst transfer to the uterus, how many eggs were present in the ovaries, the number of eggs retrieved and the thickness of the uterus lining. The program achieved the highest prediction of healthy births so far, compared to success rates listed in other studies. Artificial intelligence-aided blastocyte selection using patient and blastocyst characteristics may improve IVF success rates and reduce the number of treatment cycles patient couples undergo. Before specialists can use artificial intelligence in their clinics, they must conduct confirmatory clinical studies that enroll patient couples to compare conventional methods and artificial intelligence.

Zygote Diameter and Total Cytoplasmic Volume as Useful Predictive Tools of Blastocyst Quality.

Introduction According to the Embryo Protection Act, the selection of embryos with the greatest potential for successful implantation in Germany must be performed in the pronucleus stage. The main aim of this study was to identify morphokinetic parameters that could serve as noninvasive biomarkers of blastocyst quality in countries with restrictive reproductive medicine laws. Materials and Methods The sample comprised 191 embryos from 40 patients undergoing antagonist cycles for intracytoplasmic sperm injection. Blastocysts were cultured in an EmbryoScope chamber and video records were validated to determine the post-injection timing of various developmental stages, cleavage stages, and blastocyst formation. The Gardner and Schoolcraft scoring system was used to characterize blastocyst quality. Results Morphokinetic data showed that the zygote diameter and total cytoplasmic volume were significantly different between good and poor blastocysts quality groups, where zygotes, which formed better blastocyst quality, had smaller diameter and smaller total cytoplasmic volume. Zygotes with more rapid pronuclear disappearance developed in better-quality blastocysts. Differences between good- and poor-quality blastocysts were also observed for late-stage parameters and for the spatial arrangement of blastomere where tetrahedral embryos more frequently forming good-quality blastocyst compare to the non-tetrahedral. Conclusions The study findings could be used to enhance embryo selection, especially in countries with strict Embryo Law Regulations. Further studies, including those in which the implantation potential and pregnancy rate are considered, are warranted to confirm these preliminary results.

Low-grade inflammation is negatively associated with live birth in women undergoing IVF.

RESEARCH QUESTION: Is low-grade inflammation, detected by C-reactive protein (CRP), a marker of IVF outcome addressing both blastocyst quality and pregnancy outcome? DESIGN: This sub-study of a multicentre randomized controlled trial included 440 women undergoing IVF treatment with a gonadotrophin-releasing hormone (GnRH) antagonist protocol. Serum CRP was measured on cycle day 2-3 (baseline) and on the day of ovulation triggering. The association between CRP concentrations and reproductive outcomes (number of retrieved oocytes, number of good-quality blastocysts, pregnancy, pregnancy loss and live birth), were analysed, adjusting for relevant confounders. RESULTS: A negative association was found between higher baseline CRP concentrations and live birth rate (odds ratio [OR] 0.77, 95% confidence interval [CI] 0.62-0.96, P = 0.02) and higher CRP concentrations at baseline were associated with pregnancy loss among women who conceived (OR 1.37, 95% CI 1.07-1.76, P = 0.01). When testing for a specific cut-off, CRP concentrations above 2.34 (the highest quartile) were more likely to be associated with pregnancy loss (P = 0.02) and a lower chance of live birth (P = 0.04) compared with the lowest quartile. No associations were found between CRP concentrations and pregnancy outcomes on the day of ovulation triggering, and there were no associations between CRP concentrations and the number of good-quality blastocysts. CONCLUSIONS: Higher CRP concentrations at cycle day 2-3, before starting ovarian stimulation, are negatively associated with chance of live birth, possibly because of an increased risk of pregnancy loss. No association was found between the number of good-quality blastocysts and CRP concentration. More studies are needed to investigate the impact of low-grade inflammation.

Effect of development speed and quality of blastocyst on singleton birthweight in single frozen-thawed blastocyst transfer cycles.

Background: Assisted reproductive technology (ART) has revolutionized infertility treatment, leading to a surge in ART-conceived children. Despite its success, ART-born offspring face higher risks of preterm birth (PTB), low birth weight (LBW), and small for gestational age (SGA). The mechanisms behind these outcomes remain unclear, partly attributed to multiple embryo transfers. Recent advancements advocate single blastocyst transfers for improved outcomes. However, the influence of blastocyst quality and development speed on neonatal outcomes is underexplored. Objective: This study investigated whether blastocyst development speed and quality affect singleton birthweight when the blastocyst is selected for single frozen-thawed blastocyst transfer (FBT). Methods: Data from patients who performed an FBT cycle at our center from July 2011 to June 2021 were collected and analyzed. Based on the inclusion and exclusion criteria, 420 single FBT cycles were assessed. The women were divided into four groups, Group A (day 5, good-quality blastocysts), Group B (day 5, non-good-quality blastocysts), Group C (day 6, good-quality blastocysts), and Group D (day 6, non-good-quality blastocysts) according to the developmental speed and quality of the transferred blastocyst. Results: The birthweight was relatively the highest in Group A, which developed rapidly and transferred good quality blastocysts. However, no significant difference existed among the groups (P>0.05). The prevalence of premature birth (PTB), low birth weight (LBW), very low birth weight (VLBW), or high birth weight (HBW) was similar among the four groups (P > 0.05). No correlation existed between birth weight and blastocyst development speed or quality after adjusting for possible confounders (P > 0.05 respectively). However, the difference in the proportion of males born among the four groups was significant, especially in Group D, which was significantly lower than that in Group A (adjusted odds ratio = 0.461, 95% confidence interval: 0.230-0.921, P < 0.05). Conclusions: This retrospective cohort study suggests that the combined effect of blastocyst development speed and quality on neonatal birthweight is insignificant. The transfer of slow-growing, non-good-quality blastocysts increases the chance of a female baby being born.

Clinical Usability of Embryo Development Using a Combined Qualitative and Quantitative Approach in a Single Vitrified-Warmed Blastocyst Transfer: Assessment of Pre-Vitrified Blastocyst Diameter and Post-Warmed Blastocyst Re-Expansion Speed.

Improving the safety and efficacy of assisted reproductive technology programs has been a continuous challenge. Traditionally, morphological grading has been used for embryo selection. However, only a few studies have assessed the morphokinetic variables and morphological dynamics of blastocysts. In the present study, we aimed to perform a quantitative analysis of blastocyst diameter and re-expansion speed. This in-depth morphokinetic evaluation can correlate with currently observed pregnancy outcomes. In total, 658 single vitrified-warmed blastocyst transfer cycles were performed between October 2017 and December 2021, which were divided into four groups according to the pre-vitrified blastocyst diameter. After warming, the groups were subdivided according to the blastocyst re-expansion speed. These quantitative measurements were performed using a time-lapse system. Both diameter and speed are essential in determining the blastocyst quality, while age, day of freezing, and blastocyst quality are crucial from a clinical perspective. The application of both quantitative (diameter and speed) and qualitative (blastocyst quality scores) parameters can help evaluate the clinical usability of blastocysts. This method can prove useful for embryologists in counseling their patients and determining pregnancy patient-oriented strategies.

Blastocyst quality and perinatal outcomes of frozen-thawed single blastocyst transfer cycles.

Objective: To investigate the effects of blastocyst quality and morphological grade on the perinatal outcomes in patients undergoing frozen-thawed single blastocyst transfer cycles. Methods: This single-center retrospective cohort study included 2648 singleton neonates resulting from frozen-thawed single blastocyst transfers performed between January 2017 and September 2021. Multivariate logistic regression was performed to evaluate perinatal outcomes for their association with blastocyst quality and morphological parameters. Result: Transfer of a good-quality blastocyst in a frozen-thawed cycle was associated with a lower rate of preterm delivery (PTD, adjusted OR =0.7, 95% CI 0.5-0.9; P=0.020) and a higher likelihood of a male neonate (adjusted OR =1.2, 95%CI 1.0-1.5; P=0.048). Compared with grade C inner cell mass (ICM) blastocyst transfer, grade B ICM (adjusted OR =0.5, 95%CI 0.2-0.9; P=0.027) and grade A ICM (adjusted OR =0.6, 95%CI 0.3-1.5; P=0.290) blastocyst transfers were associated with a lower rate of PTD, which was more evident for grade B ICM. After adjusting for confounders, the likelihood of a male neonate (grade B TE, OR =1.2, 95%CI 1.0-1.5, P=0.037; grade A TE OR =1.9, 95%CI 1.3-28, P=0.002) increased with increasing trophectoderm (TE) quality. Compared with expansion stage 4, the likelihood of a male neonate was 1.5 times greater with transfer of a stage 6 blastocyst (OR =1.5, 95%CI 1.0-2.3; P=0.06), and the risk of small for gestational age (SGA) was greater with transfer of a stage 5 blastocyst (adjusted OR =3.5, 95%CI 1.5-8.0; P=0.004). The overall grading of the blastocyst, expansion stage, ICM grade, and TE grade were not associated with length at birth, birthweight, large for gestational age (LGA), or birth defects (all P>0.05). Conclusions: In frozen-thawed single blastocyst transfer cycles, transfer of a good-quality blastocyst was associated with a lower rate of PTD and a greater likelihood of a male neonate. Transfer of grade B ICM blastocysts decreased the rate of PTD, and TE quality was positively correlated with the likelihood of a male neonate.

Leukemia inhibitory factor enhances the development and subsequent blastocysts quality of yak oocytes in vitro.

Leukemia inhibitory factor (LIF) is a multipotent cytokine of the IL-6 family which plays a critical role in the maturation and development of oocytes. This study evaluated the influence of LIF on the maturation and development ability of yak oocytes, and the quality of subsequent blastocysts under in vitro culture settings. Different concentrations of LIF (0, 25, 50, and 100 ng/mL) were added during the in vitro culture of oocytes to detect the maturation rate of oocytes, levels of mitochondria, reactive oxygen species (ROS), actin, and apoptosis in oocytes, mRNA transcription levels of apoptosis and antioxidant-related genes in oocytes, and total cell number and apoptosis levels in subsequent blastocysts. The findings revealed that 50 ng/mL LIF could significantly increase the maturation rate (p < 0.01), levels of mitochondria (p < 0.01) and actin (p < 0.01), and mRNA transcription levels of anti-apoptotic and antioxidant-related genes in yak oocytes. Also, 50 ng/mL LIF could significantly lower the generation of ROS (p < 0.01) and apoptosis levels of oocytes (p < 0.01). In addition, blastocysts formed from 50 ng/mL LIF-treated oocytes showed significantly larger total cell numbers (p < 0.01) and lower apoptosis rates (p < 0.01) than the control group. In conclusion, the addition of LIF during the in vitro maturation of yak oocytes improved the quality and the competence of maturation and development in oocytes, as well as the quality of subsequent blastocysts. The result of this study provided some insights into the role and function of LIF in vitro yak oocytes maturation, as well as provided fundamental knowledge for assisted reproductive technologies in the yak.

A randomized, controlled, first-in-patient trial of choriogonadotropin beta added to follitropin delta in women undergoing ovarian stimulation in a long GnRH agonist protocol.

STUDY QUESTION: Does addition of choriogonadotropin beta (recombinant CG beta) to follitropin delta increase the number of good-quality blastocysts following ovarian stimulation in a long GnRH agonist protocol? SUMMARY ANSWER: At the doses investigated, the addition of CG beta reduced the number of intermediate follicles and related down-stream parameters including the number of oocytes and blastocysts. WHAT IS KNOWN ALREADY: CG beta is a novel recombinant hCG (rhCG) molecule expressed by a human cell line (PER.C6®) and has a different glycosylation profile compared to urinary hCG or rhCG derived from a Chinese Hamster Ovary (CHO) cell line. In the first-in-human trial, the CG beta pharmacokinetics were similar between men and women. In women, the AUC and the peak serum concentration (Cmax) increased approximately dose proportionally following single and multiple daily doses. In men, a single dose of CG beta provided higher exposure with a longer half-life and proportionately higher testosterone production than CHO cell-derived rhCG. STUDY DESIGN, SIZE, DURATION: This placebo-controlled, double-blind, randomized trial (RAINBOW) was conducted in five European countries to explore the efficacy and safety of CG beta as add-on treatment to follitropin delta in women undergoing ovarian stimulation in a long GnRH agonist protocol. Randomization was stratified by centre and age (30-37 and 38-42 years). The primary endpoint was the number of good-quality blastocysts (Grade 3 BB or higher). Subjects were randomized to receive either placebo or 1, 2, 4, 8 or 12 µg CG beta added to the daily individualized follitropin delta dose during ovarian stimulation. PARTICIPANTS/MATERIALS, SETTING, METHODS: In total, 620 women (30-42 years) with anti-Müllerian hormone (AMH) levels between 5 and 35 pmol/l were randomized in equal proportions to the six treatment groups and 619 subjects started treatment. All 619 subjects were treated with an individualized dose of follitropin delta determined based on AMH (Elecsys AMH Plus Immunoassay) and body weight. Triggering with rhCG was performed when 3 follicles were ≥17 mm but no more than 25 follicles ≥12 mm were reached. MAIN RESULTS AND THE ROLE OF CHANCE: The demographic characteristics were comparable between the six treatment groups and the overall mean age, body weight and AMH were 35.6 ± 3.3 years, 65.3 ± 10.7 kg and 15.3 ± 7.0 pmol/l, respectively. The incidence of cycle cancellation (range 0-2.9%), total follitropin delta dose (mean 112 µg) and duration of stimulation (mean 10 days) were similar across the groups. At stimulation Day 6, the number and size of follicles was similar between the treatment groups, whereas at the end-of-stimulation dose-related decrease of the intermediate follicles between 12 and 17 mm was observed in comparison to the placebo group. In contrast, the number of follicles ≥17 mm was similar between the CG beta dose groups and the placebo group. A reduced number of intermediate follicles (12 to 17 mm) and fewer oocytes (mean range 9.7 to 11.2) were observed for all doses of CG beta compared to the follitropin delta only group (mean 12.5). The mean number of good-quality blastocysts was 3.3 in the follitropin delta group and ranged between 2.1 and 3.0 across the CG beta groups. The incidence of transfer cancellation was higher in the 4, 8 and 12 µg group, mostly as no blastocyst was available for transfer. In the group receiving only follitropin delta, the ongoing pregnancy rate (10-11 weeks after transfer) was 43% per started cycle versus 28-39% in CG beta groups and 49% per transfer versus 38-50% in the CG beta groups. There was no apparent effect of CG beta on the incidence of adverse events, which was 48.1% in the placebo group and 39.6-52.3% in the CG beta dose groups. In line with the number of collected oocytes, the overall ovarian hyperstimulation syndrome incidence remained lower following follitropin delta with CG beta (2.0-10.3%) compared with follitropin delta only treatment (11.5%). Regardless of the dose, CG beta was safe and well-tolerated with low risk of immunogenicity. LIMITATIONS, REASONS FOR CAUTION: The effect of the unique glycosylation of CG beta and its associated potency implications in women were not known prior to this trial. Further studies will be needed to evaluate optimal doses of CG beta for this and/or different indications. WIDER IMPLICATIONS OF THE FINDINGS: The high ongoing pregnancy rate in the follitropin delta group supports the use of individualized follitropin delta dosing in a long GnRH agonist protocol. The addition of CG beta reduced the presence of intermediate follicles with the investigated doses and negatively affected all down-stream parameters. Further clinical research will be needed to assess the optimal dose of CG beta in the optimal ratio to follitropin delta to develop this novel combination product containing both FSH and LH activity for ovarian stimulation. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by Ferring Pharmaceuticals, Copenhagen, Denmark. B.M. and P.L. are employees of Ferring Pharmaceuticals. M.F.S., H.V., C.Y.A., M.F., C.B., A.P. and Y.K. have received institutional clinical trial fees from Ferring Pharmaceuticals. C.B. has received payments for lectures from Organon, Ferring Pharmaceuticals, Merck A/S and Abbott. M.F.S. has received payment for lectures from Ferring Pharmaceuticals. Y.K. has received payment for lectures from Merck and travel support from Gedeon Richter. H.V. has received consulting fees from Oxo and Obseva and travel support from Gedeon Richter, Ferring Pharmaceuticals and Merck. C.Y.A. has received payment for lectures from IBSA, Switzerland. M.F and C.Y.A. were reimbursed as members of the Data Monitoring Board in this trial. M.F. has an issued patent about unitary combination of FSH and hCG (EP1633389). TRIAL REGISTRATION NUMBER: 2017-003810-13 (EudraCT Number). TRIAL REGISTRATION DATE: 21 May 2018. DATE OF FIRST PATIENT'S ENROLMENT: 13 June 2018.

Live birth rate following frozen-thawed blastocyst transfer is higher in high-grade day 6 blastocysts than in low-grade day 5 blastocysts.

Background: Day 5 (D5) blastocysts are generally given priority to transfer than day 6 (D6) blastocysts; however, which one should be prioritized to transfer when only low-grade D5 and high-grade D6 blastocysts are available? Methods: A large retrospective cohort study was carried out to evaluate the live birth rate (LBR) following D5 and D6 blastocysts in single frozen-thawed blastocyst transfer (FBT) during January 2014 and December 2018. A multivariate logistic regression was conducted to evaluate the combined impact of expansion day (D5 and D6) and blastocyst quality (high grade/low grade) on LBR, accounting for the potential confounding factors. The biopsied blastocysts from a consecutive PGT-A case series during February 2013 to December 2021 were analyzed in a supplementary study. Results: The LBR achieved in high-grade D6 blastocyst transfer was significantly higher than that in low-grade D5 blastocyst transfer (50.43% vs. 40.70%, aOR 1.54, 95% CI 1.05-2.26, p = 0.027). There were no significant differences in preterm birth rate, very preterm birth rate, mean live birth weight, and birth weight <1,500 g and >4,000 g between the two cohorts. As for aneuploidy analysis in PGT, there were 54.55% of euploid blastocysts (30/55) among high-grade D6 blastocysts, significantly higher than the 41.39% of euploid blastocysts (565/1,365) among low-grade D5 blastocysts (p < 0.001). Conclusions: Our data suggest that D6 blastocysts with high morphology grading are preferred than D5 blastocysts with low morphology grading when selecting blastocyst transfer to shorten the time of conception.

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.

Does longer storage of blastocysts with equal grades in a cryopreserved state affect the perinatal outcomes?

AIM: Although mammalian embryos could be preserved in liquid nitrogen for thousands of years in theoretical models, the viability of cryopreserved blastocyst with varying grades remains to be speculated. In this study, we aimed to determine whether the longer storage time of blastocysts with equal grades could negatively affect the perinatal outcomes. MATERIALS AND METHODS: Single vitrified-warmed blastocyst was divided into four grades (AA, AB/BA, BB, BC/CB) according to the blastocyst score when freezing, and each grade of blastocyst was categorized into four storage duration categories: 28 days-1 year, 1-3 years, 3-5 years, and ≥5 years. Then the perinatal outcomes with different storage time were analyzed. RESULTS: Our results revealed that for blastocysts with the same grade, the length of storage time had no statistical effect on blastocyst survival rate, clinical pregnancy/implantation rate, live birth rate, and abortion rate. In addition, more advanced developmental blastocyst could obtain better pregnancy outcomes regardless of the cryopreservation length. Similar neonatal outcomes were obtained over time. CONCLUSIONS: Cryopreservation time could not negatively affect the perinatal outcomes of blastocysts with equal grades. Efficient blastocyst cryopreservation technology by vitrification can help older women obtain high-quality embryos at a young age.

CHIR99021 and rpIL6 promote porcine parthenogenetic embryo development and blastocyst quality.

Signaling pathways and transcription factors are involved in porcine embryonic development. Here, we demonstrate that glycogen synthase kinase-3 (GSK3) inhibitor, CHIR99021 and recombinant porcine interleukin-6 (rpIL6) significantly promote porcine parthenogenetic blastocyst formation (49.23 ± 8.40% vs 32.34 ± 4.15%), with increased inner cell mass (ICM) cell numbers (7.72 ± 2.30 vs 4.28 ± 1.60) and higher expression of pluripotent genes, such as OCT4, SOX2 and NANOG. Furthermore, CHIR99021 and rpIL6 improve blastocyst quality with increased blastocyst hatching percentage (16.19 ± 1.96% vs 10.25 ± 1.12%) and subsequently porcine pluripotent stem cells (pPSCs) derivation efficiency. These results advance the understanding of porcine pre-implantation development and provide evidences in improving the blastocyst quality.

Live birth rate and neonatal outcomes of different quantities and qualities of frozen transferred blastocyst in patients requiring whole embryo freezing stratified by age.

BACKGROUND: Multiple pregnancies are associated with significant complications and health risks for both mothers and infants. Single blastocyst transfer (SBT) is a logical and effective measure to reduce the incidence of multiple pregnancy with assisted reproductive technology (ART). Whether it is suitable for everyone undergoing SBT was inconclusive, in view of the consideration of embryo quality and patients' age. Therefore, this study aimed to explore live birth rate (LBR) and neonatal outcomes of different quantities and qualities of blastocysts in patients stratified by age, using a cutoff of 35 years, who required whole embryo freezing and underwent a subsequent frozen thawed transfer (FET) cycle. METHODS: Atotal of 3,362 patients were divided into five groups: group A (n=1569) received a single good-quality blastocyst, group B (n=1113) received two good-quality blastocysts, group C (n=313) received one good-and one average-quality blastocyst, group D (n=222) received two average-quality blastocysts, and group E (n=145) received one average-quality blastocyst. RESULTS: For patients who received good-quality blastocysts, irrespective of age, the LBR of double blastocyst transfer (DBT) was about 50-65% and the multiple pregnancy rate (MPR) was 40-60%; however, the LBR of SBT was 40-55%, and the MPR was 3.5-6.3%. For patients who only had average-quality blastocysts, the MPR of double average-quality blastocyst transfer was as high as 30-50%. Moreover, about 70-90% of preterm births resulted from multiple pregnancies, and about 85-95% of low birth weight babies come from multiple pregnancies. The neonatal outcomes (gestational age, birth weight, and birth height) of DBT were significantly lower than those of SBT regardless of age, and this statistical difference disappeared if the patients were subgrouped by singleton or twin. There is no significant difference in neonatal outcomes between single good-quality blastocyst and single average-quality blastocyst transfer. CONCLUSIONS: SBT is a preferable option for patients regardless of age when good-quality blastocysts are available. For patients who only had average-quality blastocysts, they should be informed that DBT was associated with higher multiple pregnancy and adverse neonatal outcomes when compared with SBT regardless of age, suggesting that the practice of SBT is also feasible for these patients.

The effect of Day 3 cell number on pregnancy outcomes in vitrified-thawed single blastocyst transfer cycles.

STUDY QUESTION: Does cell number on Day 3 have an impact on pregnancy outcomes in vitrified-thawed single blastocyst transfer cycles? SUMMARY ANSWER: A low Day 3 cell number (≤5 cells) was independently associated with decreased live birth rate (LBR) during single blastocyst transfer cycles in young women. WHAT IS KNOWN ALREADY: Day 3 cell number is an effective predictor of IVF success rates when transferring cleavage stage embryos. However, the association between Day 3 blastomere number and pregnancy outcomes after blastocyst transfer is still unknown. STUDY DESIGN, SIZE, DURATION: A retrospective cohort study of 3543 patients who underwent frozen-thawed single blastocyst transfers from January 2013 to June 2018 at a tertiary-care academic medical center. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients were grouped into six groups according to the Day 3 cell number: ≤4 cells, 5 cells, 6 cells, 7 cells, 8 cells and >8 cells. The primary outcome measure was LBR. A logistic regression analysis was performed to explore the independent association between Day 3 blastomere number and LBR after adjustment for some potential confounders. MAIN RESULTS AND THE ROLE OF CHANCE: In women <35 years old, the LBR varied significantly according to Day 3 cell number, with the rate of 31.2%, 34.4%, 41.9%, 45.1%, 48.1% and 48.2% for the ≤4-cell, 5-cell, 6-cell, 7-cell, 8-cell and >8-cell groups, respectively (P < 0.001). This significant difference was also observed in the high- and low-quality blastocyst subgroups of young women. However, for women ≥35 years old, the rate of live birth was similar between groups. Furthermore, after accounting for confounding factors, the LBR was significantly decreased in the ≤4-cell (adjusted odds ratio (aOR): 0.62, 95% CI: 0.48-0.80, P < 0.001) and 5-cell (aOR: 0.73, 95% CI: 0.57-0.92, P = 0.009) groups as compared to the 8-cell group. Likewise, the blastocysts arising from ≤4-cell (aOR: 0.73, 95% CI: 0.57-0.93, P = 0.010) or 5-cell (aOR: 0.77, 95% CI: 0.61-0.97, P = 0.024) embryos were associated with lower clinical pregnancy rate than those from 8-cell embryos. No significant differences were observed in biochemical pregnancy rate and miscarriage rate. LIMITATIONS, REASONS FOR CAUTION: A limitation of the current study was its retrospective design. Future prospective studies are needed to confirm our findings. WIDER IMPLICATIONS OF THE FINDINGS: Our observations suggested that a low Day 3 cell number was related to decreased LBR after blastocyst transfer in young women, which provided vital information for clinicians in selecting blastocyst during IVF treatment. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Natural Science Foundation of China (NSFC) (31770989 to Y.W.; 81671520 to Q.C.) and the Shanghai Ninth People's Hospital Foundation of China (JYLJ030 to Y.W.). The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

The frequency of collapse as a predictor of feline blastocyst quality.

Domestic cats are frequently used as a research model for felid species that are threatened with extinction. Until now, the development of feline embryos has been evaluated using both classical observation methods and time-lapse monitoring (TLM). Blastocyst collapse observed using time-lapse cinematography is used as a predictor of blastocyst quality and is closely related to implantation potential. The aim of this study was to determine the relationship between the quality of domestic cat blastocysts obtained after in vitro fertilization and the frequency and duration of collapse, and of hatching. There was a significant difference in the average number of collapses and weak contractions between good and poor quality blastocysts. There was no significant difference between hatching and non-hatching blastocysts in terms of blastocyst cavity formation time or average number and duration of collapse. These results showed that the time of cavity formation was not related to blastocyst quality. The number of collapses and the occurrence of hatching were positively related to blastocyst quality, and poor quality blastocysts have, as a consequence, a reduced potential for implantation. TLM plays a significant role in cat embryo evaluation.