Maternal RNF114-mediated target substrate degradation regulates zygotic genome activation in mouse embryos.

Zygotic genomic activation (ZGA) is a landmark event in the maternal-to-zygotic transition (MZT), and the regulation of ZGA by maternal factors remains to be elucidated. In this study, the depletion of maternal ring finger protein 114 (RNF114), a ubiquitin E3 ligase, led to developmental arrest of two-cell mouse embryos. Using immunofluorescence and transcriptome analysis, RNF114 was proven to play a crucial role in major ZGA. To study the underlying mechanism, we performed protein profiling in mature oocytes and found a potential substrate for RNF114, chromobox 5 (CBX5), ubiquitylation and degradation of which was regulated by RNF114. The overexpression of CBX5 prevented embryonic development and impeded major ZGA. Furthermore, TAB1 was abnormally accumulated in mutant two-cell embryos, which was consistent with the result of in vitro knockdown of Rnf114. Knockdown of Cbx5 or Tab1 in maternal RNF114-depleted embryos partially rescued developmental arrest and the defect of major ZGA. In summary, our study reveals that maternal RNF114 plays a precise role in degrading some important substrates during the MZT, the misregulation of which may impede the appropriate activation of major ZGA in mouse embryos.

Prioritized single vitrified blastocyst to be warmed between grades 3 or 4 blastocyst on day 5 transfer cycles.

PURPOSE: Selecting the optimal blastocyst to implant during cryopreservation and warming is critial for in vitro fertilization success. Therefore, the aim of this study was to explore which blastocyst should be prioritized to be thawed when facing a single vitrified blastocyst on day 5 transfer. METHODS: A retrospective study including 1,976 single vitrified-warmed blastocyst transfer cycles was conducted from January 2016 to December 2020. RESULTS: We found that grade 4 vitrified blastocyst had a higher clinical pregnancy (60.64% vs. 49.48%, P < 0.001) and live birth rates (50.12% vs 39.59%, P < 0.001) than the grade 3 vitrified blastocyst. However, no statistical difference was found between groups in miscarriage rate, birth weight, or gestational age. Besides, the grade 4 vitrified-thawed blastocyst had significant potential to develop into grade 6 blastocyst after further culturing for 16 h (73.68% vs. 48.60%, P < 0.001). The grade 6 transferred blastocyst was markedly higher in both clinical pregnancy rate (61.88% vs. 51.53%, P < 0.001) and live birth rate (50.91% vs. 40.46%, P < 0.001) compared to grade 5 transferred blastocyst. CONCLUSIONS: Grade 4 vitrified blastocyst is recommended when facing single vitrified blastocyst on day 5 transfer. More importantly, the "embryonic escape hypothesis" was firstly proposed to reveal the findings.

Molecular characterization of extracellular vesicles derived from follicular fluid of women with and without PCOS: integrating analysis of differential miRNAs and proteins reveals vital molecules involving in PCOS.

PURPOSE: To elucidate the characterization of extracellular vesicles (EVs) in the follicular fluid-derived extracellular vesicles (FF-EVs) and discover critical molecules and signaling pathways associating with the etiology and pathobiology of PCOS, the differentially expressed miRNAs (DEmiRNAs) and differentially expressed proteins profiles (DEPs) were initially explored and combinedly analyzed. METHODS: First, the miRNA and protein expression profiles of FF-EVs in PCOS patients and control patients were compared by RNA-sequencing and tandem mass tagging (TMT) proteomic methods. Subsequently, Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes were used to analyze the biological function of target genes of DEmiRNAs and DEPs. Finally, to discover the functional miRNA-target gene-protein interaction pairs involved in PCOS, DEmiRs target gene datasets and DEPs datasets were used integratedly. RESULTS: A total of 6 DEmiRNAs and 32 DEPs were identified in FF-EVs in patients with PCOS. Bioinformatics analysis revealed that DEmiRNAs target genes are mainly involved in thiamine metabolism, insulin secretion, GnRH, and Apelin signaling pathway, which are closely related to the occurrence of PCOS. DEPs also closely related to hormone metabolism processes such as steroid hormone biosynthesis. In the analysis integrating DEmiRNAs target genes and DEPs, two molecules, GRAMD1B and STPLC2, attracted our attention that are closely associated with cholesterol transport and ceramide biosynthesis, respectively. CONCLUSION: Dysregulated miRNAs and proteins in FF-EVs, mainly involving in hormone metabolism, insulin secretion, neurotransmitters regulation, adipokine expression, and secretion, may be closely related to PCOS. The effects of GRAMD1B and STPLC2 on PCOS deserve further study.

Effect of unplanned spontaneous follicular growth and ovulation on pregnancy outcomes in planned artificial frozen embryo transfer cycles: a propensity score matching study.

STUDY QUESTION: Does unplanned spontaneous follicular growth and ovulation affect clinical outcomes after planned artificial frozen-thawed embryo transfer (AC-FET) cycles? SUMMARY ANSWER: AC-FET and spontaneous follicular growth and ovulation events resulted in notably better pregnancy outcomes with a significantly higher implantation rate (IR), clinical pregnancy rate (CPR), ongoing pregnancy rate (OPR) and live birth rate (LBR) and a significantly lower miscarriage rate. WHAT IS KNOWN ALREADY: The AC-FET protocol without GnRH agonist administration is associated with a low incidence of follicular growth and ovulation. In the literature, authors often refer to these types of cycles with concern due to possibly impaired FET outcomes. However, the real impact of such cycles has yet to be elucidated due to the lack of existing data. STUDY DESIGN, SIZE, DURATION: This was a retrospective clinical study involving 2256 AC-FET cycles conducted between January 2017 and August 2019. Propensity score (PS) matching was used to control for confounding variables. PARTICIPANTS/MATERIALS, SETTING, METHODS: Subjects were divided into two groups: a study group: cycles with spontaneous follicular growth and ovulation (the maximum diameter of follicles in any ovary was ≥14 mm and ovulation was confirmed by consecutive ultrasound examinations) and a control group featuring cycles without growing follicles (the maximum diameter of follicles in both ovaries were <10 mm). The study group was matched by PS with the control group at a ratio of 1:2. The study group consisted of 195 patients before PS matching and 176 patients after matching. The numbers of participants in the control group before and after PS matching were 2061 and 329, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: This analysis showed that patient age (adjusted odds ratio [aOR] 1.05; 95% CI 1.01-1.09; P=0.010) and basal FSH level (aOR 1.06; 95% CI 1.01-1.11; P=0.012) were significantly and positively related with the spontaneous follicular growth and ovulation event. In addition, this event was negatively correlated with BMI (aOR 0.92; 95% CI 0.87-0.97; P=0.002), AMH level (aOR 0.66; 95% CI 0.59-0.74; P<0.001) and a high starting oestrogen dose (aOR 0.53; 95% CI 0.38-0.76 for 6 mg vs. 4 mg; P<0.001). Baseline characteristics were similar between groups after PS matching. Patients in the study group had a significantly higher IR (28.8% vs. 21.8%, P=0.016), CPR (44.9% vs. 33.4%, P=0.011), OPR (39.2% vs. 26.1%, P=0.002) and LBR (39.2% vs. 24.9%, P=0.001) and a lower miscarriage rate (12.7% vs. 25.5%, P=0.030), compared with those in the control group. LIMITATIONS, REASONS FOR CAUTION: This was a retrospective study carried out in a single centre and was therefore susceptible to bias. In addition, we only analysed patients with normal ovulation patterns and excluded those with follicular growth but without ovulation. Further studies remain necessary to confirm our results. WIDER IMPLICATIONS OF THE FINDINGS: It is not necessary to cancel cycles that experience spontaneous follicular growth and ovulation. Our data support promising clinical outcomes after this event. Our findings are important as they can better inform clinicians and patients. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by National Natural Science Foundation of China (grant no. 81701507, 81801404, 81871210, 82071648), Natural Science Foundation of Jiangsu Province (grant no. BK20171126, BK20201123) and Jiangsu Province '333' project. The authors declare that they have no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Peptidomic analysis of blastocyst culture medium and the effect of peptide derived from blastocyst culture medium on blastocyst formation and viability.

High-quality in vitro human embryo culture medium can improve the blastocyst formation rate and blastocyst quality and be beneficial for the clinical application of single blastocyst transfer. Mammalian embryos can secrete protein products into the surrounding medium. As a group of bioactive molecules and degraded proteins, peptides have been shown to participate in various biological processes. Using liquid chromatography-tandem mass spectrometry, we performed comparative peptidomic analysis of human culture medium in blastocyst formation and nonblastocyst-formation groups. A total of 201 differentially expressed peptides originating from 157 precursor proteins were identified. Among these, a peptide derived from HERC2 (peptide derived from blastocyst culture medium [PDBCM]) passed through the zona pellucida, was distributed on the perivitelline space, was absent in arrest embryos and highly expressed in high-quality blastocysts compared with low-quality blastocysts, and significantly promoted blastocyst formation in a concentration-dependent manner. These results indicate that PDBCM may be a novel biomarker for predicting blastocyst formation and viability. The mechanism remains unclear and needs to be explored in the future.

Retrospective clinical analysis of two artificial shrinkage methods applied prior to blastocyst vitrification on the outcome of frozen embryo transfer.

PURPOSE: Vitrification significantly improves the rates of blastocyst survival and clinical pregnancy following frozen embryo transfer (FET). However, ice crystal formation during the freezing process reduces the blastocyst survival rate. Artificial shrinkage (AS) prior to blastocyst vitrification decreases the formation of ice crystals, increasing the blastocyst survival rate. The aim of this study was to identify an efficient AS method to improve blastocyst survival rates following vitrification. METHOD: Use of the 29-gauge needle AS and Laser pulse AS methods prior to vitrification was compared in terms of the impacts on the rates of blastocyst survival in FET cycles, blastocyst hatching, clinical pregnancy after transfer, embryo implantation, abortion, gestational duration and birth weight. RESULT: In total, 438 blastocysts in 219 cycles were thawed, resulting in survival of 407 (92.9 %). Of these, 213 cycles were transferred, resulting in 129 clinical pregnancies (60.6 %) and 140 successful births. There were no differences between the two methods in the rates of blastocyst survival, clinical pregnancy, embryo implantation and abortion. However, the 29-gauge needle AS group was associated with a significantly lower blastocyst hatching rate (83.6 % vs. 91.2 %), shorter average gestational duration (37.36 ± 2.34 vs. 38.06 ± 1.76), and higher premature birth rate (40.00 % vs. 21.15 %) compared with Laser pulse AS group. CONCLUSION: No significant differences in the effectiveness of the two methods applied prior to blastocyst vitrification were observed before birth, while after birth, a significantly improved clinical outcome was obtained with laser pulse AS indicating that this is a more effective pre-processing method for blastocyst vitrification.

Trophectoderm morphology predicts outcomes of pregnancy in vitrified-warmed single-blastocyst transfer cycle in a Chinese population.

PURPOSE: In this study, we estimated the effect of blastocoele expansion, ICM and TE quality after warming and culture on the rates of clinical pregnancy, live birth and miscarriage in vitrified-warmed single-blastocyst transfer cycle in a Chinese population. METHODS: A retrospective analysis of 263 cycles of vitrified-warmed single-blastocyst transfers was performed. RESULTS: The blastocysts with higher TE grade significantly increased the rates of clinical pregnancy (OR = 0.59, 95 % CI, 0.35-0.99, P = 0.045, grade (A + B) vs grade C) and live birth (OR = 0.55, 95 % CI, 0.32-0.94, P = 0.029, grade (A + B) vs grade C). And the association between TE grade and the rate of live birth didn't change after the number of repeated cycles was adjusted (OR = 0.55, 95 % CI, 0.32-0.95, P = 0.033, grade (A + B) vs grade C). The number of repeated cycles was a confounding factor significantly different between the live birth and no live birth groups. By contrast, neither blastocoele expansion nor inner cell mass was statistically related to the rates of clinical pregnancy, live birth and miscarriage. CONCLUSIONS: Our data firstly provided the evidence that TE grading, but not ICM grading, was significantly associated with the clinical pregnancy rate and live birth rate in vitrified-warmed blastocyst transfer cycles in a Chinese population. TE morphology may help predict outcomes of pregnancy in single-blastocyst transfer.

Influence of post-thaw culture duration on pregnancy outcomes in frozen blastocyst transfer cycles.

In this study, we aimed to evaluate whether post-thaw culture duration affected the clinical outcomes of frozen blastocyst transfer. This retrospective cohort study included 3,901 frozen-thawed blastocyst transfer cycles. The cohorts were divided into two groups based on the developmental stage (day 5 [D5] and day 6 [D6]) and culture duration after thawing (short culture, 2-6 h; long culture, 18-20 h). Women in the short culture group following D6 blastocyst transfer were further divided into three subgroups depending on the post-thaw culture period (2, 4, and 6 h). The main outcomes, namely live birth rate (LBR), implantation rate (IR), clinical pregnancy rate (CPR), and abortion rate (AR), showed no statistical differences within the groups following D5 blastocyst transfer. Patients in the long culture group had significantly lower IR (35.5 vs. 45.8%, p < 0.001), CPR (45.3 vs. 56.6%, p = 0.001), and LBR (35.5 vs. 48.5%, p < 0.001) but a significantly higher AR (21.6 vs. 14.3%, p = 0.049) following D6 blastocyst transfer than those in the short culture group. However, the data failed to present the superiority of any short culture duration over another on the live birth outcome for embryos vitrified on D6 (adjusted odds ratio [aOR]: 0.96, 95% confidence interval [95% CI]: 0.53-1.73, p = 0.881, for the 4-h vs. 2-h subgroup; aOR: 1.01, 95% CI: 0.68-1.49, p = 0.974, for the 6-h vs. 2-h subgroup). Both post-thaw protocols can be applied to patients with D5 blastocysts. To optimize the pregnancy outcomes following D6 blastocyst transfer, a short culture period is recommended. Any of the three short culture durations (2, 4, and 6 h) can be applied, depending on the workflow of the laboratory.

Functional Ovarian Cysts in Artificial Frozen-Thawed Embryo Transfer Cycles With Depot Gonadotropin-Releasing Hormone Agonist.

Objectives: To investigate the incidence of functional ovarian cysts, its influence on clinical rates, and proper management after depot gonadotropin-releasing hormone (GnRH) agonist pretreatment in artificial frozen-thawed embryo transfer cycles (AC-FET). Methods: This retrospective cohort study involved 3375 AC-FET cycles with follicular-phase depot GnRH agonist administration between January 2017 and December 2020. Subjects were divided into a study group (cycles with cyst formation) and a control group (cycles without cyst formation). The study group was matched by propensity scoring matching with the control group at a ratio of 1:2. For patients with ovarian cyst formation, two major managements were used: a conservative approach (i.e., expectant treatment) and a drug approach (i.e., continued agonist administration). The primary outcome was live birth rate (LBR). Results: The incidence of functional ovarian cysts following pituitary downregulation is 10.1% (341/3375). The study group exhibited a LBR similar to the control group (54.5% vs. 50.1%, adjusted odds ratio [aOR] 1.17, 95% confidence interval [CI] 0.88-1.56, P = 0.274). Patients with a lower body mass index and anti-Müllerian hormone, and a higher basal estradiol level were more susceptible to developing functional ovarian cysts. The LBR decreased after the drug approach compared with the conservative approach, but not significantly (aOR 0.63, 95% CI 0.35-1.14, P = 0.125). Following the conservative approach, cycles arrived at live births had a significantly shorter duration from the detection of functional cysts to the start of endometrium preparation (15.7 ± 5.1 days vs. 17.4 ± 5.3 days, P = 0.009) and a significantly higher proportion of ovarian cysts on the initial day of exogenous hormone supplementation (51.4% vs. 30.3%, P = 0.001). After controlling for all confounders, the differences remained statistically significant. Conclusions: It is unnecessary to cancel cycles that experience functional ovarian cyst formation. Conservative management and further agonist suppression protocol had similar pregnancy rates. However, a conservative approach was recommended due to its lower cost and fewer side effects. Our findings support a shorter waiting period when choosing the conservative protocol.

Effect of Embryo Developmental Stage, Morphological Grading, and Ploidy Status on Live Birth Rate in Frozen Cycles of Single Blastocyst Transfer.

To determine whether embryo developmental stage or morphological grading can predict live birth rate (LBR) from a single blastocyst in nonbiopsied and biopsied frozen embryo transfer (FET) cycles. This retrospective study included 1336 nonbiopsied and 360 euploid FET cycles. Blastocysts were divided according to developmental stage (day 5 [D5] and day 6 [D6]) and morphology (good quality and low quality). Nonbiopsied cycles in which D5 blastocysts were transferred were associated with a significantly higher LBR than those in the D6 group (48.5 vs. 24.3%; p < 0.001), as well as in good-quality embryo transfer cycles than that in low-quality embryo cycles (52.6 vs. 25.3%; p < 0.001). Embryos reaching good-quality blastocysts on D5 yielded significantly higher LBR than those similar quality blastocysts on D6. The same trend was seen in low-quality embryos. Concerning only D5 or D6 blastocyst transfer, the LBRs of good-quality embryos were still superior to those of low-quality embryos. In the case of euploid embryo transfers, the LBR (48.9 vs. 44.9%, p = 0.444) of D5 blastocysts did not significantly differ from that of D6 blastocysts. Good-quality embryos showed a higher LBR than low-quality embryos (51.6 vs. 40.0%, p = 0.030); the adjusted odds ratio remained insignificant after controlling for confounders (aOR 1.56; 95% CI 0.99-2.45; p = 0.056). The LBRs in the same developmental stage or morphology subgroups were not statistically significant. Embryo developmental stage and morphological grade are useful predictors of LBR in nonbiopsied FET cycles. However, no association was found in euploid transfer cycles.

Vitrified-warmed blastocyst transfer cycles yield higher pregnancy and implantation rates compared with fresh blastocyst transfer cycles--time for a new embryo transfer strategy?

OBJECTIVE: To compare the clinical outcome of fresh versus vitrified-warmed blastocyst transfer (BT) cycles. DESIGN: Retrospective study. SETTING: Medical university affiliated hospital. PATIENT(S): Women aged less than 40 years undergoing BT cycles. INTERVENTION(S): Vitrification and warming of blastocyst with the Cryotop system. MAIN OUTCOME MEASURE(S): Clinical pregnancy rate (CPR), implantation rate (IR), and multiple pregnancy rate (MPR). RESULT(S): In 110 fresh BT cycles versus 136 vitrified-warmed BT cycles performed from January 2007 to March 2010, the IR and CPR of vitrified-warmed BT cycles were 37.0% and 55.1%, respectively, which were statistically significantly higher than the corresponding values of 25.2% and 36.4% obtained for fresh BT cycles. Additionally, the MPR was not statistically significantly different between vitrified-warmed and fresh BT cycles when a similar number of blastocysts was transferred to patients. CONCLUSION(S): Vitrified-warmed BT cycles resulted in statistically significantly higher CPR and IR compared with fresh BT cycles. A new embryo transfer strategy is therefore proposed whereby fresh BT would be avoided in the initial ovarian stimulation cycle. Instead, all the patient's available blastocysts would be vitrified-warmed and transferred in subsequent cycles.