Background: To determine whether the embryo developmental stage affects biochemical or clinical pregnancy loss in young women undergoing frozen-thawed embryo transfer (FET) and to investigate the underlying mechanism.Methods: This was a retrospective study including a total of 18,34 β-HCG (human chorionic gonadotropin)-positive FET cycles. According to the morphological appearance (MA) of transferred blastocysts, FET cycles with blastocysts were divided into two groups: Group A: morphologically good (MG) blastocysts only, and Group B: at least one morphologically non-good (MNG) blastocyst. FET cycles with day 3 cleavage embryos were assigned as Group C. Biochemical and clinical pregnancy loss were the main outcome measures.Result(s): We predicted 78% in vivo-formed MG and 53.9% in vivo-formed day 5 blastocysts in Group C. (a) Including cases in Group A and Group B for binary logistic regression, we showed that Group B and day 6 blastocysts had significantly higher rates of BPL and CPL than Group A and day 5 blastocysts, respectively. (b) Including cases in Group A, Group B, and Group C for binary logistic regression, we showed that Group C had a significantly higher rate of BPL than Group A and day 5 blastocysts and a similar rate of BPL as Group B and day 6 blastocysts. Group C had a higher rate of CPL than Group A (p=0.071) and day 5 blastocysts (p=0.039), and a lower rate of CPL than Group B (p=0.199) and day 6 blastocysts (p=0.234).Conclusion(s): (1) MA and days of usable blastocysts could serve as independent factors affecting the occurrence of BPL and CPL. (2) Transfer of day 3 cleavage embryos may produce “unusable blastocysts” in vivo, which significantly increased the rate of BPL. (3) The rate of CPL resulting from the transfer of day 3 embryos may depend on the rate of in vivo-formed MG or day 5 blastocysts. Our study indicated that the difference in the BPL or CPL between transfer of blastocysts and day 3 cleavage embryos may largely depend on the quality of embryos transferred.

Previous studies have reported that senolytic drugs can reverse obesity-mediated accumulation of senescent cells in the ovary and protect against cisplatin-induced ovarian injury by removing senescent cells. Early intervention with ABT-263 has been shown to mitigate ovarian aging. However, it remains unknown whether treatment with ABT-263 could rejuvenate the aged ovary in reproductively old females. Therefore, the current study was aimed to investigate whether advanced age intervention with ABT-263 could ameliorate age-related decline in ovarian function. Fourteen 16-month-old mice with a C57/BL6 background were treated with ABT-263 (N = 7) or vehicle (N = 7) for two weeks. Mice were initially treated with ABT-263 (60 mg/kg/d) or vehicle for 7 consecutive days. After a 7-day break, the treatment was repeated for another 7 consecutive days. Six 2-month-old mice with C57BL/6 were used as a young control. The hormonal levels, estrus cycles, ovarian reserve, ovarian cell proliferation and apoptosis, ovarian fibrosis, and steroidogenic gene expression of ovarian stromal cells were evaluated. ABT-263 treatment did not rescue abnormal estrus cycles and sex hormonal levels, or inhibit the formation of multinucleated giant cells and ovarian stromal cell apoptosis in aged ovaries. However, it reduced ovarian fibrosis and preserved the steroidogenic gene expression of ovarian stromal cells in aged ovaries. Importantly, ABT-263 treatment further depleted ovarian follicles in aged mice. In conclusion, ABT-263 treatment accelerated the depletion of ovarian follicles in aged mice, suggesting that senolytic drugs for reproductively old female may adversely affect female fertility.

Background: Blastocyst transfer has been associated with shorter leukocyte telomere length in ART-conceived children, suggesting that extended embryo culture may accelerate aging in offspring. Selecting Day 3 embryos with high developmental potential for transfer could address this issue. The aim of this study is to investigate whether machine learning combined with Raman spectroscopy of spent Day 3 culture medium can serve as a potential method for predicting extended embryo culture outcomes, thereby enabling embryo selection at the cleavage stage.Methods: This prospective study analyzed 172 Day 3 culture medium samples with known extended culture outcomes from 78 couples collected between February 2020 and February 2021. Samples were categorized into three groups based on extended culture outcomes: morphologically good blastocysts (group A), morphologically non-good blastocysts (group B), and clinically non-useful embryos (group C). For each sample, 30–40 Raman spectra were acquired. Machine learning analyses (both unsupervised and supervised) were performed for data visualization and clustering. Eighty percent of the samples from each group were used as training data, while the remaining 20% served as the test set. Twelve machine learning models, including both deep learning and traditional approaches, were independently trained and evaluated. Accuracy, sensitivity, and specificity were calculated for each model. Finally, the best four top-performing models were further combined using a stacking strategy for final prediction.Results: The study included good-prognosis females (average age: 29.55 ± 2.94 years) with an adequate number of Day 3 embryos (median: 9 [7, 11]). Supervised machine learning of labeled Raman spectra revealed distinct clusters for each group. The best-performing models were multilayer perceptron, artificial neural network, gated recurrent unit, and linear discriminant analysis. Using the stacking strategy, two samples were misclassified, and 33 were correctly predicted. Sensitivity for A, B, and C predictions was 0.92, 1.00, and 0.94, respectively. Specificity for A, B, and C predictions was 1.00, 0.93, and 1.00, respectively. The overall accuracy, sensitivity, and specificity were 0.94, 0.93, and 0.97, respectively.Conclusion: Our preliminary study suggests that machine learning combined with Raman spectra of spent Day 3 culture medium represents a promising non-invasive approach for embryo selection at the cleavage stage.

Background: Recent studies have focused on investigating the role of cellular senescence in ovarian aging. Targeting cellular senescence has been proposed as a potential strategy to improve ovarian aging. p16 and p21 are classical molecules involved in mediating cellular senescence. In our previous study, we demonstrated that ablation of p16 is dispensable for premature ovarian aging induced by alkylating agents. In the present study, we investigated whether p21 deficiency could mitigate ovarian aging caused by alkylating agents.Methods: Eight-week-old wild-type (WT, n=7) and p21 knockout (KO, n=7) female mice received a single injection of busulfan (BUL, 30 mg/kg) and cyclophosphamide (CTX, 120 mg/kg) to induce premature ovarian insufficiency (POI). Untreated WT (n=4) and p21 KO (n=4) mice served as controls. Ovaries were analyzed thirteen weeks after treatment. Ovarian reserve, folliculogenesis, cell proliferation, apoptosis and senescence, multinucleated giant cells (MGCs) and their characteristics, pro-inflammatory factors, fibrosis, ovarian stromal cell properties, and the expression of cell cycle inhibitors, including p16, p19, p27, and p53, were evaluated.Results: Female mice treated with alkylating agents exhibited typical features of POI, including a dramatic reduction in the number of primordial and growing follicles; defective folliculogenesis characterized by growth arrest in early-stage follicles, extensive atresia in mid-stage follicles, dysregulated FSH receptor (FSHr) expression in antral follicles, and abnormal over-activation of primordial follicles; the presence of hemosiderin-laden MGCs and fibrosis in the ovarian cortical region. p21 deficiency did not significantly mitigate these phenotypes. There were no significantly differences in the expression of pro-inflammatory factors, folliculogenesis-regulating factors, or steroidogenesis-related factors and cell cycle inhibitors between WT and p21 KO mice treated with alkylating agents. In addition, p21 deficiency did not prevent alkylating agent-induced cellular senescence.Conclusion: These results demonstrated that p21 is dispensable for POI caused by alkylating agents, suggesting that targeting p21 alone may not mitigate ovarian aging caused by alkylating agents.

The aim of this study was to verify the effectiveness of the split insemination (IVF+ICSI) in patients with borderline semen for the first cycle, we furthermore compared the treatment outcome of the conventional IVF and ICSI on sibling oocytes with the rate of blastocyst formation in different days (day 5 or day 6) as primary outcome and pregnancy as secondary outcome to provide theoretical support for embryo selection. Between January 2017 and November 2021,190 couples undergoing the split insemination (IVF+ICSI) cycle were enrolled in the study with at least eight oocytes and the borderline semen in first cycle to analyze the basic characteristics and clinical outcome. The remaining 157 patients were analyzed in this study to compare the IVF and ICSI after excluding those who were completely unfertilized by IVF (n=33) including a patient who was completely unfertilized by IVF and ICSI. Present study showed that about 32(32/190,16.8%) patients with borderline semen in first cycle were completely unfertilized performing the conventional IVF and only 1(1/190,0.53%) patient was completely unfertilized using the IVF and ICSI in the split insemination (IVF+ICSI), the rate of total fertilization failure (TFF) was significantly decrease by the ICSI treatment (16.8%&0.53%,P<0.0001). By the split oocytes, the fertilization rate was significantly superior in ICSI(729/982,74.2%) compared to IVF (486/940, 51.7%, P<0.0001), the usable blastocyst and high-quality blastocyst rate on the fifth day were statistically superior in IVF compared to ICSI(31.3% &22.8%,P=0.009) (27.3%&20.6%,P=0.03), The pregnancy rate, implantation rate and live birth rate in the IVF first cycle were higher than the ICSI(75.9%&64%, respectively) (52.3%&41.8%, respectively)(64.8%&54.7%, respectively),although there was no statistical difference,it is also about ten percentage points difference. The treatment of sibling oocyte with both IVF and ICSI could be an appropriate choice to prevent TFF and preserve the embryo development potential for the patients with the borderline semen in present study. Furthermore, the embryo development potential from conventional IVF was better than the embryo from the ICSI technology, the ICSI technology may have a negative effect on the embryo development.

Background: This study aimed to compare the effects of different gamete co-incubation times on fertilization, embryo development potential, along with clinical outcomes. Methods: The study included 530 cycles with short co-incubation times (4–6 hours), referred to as the S group, and 1653 cycles with long co-incubation times (16–24 hours), referred to as the L group, all undergoing in vitro fertilization (IVF) without rescue intracytoplasmic sperm injection (R-ICSI). The study analyzed the basic characteristics and clinical outcomes of these two groups. Additionally, the study stratified the cycles based on the number of oocytes retrieved and performed multivariate logistic regression analysis with the normal fertilization (two pronuclei, 2PN) rate as the dependent variable and co-incubation time as the main independent variable. The study also examined 79 partial short co-incubation cycles, dividing them into a short co-incubation part (10 oocytes) and a long co-incubation part (the remaining oocytes), to analyze embryo developmental parameters. Results: There were no significant differences in the rates of top-quality blastocyst formation, clinical pregnancy, and implantation between the two groups (p > 0.05). 2PN rate in S group was slightly lower than in L group (68.06% vs. 71.08%, p < 0.01). After stratifying by the number of oocytes obtained, multiple logistic regression analysis demonstrated no significant correlation between co-incubation time and 2PN rate (p > 0.05). In the 79 partial short co incubation cycles, there were no significant differences in the rates of 2PN oocytes, ≥3PN (3 pronuclei) oocytes, top-quality cleavage embryos, and top-quality blastocysts on Day 5/Day 6 between the two parts (p > 0.05). Conclusions: Short oocyte-sperm co-incubation is an effective strategy in preventing fertilization failure. Short and long oocyte-sperm co-incubation times have similar outcomes on in vitro development.