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: Chemical agents such as alkylating agents (AAs) that are commonly used for the treatment of cancer cause great damage to the ovaries, thereby significantly increasing the risk of premature ovarian insufficiency (POI). However, the exact molecules underlying AA-induced POI remain largely obscure. Upregulation of the p16 gene may contribute to the progression of POI. As yet, no in vivo data from p16-deficient (KO) mice are available to demonstrate a critical role of p16 in POI. In the present study, we employed p16 KO mice to investigate whether loss of p16 could protect against POI caused by AAs. Methods: WT mice and their p16 KO littermates received a single dose of BUL + CTX to establish an AA-induced POI mouse model. One month later, oestrous cycles were monitored. Three months later, some of the mice were sacrificed to collect sera for measurements of hormone levels and ovaries for measurements of follicle counts, the proliferation and apoptosis of granulosa cells, ovarian stromal fibrosis and vessels. The remaining mice were mated with fertile males for the fertility test. Results: Our results showed that treatment with BUL + CTX significantly disrupted the oestrous cycles, increased the levels of FSH and LH while decreasing the levels of E2 and AMH, decreased the counts of primordial follicles and growing follicles while increasing the counts of atretic follicles, reduced the vascularized area in the ovarian stroma, and decreased fertility. All of these results were comparable between WT and p16 KO mice treated with BUL + CTX. In addition, ovarian fibrosis was not increased significantly in WT and p16 KO mice treated with BUL + CTX. Growing follicles with normal appearance had normally proliferating granulosa cells (without apparent apoptosis). Conclusion: We concluded that genetic ablation of the p16 gene did not attenuate ovarian damage or help preserve the fertility of mice challenged by AAs. This study demonstrated for the first time that p16 is dispensable for AA-induced POI. Our preliminary findings suggest that targeting p16 alone may not preserve the ovarian reserve and fertility of females treated with AAs.

Background: Intrauterine infection is a major cause of preterm birth, fetal demise, and growth restriction. Placental damage resulting from such infections plays a central role in mediating these adverse outcomes. Pyrroloquinoline quinone (PQQ) is a naturally occurring nutrient known for its antioxidant, anti-inflammatory, and mitochondrial-supporting properties. This study aimed to investigate whether pre-conditioning with PQQ during pregnancy could mitigate adverse effects induced by lipopolysaccharide (LPS)-mediated inflammation in mice.Methods: Pregnant mice were randomly assigned to three groups: control, LPS, and LPS + PQQ. On gestational day (GD) 16.5, mice in the LPS groups were intraperitoneally injected with either a single dose of 3 μg/mouse (moderate inflammation) or two doses of 3ug/mouse (severe inflammation) of LPS. In the LPS + PQQ group, PQQ was administered daily from GD 0.5. Outcomes assessed included labor time, fetal survival, fetal and placental weights. Placental structure, vascular networks, inflammation, oxidative stress, and gene expression profiles were evaluated using H&E staining, immunohistochemistry, Prussian blue staining, and RNA sequencing.Results: Pre-conditioning with PQQ significantly alleviated LPS-induced fetal demise and reduced fetal and placental growth. PQQ also improved placental morphology, restored vascular integrity, and normalized aberrant gene expression profiles. Furthermore, PQQ treatment markedly reduced placental inflammation and oxidative stress in mice exposed to moderate LPS. However, under high-dose LPS conditions, PQQ failed to confer significant protective effects.Conclusion: Our findings suggest that Pre-conditioning with PQQ during pregnancy can protect against inflammation-induced placental damage and improve fetal survival and growth under moderate inflammatory conditions. This study provides compelling proof-of-concept that PQQ buffers the placenta against maternal systemic inflammatory insults. However, its efficacy appears limited in the context of severe inflammation.

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: To investigate whether double embryo transfer (DET) increases the risk of spontaneous clinical pregnancy loss (CPL) in singleton pregnancies following frozen-thawed embryo transfer (FET), compared to single embryo transfer (SET).Methods: This retrospective cohort study included 2,448 females with singleton pregnancies (excluding vanishing twin cases) resulting from frozen-thawed single or double embryo transfers between January 2017 and September 2022. The CPL rate was the sole outcome measure. We compared CPL rates between SET and DET across three populations with increasing embryo developmental potential using binary logistic regression analysis: P1, comprising transfers of Day 3 cleavage-stage embryos; P2, comprising transfers of blastocysts; and P3, comprising transfers of top-quality blastocysts, defined as morphologically good Day 5 blastocysts.Results: After adjusting for confounding factors, the comparison between SET and DET revealed the following findings: in P1, DET had a slightly higher CPL rate compared to SET [OR (95% CI): 1.18 (0.74-1.90), p=0.46]; In P2, DET showed a moderately higher CPL rate [OR (95% CI): 1.34 (0.96-1.87), p=0.08]; In P3, DET had a significantly higher CPL rate [OR (95% CI): 1.55 (1.02-2.37), p=0.04]. A combined analysis indicated that as the developmental potential of the transferred embryo increased (from P1 to P2 and further to P3), the impact of DET on CPL also increased, as reflected by the rising OR values and decreasing p-values. We proposed that in singleton pregnancies resulting from DET, the loss of a non-viable embryo at a later stage, when it has a larger cell mass, may trigger excessive intrauterine inflammation, thereby increasing the risk of CPL for the remaining full developmental potential embryo. In singleton pregnancies resulting from DET, a higher-quality embryo that fails is more likely to die at a later stage. This could explain why the impact of DET on CPL increases with the developmental potential of the embryo.Conclusion: Since a significant difference in CPL between SET and DET was only observed in P3 population. Therefore, we concluded that compared to SET, the transfer of double morphologically good Day 5 blastocysts is associated with increased clinical pregnancy loss in singleton pregnancies following FET.

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.