Application of amphiregulin in IVM culture of immature human oocytes and pre-insemination culture for COCs in IVF cycles.

Background: Amphiregulin (AR) is a growth factor that resembles the epidermal growth factor (EGF) and serves various functions in different cells. However, no systematic studies or reports on the role of AR in human oocytes have currently been performed or reported. This study aimed to explore the role of AR in human immature oocytes during in vitro maturation (IVM) and in vitro fertilization (IVF) in achieving better embryonic development and to provide a basis for the development of a pre-insemination culture medium specific for cumulus oocyte complexes (COCs). Methods: First, we examined the concentration of AR in the follicular fluid (FF) of patients who underwent routine IVF and explored the correlation between AR levels and oocyte maturation and subsequent embryonic development. Second, AR was added to the IVM medium to culture immature oocytes and investigate whether AR could improve the effects of IVM. Finally, we pioneered the use of a fertilization medium supplemented with AR for the pre-insemination culture of COCs to explore whether the involvement of AR can promote the maturation and fertilization of IVF oocytes, as well as subsequent embryonic development. Results: A total of 609 FF samples were examined, and a positive correlation between AR levels and blastocyst formation was observed. In our IVM study, the development potential and IVM rate of immature oocytes, as well as the fertilization rate of IVM oocytes in the AR-added groups, were ameliorated significantly compared to the control group (All P < 0.05). Only the IVM-50 group had a significantly higher blastocyst formation rate than the control group (P < 0.05). In the final IVF study, the maturation, fertilization, high-quality embryo, blastocyst formation, and high-quality blastocyst rates of the AR-added group were significantly higher than those of the control group (All P < 0.05). Conclusion: AR levels in the FF positively correlated with blastocyst formation, and AR involvement in pre-insemination cultures of COCs can effectively improve laboratory outcomes in IVF. Furthermore, AR can directly promote the in vitro maturation and developmental potential of human immature oocytes at an optimal concentration of 50 ng/ml.

[Comparative Study of the Embryo Development and Clinical Outcomes of 3 Ovarian Stimulation Protocols in Different Age Groups]. / 不同年龄段人群三种促排卵方案胚胎发育及临床结局比较.

Objective: The main purpose of this study is to compare the embryo development and clinical outcomes of women in different age groups undergoing in vitro fertilization (IVF) processes using gonadotrophin-releasing hormone (GnRH) antagonist protocol, GnRH agonist long protocol, and early follicular phase protocol. We aim to provide reliable reference for future clinical treatments. Methods: We conducted a detailed analysis of patients who underwent treatment between January 2021 and February 2023. 1) In the overall patient population, we comprehensively compared the basic characteristics, the embryo development, and the clinical outcomes of patients treated with three different ovarian stimulation protocols, including the GnRH antagonist protocol group (n=4173), the agonist long protocol group (n=2410), and the early follicular phase long protocol group (n=341). 2) We divided the overall population into three age groups, one group for patients under 30 years old (n=2576), one for patients aged 30-35 (n=3249), and one for patients older than 35 years old (n=1099). Then, we compared the three stimulation protocols based on the group division. We separately compared the embryo development and clinical outcomes of patients using the three stimulation protocols in the under 30 years old, the 30-35 years old, and the over 35 years old age groups. With this analysis, we aimed to explore the response of different age groups to different stimulation protocols and their impact on the success rate of IVF. Results: 1) In the overall population, we found that the average number of oocytes retrieved in the GnRH agonist long protocol group was significantly higher than that in the GnRH antagonist protocol group ([13.85±7.162] vs. [13.36±7.862], P=0.0224), as well as the early follicular phase long protocol group ([13.85±7.162] vs. [11.86±6.802], P<0.0001). Patients in the GnRH antagonist protocol group not only had a significantly lower starting dose of gonadotrophin (Gn) compared to the other two groups (P<0.05) but also had a significantly lower number of days of Gn use (P<0.05). The blastocyst formation rate in the GnRH antagonist protocol group was the highest among the three groups, significantly higher compared to the GnRH agonist long protocol group (64.91% vs. 62.35%, P<0.0001) and the early follicular phase long protocol group (64.91% vs. 61.18%, P=0.0001). However, there were no significant differences in the clinical pregnancy rates or the live birth rates among the three groups treated with different ovarian stimulation protocols (P>0.05). 2) In the <30 age group, the blastocyst formation rate in the GnRH antagonist protocol group was the highest among the three groups, significantly higher compared to the GnRH agonist long protocol group (66.12% vs. 63.33%, P<0.0001) and the early follicular phase long protocol group (66.12% vs. 62.13%, P=0.0094). In the 30-35 age group, the blastocyst formation rate in the GnRH antagonist protocol group was the highest among the three groups, significantly higher compared to the GnRH agonist long protocol group (64.88% vs. 62.93%, P=0.000 9) and the early follicular phase long protocol group (64.88% vs. 60.39%, P=0.0011). In the >35 age group, the blastocyst formation rate in the GnRH antagonist protocol group was significantly higher than that in the GnRH agonist long protocol group (59.83% vs. 56.51%, P=0.0093), while there was no significant difference compared to that of the early follicular phase long protocol group (P>0.05). In the three age groups, we found that there were no significant differences in clinical pregnancy rate, live birth rate, and neonatal outcome indicators (fetal weight and Apgar score) among the three stimulation protocols (antagonist protocol, GnRH agonist long protocol, and early follicular phase long protocol) (P>0.05). The findings showed no significant differences between clinical and neonatal outcomes in patients of all ages, regardless of the ovarian stimulation protocol, suggesting that the three ovarian stimulation protocols have similar therapeutic effects in patients of different ages. The results of this study have important implications for the selection of an appropriate ovarian stimulation protocol and the prediction of treatment outcomes. Conclusion: In the younger than 30 and 30-35 age groups, the GnRH antagonist protocol showed a more significant advantage over the GnRH agonist long protocol and the early follicular phase long protocol. This suggests that for younger and middle-aged patients, the antagonist protocol may lead to better outcomes during ovarian stimulation. In the older than 35 age group, while the antagonist protocol still outperformed the GnRH agonist long protocol, there was no significant difference compared to the early follicular phase long protocol. This may imply that with increasing age, the early follicular phase long protocol may have effects similar to the antagonist protocol to some extent. The advantages of the antagonist protocol lie in its ability to reduce stimulation duration and the dosage of GnRH, while enhancing patient compliance with treatment. This means that patients may find it easier to accept and adhere to this treatment protocol, thereby improving treatment success rates. Particularly for older patients, the use of the antagonist protocol may significantly increase the blastocyst formation rate, which is crucial for improving the success rates. Although there were no significant differences in the clinical outcomes of patients treated with the three protocols in each age group, further research is still needed to validate these findings. Future multicenter studies and increased sample sizes may help comprehensively assess the efficacy of different stimulation protocols. Additionally, prospective studies are needed to further validate these findings and determine the optimal treatment strategies.

Melatonin application during cryopreservation improves the development and clinical outcomes of human vitrified-warmed oocytes.

In this clinical study, we investigated the potential of melatonin (MT) supplementation in the freeze-thaw medium used for cryopreserved human oocytes. In total, 152 patients who underwent in vitro fertilization between January 2020 and December 2022 were included and categorized into different groups as follows: the donor group, comprising 108 patients who donated their oocytes, with 34 patients using a vitrification and warming medium supplemented with MT (D-MT subgroup) and 74 patients using conventional medium without MT (D-0 subgroup); and the autologous group, comprising 38 patients who used their own oocytes, with 19 patients using medium supplemented with MT (A-MT subgroup) and 19 patients using medium without MT (A-0 subgroup). After thawing, the surviving oocytes in the D-MT and A-MT subgroups and D-0 and A-0 subgroups were cultured in a fertilization media with and without 10-9 MMT for 2.5 h, respectively, followed by intracytoplasmic sperm injection insemination, embryo culture, and transfer. The survival, cleavage, high-quality embryo, clinical pregnancy, ongoing pregnancy, and implantation rates were significantly higher in the D-MT subgroup than in the D-0 subgroup (all P < 0.05). Similarly, the survival, fertilization, high-quality embryo, and high-quality blastocyst rates were significantly higher in the A-MT subgroup than in the A-0 subgroup (all P < 0.05). These findings indicate that MT addition during cryopreservation can enhance the development of vitrified-warmed human oocytes and improve clinical outcomes.

Melatonin enhances the developmental potential of immature oocytes from older reproductive-aged women by improving mitochondrial function.

Aims: To evaluate whether melatonin (MT) supplementation during in vitro maturation (IVM) of human oocytes can reverse the age-related decline in oocyte quality. Main methods: We enrolled 172 patients aged ≥35 years (older reproductive-aged women) and 83 patients aged <35 years (young women) who underwent in vitro fertilization between 2019 and 2022. We conducted IVM with and without 10 µM MT in immature oocytes of different ages. Oocyte fertilization and embryo development were observed using a stereomicroscope. We assessed the immunofluorescence intensity of mitochondrial function, measured the copy number of mitochondrial DNA (mtDNA), and examined the spindle and chromosome composition in in vitro mature stage II (IVM-MII) oocytes using immunofluorescence and second-generation sequencing. Key findings: MT supplementation significantly improved the redox level in the IVM medium and IVM-MII oocytes in older reproductive-aged women. It also significantly increased the proportion of circular mtDNA and the adenosine triphosphate content in IVM-MII oocytes. In addition, the IVM-MII oocytes obtained with MT supplementation showed a significant improvement in the normal composition of the spindle and chromosomes. Thus, the aged immature oocytes also showed significantly improved maturation and blastocyst formation rates owing to the role of MT. Significance: Supplementation with 10 µM MT in the IVM medium reverses the age-related decline in oocyte quality. Our findings provide a viable solution for enhancing fertility in older reproductive-aged women.

Blastocyst Transplantation Strategies in Women of Different Ages.

(1) Background: Single blastocyst transfers (SBT) and double blastocyst transfers (DBT) are widely used in clinical practice. The objective of this study was to investigate the application of these two strategies in women of different ages. (2) Methods: Analysis was carried out on 5477 frozen embryo transfer cycles of women in different ages. All the cycles were divided into three groups according to the age of the included women: <35, 35-39, and >39. The live birth rate (LBR) and multiple birth rate (MBR) between the SBT and DBT among these groups, respectively, were compared. (3) Results: In the women < 35 group, the LBR was similar in SBT and DBT, but the MBR was higher in DBT than SBT. In women 35-39, with >10 oocytes retrieved, the MBR in DBT was significantly higher than SBT, but there was no significant difference in LBR between the two groups; with ≤10 oocytes retrieved, the LBR in DBT were significantly higher than SBT, but the MBT was not significantly different between the two groups. In women > 39, the LBR and MBR were lower in the SBT than DBT, but the differences were not statistically significant. (4) Conclusions: Selective SET is appropriate for most young women, whereas older women are advised to make individualized choices based on the number of oocytes retrieved and blastocyst quality.

Comparative transcriptomics analysis reveals a calcineurin B-like gene to positively regulate constitutive and acclimated freezing tolerance in potato.

Freezing stress is a major limiting factor in crop production. To increase frost-hardiness of crops via breeding, deciphering the genes conferring freezing-tolerance is vital. Potato cultivars (Solanum tuberosum) are generally freezing-sensitive, but some potato wild species are freezing-tolerant, including Solanum commersonii, Solanum malmeanum and Solanum acaule. However, the underlying molecular mechanisms conferring the freezing-tolerance to the wild species remain to be deciphered. In this study, five representative genotypes of the above-mentioned species with distinct freezing-tolerance were investigated. Comparative transcriptomics analysis showed that SaCBL1-like (calcineurin B-like protein) was upregulated substantially in all of the freezing-tolerant genotypes. Transgenic overexpression and known-down lines of SaCBL1-like were examined. SaCBL1-like was shown to confer freezing-tolerance without significantly impacting main agricultural traits. A functional mechanism analysis showed that SaCBL1-like increases the expression of the C-repeat binding factor-regulon as well as causes a prolonged higher expression of CBF1 after exposure to cold conditions. Furthermore, SaCBL1-like was found to only interact with SaCIPK3-1 (CBL-interacting protein kinase) among all apparent cold-responsive SaCIPKs. Our study identifies SaCBL1-like to play a vital role in conferring freezing tolerance in potato, which may provide a basis for a targeted potato breeding for frost-hardiness.

Salicylic Acid Improves the Constitutive Freezing Tolerance of Potato as Revealed by Transcriptomics and Metabolomics Analyses.

Freezing severely impacts potato production. Deciphering the pathways and metabolites that regulate the freezing tolerance of potato is useful in cultivation and breeding for hardiness. In the present study, Solanum acaule was identified to be more freezing tolerant than S. tuberosum. Furthermore, the two genotypes before/after exposure to 4 °C for 7 d with additional -1 °C for 12 h were analysed by RNA-seq and metabolomics, and the results were compared with the previous -1 °C for 12 h. The results showed that S. acaule activated numerous genes that differed from those of S. tuberosum. Among the genes, five pathways, such as the hormone signalling pathway, which includes salicylic acid, were enriched. Further metabolomics analysis showed that the content of salicylic acid was improved in S. acaule in response to -1 °C for 12 h. Moreover, exogenous application of 0.1 mM salicylic acid to potato was shown to improve constitutive freezing tolerance and increase the expression of HSFC1. Following transcriptome and metabolome analyses, it was documented that the content of SA that increased in freezing-tolerant S. acaule after exposure to cold condition, associated with the SA signalling pathway, enhanced potato freezing tolerance, probably through HSFC1.