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.

Voltage dependence of M2 muscarinic receptor antagonists and allosteric modulators.

Muscarinic receptors are G protein-coupled receptors (GPCRs) that play a role in various physiological functions. Previous studies have shown that these receptors, along with other GPCRs, are voltage-sensitive; both their affinity toward agonists and their activation are regulated by membrane potential. To our knowledge, whether the effect of antagonists on these receptors is voltage-dependent has not yet been studied. In this study, we used Xenopus oocytes expressing the M2 muscarinic receptor (M2R) to investigate this question. Our results indicate that the potencies of two M2R antagonists, atropine and scopolamine, are voltage-dependent; they are more effective at resting potential than under depolarization. In contrast, the M2R antagonist AF-DX 386 did not exhibit voltage-dependent potency.Furthermore, we discovered that the voltage dependence of M2R activation by acetylcholine remains unchanged in the presence of two allosteric modulators, the negative modulator gallamine and the positive modulator LY2119620. These findings enhance our understanding of GPCRs' voltage dependence and may have pharmacological implications.

Factors affecting age at menopause and their relationship with ovarian reserve: a comprehensive review.

OBJECTIVE: The aim of this article was to discuss all the factors affecting the age at menopause and their correlation with ovarian reserve. MATERIALS AND METHODS: A narrative review of original articles was performed using PubMed until December 2023. The following keywords were used to generate the list of citations: 'menopause', 'ovarian reserve' 'oocytes quality and quantity', 'ovarian ageing'. RESULTS: Menopause is the final step in the process of ovarian ageing and is influenced by the oocyte pool at birth. Conditions that accelerate follicle depletion during the reproductive lifespan lead to premature ovarian insufficiency (POI) and premature ovarian failure (POF), while a higher ovarian reserve is associated with a delayed time to menopause. Reproductive history, sociodemographic, lifestyle and iatrogenic factors may impact ovarian reserve and the age at menopause. CONCLUSIONS: Some factors affecting the age at menopause are modifiable and the risks of early menopause may be preventable. We hypothesise that by addressing these modifiable factors we may also preserve ovarian reserve. However, further interventional studies are needed to evaluate the effects of the described strategies on ovarian reserve.

The age of menopause is determined by the process of follicle depletion, which leads to a decrease in the quantity and quality of oocytes. Various factors such as demographics, menstrual patterns, reproductive history, family history, genetics, and lifestyle choices appear to influence the age at which natural menopause occurs. Some of these factors can be modified. Considering the relationship between ovarian reserve and the age of natural menopause, is it possible to intervene on these modifiable factors to preserve ovarian reserve?

Pretreatment with luteal estradiol for programming antagonist cycles compared to no pretreatment in advanced age women stimulated with corifollitropin alfa: a non-inferiority randomized controlled trial.

STUDY QUESTION: Does luteal estradiol (E2) pretreatment give a similar number of retrieved oocytes compared to no-pretreatment in advanced-aged women stimulated with corifollitropin alfa in an antagonist protocol? SUMMARY ANSWER: Programming antagonist cycles with luteal E2 gave similar number of retrieved oocytes compared to no-pretreatment in women aged 38-42 years. WHAT IS KNOWN ALREADY: Programming antagonist cycles with luteal E2 pretreatment is a valuable tool to organize the IVF procedure better and is safe without any known impact on cycle outcome. However, variable effects were observed on the number of retrieved oocytes depending on the treated population. In advanced-age women, recruitable follicles tend to decrease in number and to be more heterogeneous in size but it remains unclear if estradiol pretreatment could change the oocyte yield through its negative feed-back effect on FSH intercycle rise. STUDY DESIGN, SIZE, DURATION: This non-blinded randomized controlled non-inferiority trial was conducted between 2016 and 2022 with centrally computerized randomization and concealed allocation. Participants were 324 women aged 38-42 years undergoing IVF treatment. The primary endpoint was the total number of retrieved oocytes. Statistical analysis was performed with one-sided alpha risk of 2.5% and 95% confidence interval (CI) with the non-inferiority of E2 pretreatment proved by a P value <0.025 and a lower delta margin of the CI within two oocytes compared to no pretreatment. Secondary endpoints were duration and total dosage of recombinant FSH, cancellation rate, percentage of oocyte pick-up (OPU) on working days, total number of metaphase II oocytes and obtained embryos, fresh transfer live birth rate, and cumulative live birth rate. PARTICIPANTS/MATERIALS, SETTING, METHODS: This multicentric study enrolled women with regular cycles, weight >50 kg and body mass index <32, IVF cycle 1-2. According to randomization, micronized estradiol 2 mg twice a day was started on days 20-24 and continued until Wednesday beyond the onset of menses followed by administration of corifollitropin alfa on Friday, i.e. stimulation (S)1 or from D1-3 of a natural cycle in unpretreated patients. GnRH antagonist was started at S6 and additional FSH at S8. MAIN RESULTS AND THE ROLE OF CHANCE: Basal characteristics were similar in patients randomized in E2 pretreated (n = 164) and non-pretreated (n = 160) groups (intended to treat (ITT) population). A total of 291 patients started treatment (per protocol (PP) population), 147 in E2 pretreated group with a mean number [SD] of pre-treatment days 9.8 [2.6] and 144 in the non-pretreated group. Despite advanced age, oocyte yields ranged from 0 to 29 in both groups with a median number of 6 retrieved oocytes in accordance with a mean anti-Müllerian hormone (AMH) level above 1.2 ng/ml. We demonstrated the non-inferiority of E2 pretreatment with a mean difference of -0.1 oocyte 95% CI [-1.5; 1.3] P = 0.004 in the PP population and a mean difference of -0.44 oocyte [-1.84; 0.97] P = 0.014 in the ITT population. Oocyte retrieval was more often on working days in E2 pretreated patients (91.9 versus 74.2%, P < 0.001). In patients reaching OPU, the duration of stimulation was statistically significantly longer (11.7 [1.7] versus 10.8 [1.8] days, P < 0.001) and the extra FSH dosage in addition to corifollitropin alfa was statistically significantly higher (1040 [548] versus 778 [504] IU, P < 0.001) in E2 pretreated than non-pretreated patients. We did not observe any significant differences in the number of retrieved oocytes (8.4 [6.1] versus 9.1 [6.0]), in the number of Metaphase 2 oocytes (7 [5.5] versus 7.3 [5.2]) nor in the number of obtained embryos (5 [4.6] versus 5.2 [4.2]) in E2 pretreated patients compared to non-pretreated patients. The live birth rate after fresh transfer (16.2% versus 18.5%, respectively), and the cumulative live birth rate per patient (17.7% versus 22.9%, respectively) were similar in both groups. Among the PP population, 31.6% of patients fulfilled the criteria for group 4 of Poseïdon classification (AMH <1.2 ng/ml and/or antral follicle count <5). In this sub-group of patients, we observed in contrast a statistically higher number of retrieved oocytes in E2 pretreated patients compared to non-pretreated (5.1 [3.8] versus 3.4 [2.7], respectively, the mean difference of +1.7 oocyte [0.2; 3.2] P = 0.022) but without significant difference in the cumulative live birth rate per patient (15.7% versus 7.3%, respectively). LIMITATIONS, REASONS FOR CAUTION: Our stimulated women older than 38 years obtained a wide range of collected oocytes suggesting very different stages of ovarian aging in both groups. E2 pretreatment is more likely to increase oocyte yield at the stage of ovarian aging characterized by asynchrony of a reduced follicular cohort. Another limitation is the sample size in sub-group analysis of patients with AMH <1.2 ng/ml. Finally, the absence of placebo for pretreatment could also introduce possible bias. WIDER IMPLICATIONS OF THE FINDINGS: Programming antagonist cycles with luteal E2 pretreatment seems a useful tool in advanced age women to better schedule oocyte retrievals on working days. However, the potential benefit of the number of collected oocytes remains to be demonstrated in a larger population displaying the characteristics of decreased ovarian reserve encountered in Poseïdon classification. STUDY FUNDING/COMPETING INTEREST(S): Research grant from (MSD) Organon, France. I.C., S.D., B.B., X.M., S.G., and C.J. have no conflict of interest with this study. I.C.D. declares fees as speaker from Merck KGaA, Gedeon Richter, MSD (Organon, France), Ferring, Theramex, and IBSA and participation on advisory board from Merck KGaA. I.C.D. also declares consulting fees, and travel and meeting support from Merck KGaA. N.M. declares grants paid to their institution from MSD (Organon, France); consulting fees from MSD (Organon, France), Ferring, and Merck KGaA; honoraria from Merck KGaA, General Electrics, Genevrier (IBSA Pharma), and Theramex; support for travel and meetings from Theramex, Merck KGaG, and Gedeon Richter; and equipment paid to their institution from Goodlife Pharma. N.C. declares grants from IBSA Pharma, Merck KGaA, Ferring, and Gedeon Richter; support for travel and meetings from IBSA Pharma, Merck KGaG, MSD (Organon, France), Gedeon Richter, and Theramex; and participation on advisory board from Merck KGaA. A.G.L. declares fees as speaker from Merck KGaA, Gedeon Richter, MSD (Organon, France), Ferring, Theramex, and IBSA. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov NCT02884245. TRIAL REGISTRATION DATE: 29 August 2016. DATE OF FIRST PATIENT'S ENROLMENT: 4 November 2016.

Impaired embryo development potential associated with thyroid autoimmunity in euthyroid infertile women with diminished ovarian reserve.

Purpose: The aim of this study was to evaluate the associations of thyroid autoimmunity (TAI) with the number of oocytes retrieved (NOR), fertilization rate (FR), and embryo quality (EQ) in euthyroid women with infertility and diminished ovarian reserve (DOR). Methods: This retrospective cohort study involved 1,172 euthyroid women aged 20-40 years with infertility and DOR who underwent an oocyte retrieval cycle. TAI was diagnosed in the presence of serum thyroperoxidase antibody (TPOAb) concentrations higher than 34 IU/ml and/or serum thyroglobulin antibody (TgAb) concentrations exceeding 115.0 IU/ml. Among these women, 147 patients with TAI were classified as the TAI-positive group, while 1,025 patients without TAI were classified as the TAI-negative group. Using generalized linear models (GLMs) adjusted for confounding factors, we evaluated the associations of TAI and the serum TPOAb and TgAb concentrations and NOR, FR, and EQ in this study's subjects. The TPOAb and TGAb values were subjected to log10 transformation to reduce skewness. Logistic regression models were used to estimate the effects of TPOAb and TgAb concentrations on the probabilities of achieving a high NOR (≥7) and high FR (>60%). Results: For the whole study population, women with TAI had a significantly lower NOR and poorer EQ than women without TAI (P < 0.001 for both). Interestingly, in the TSH ≤2.5 subgroup, the TAI-positive group also had a significantly lower NOR and poorer EQ than the TAI-negative group (P < 0.001 for both). Furthermore, negative associations were observed between log10(TPOAb) concentrations and NOR and the number of high-quality embryos and available embryos (P < 0.05 for all). The log10(TgAb) concentrations were inversely associated with NOR and the number of high-quality embryos (P < 0.05 for all). In the regression analysis, the log10(TPOAb) concentrations had lower probabilities of achieving a high NOR [adjusted odds ratio (aOR): 0.56; 95% confidence interval (95% CI) 0.37, 0.85; P = 0.007]. Conclusions: TAI and higher TPOAb and TgAb concentrations were shown to be associated with reductions in the NOR and EQ in the study population. Our findings provide further evidence to support systematic screening and treatment for TAI in euthyroid women with infertility and DOR.

Docosahexaenoic acid supplementation during porcine oocyte in vitro maturation improves oocyte quality and embryonic development by enhancing the homeostasis of energy metabolism.

Although supplementation with docosahexaenoic acid (DHA) during porcine oocyte IVM is well-established, the available data are limited due to the lack of consistency. Moreover, to our knowledge, the anti-oxidant effects of DHA on porcine oocytes have not been reported. Hence, this study aimed to examine the effects of DHA supplementation on the regulation of energy metabolism during porcine oocyte maturation to improve oocyte maturation and embryonic development. By supplementing the IVM medium with various DHA concentrations, 25 µM DHA was identified as the optimal concentration which improved intraoocyte glutathione content and enhanced embryonic development after parthenogenesis. Compared to embryos derived from the control group, those derived from SCNT or IVF showed significantly improved blastocyst formation upon DHA supplementation during IVM. In addition, various transcription factors associated with oocyte development and apoptosis in mature oocytes were beneficially regulated in the DHA-treated oocytes. Moreover, DHA improved the AMP-activated protein kinase (AMPK)-regulatory ability of porcine oocytes and ameliorated nuclear maturation and embryonic development, which were decreased by artificially downregulating AMPK. To our knowledge, this is the first study to examine the effects of DHA as an AMPK regulator on oocyte maturation and embryo development in pigs. Furthermore, DHA addition to the IVM medium upregulated the relative expression of genes associated with mitochondrial potential and lipid metabolism. Therefore, the membrane potential of mitochondria (evaluated based on the JC-1 aggregate/JC-1 monomer ratio) and the levels of fatty acids and lipid droplets in matured oocytes increased, resulting in increased ATP synthesis. In conclusion, the DHA treatment of porcine oocytes with 25 µM DHA during IVM enhances the homeostasis of energy metabolism by improving mitochondrial function and lipid metabolism, leading to improved quality of matured oocytes and enhanced embryonic developmental potential of in vitro produced (IVP) embryos. Thus, 25 µM DHA supplementation could serve as a tool for improving the quality of IVP embryos. The study findings provide a basis for further research on improving the production efficiency of cloned animals by securing high-quality matured oocytes and enhancing energy metabolism in mammalian oocytes, including those of pigs.

Effect of single versus grouped culture of human cumulus-oocyte complexes in PCOS women treated with biphasic in vitro maturation: A sibling oocyte pilot study.

Purpose: This study investigated the differences in the maturation rate of single versus grouped cumulus-oocyte complexes (COCs) culture methods for capacitation in vitro maturation (CAPA-IVM) in women with polycystic ovary syndrome (PCOS). Methods: This study was performed at My Duc Phu Nhuan Hospital, Vietnam from October 1, 2020 to October 24, 2021. Women aged 18-37 years with a diagnosis of PCOS were recruited. COCs from each woman were randomly divided into two groups: single or grouped culture during CAPA-IVM culture. The primary outcome was the maturation rate. Results: A total of 322 COCs from 15 eligible women included were randomly assigned to the two study groups. The maturation rate was comparable between the single and grouped culture groups (61.3% vs. 64.8%; p = 0.56). There were no significant differences in the number of 2-pronuclei fertilized oocytes, number of day-3 embryos, and number of good-quality embryos in the two culture method groups. In the single culture group, COCs morphology was associated with the day-3 embryo formation rate but not the maturation rate. Conclusions: Comparable oocyte maturation and embryology outcomes between single and grouped COCs culture utilizing sibling COCs derived from women with PCOS suggest the feasibility of both methods for CAPA-IVM culture.

Screening and Mechanism Study of Three Antagonistic Drugs, Oxysophoridine, Rutin, and Phellodendrine, against Zearalenone-Induced Reproductive Toxicity in Ovine Oocytes.

Zearalenone (ZEN) is a common fungal toxin with reproductive toxicity in various grains. It poses a serious threat to ovine and other animal husbandry industries, as well as human reproductive health. Therefore, investigating the mechanism of toxicity and screening antagonistic drugs are of great importance. In this study, based on the natural compound library and previous Smart-seq2 results, antioxidant and anti-apoptotic drugs were selected for screening as potential antagonistic drugs. Three natural plant compounds (oxysophoridine, rutin, and phellodendrine) were screened for their ability to counteract the reproductive toxicity of ZEN on ovine oocytes in vitro using quantitative polymerase chain reaction (qPCR) and reactive oxygen species detection. The compounds exhibited varying pharmacological effects, notably impacting the expression of antioxidant (GPX, SOD1, and SOD2), autophagic (ATG3, ULK2, and LC3), and apoptotic (CAS3, CAS8, and CAS9) genes. Oxysophoridine promoted GPX, SOD1, ULK2, and LC3 expression, while inhibiting CAS3 and CAS8 expression. Rutin promoted SOD2 and ATG3 expression, and inhibited CAS3 and CAS9 expression. Phellodendrine promoted SOD2 and ATG3 expression, and inhibited CAS9 expression. However, all compounds promoted the expression of genes related to cell cycle, spindle checkpoint, oocyte maturation, and cumulus expansion factors. Although the three drugs had different regulatory mechanisms in enhancing antioxidant capacity, enhancing autophagy, and inhibiting cell apoptosis, they all maintained a stable intracellular environment and a normal cell cycle, promoted oocyte maturation and release of cumulus expansion factors, and, ultimately, counteracted ZEN reproductive toxicity to promote the in vitro maturation of ovine oocytes. This study identified three drugs that antagonize the reproductive toxicity of ZEN on ovine oocytes, and compared their mechanisms of action, providing data support and a theoretical basis for their subsequent application in the ovine breeding industry, reducing losses in the breeding industry, screening of ZEN reproductive toxicity antagonists and various toxin antagonists, improving the study of ZEN reproductive toxicity mechanisms, and even protection of human reproductive health.

Cytokine-Supplemented Maturation Medium Enhances Cytoplasmic and Nuclear Maturation in Bovine Oocytes.

Bovine in vitro oocyte maturation (IVM) is an easy way to obtain oocytes for subsequent assisted reproductive techniques but is inefficient compared to in vivo maturation. Supplementation of three cytokines, fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1), or FLI, has increased oocyte maturation and embryo development in multiple species, but studies have not explored the oocyte differences caused by FLI IVM supplementation. This study aimed to assess important nuclear and cytoplasmic maturation events in high-quality oocytes. FLI-supplemented oocytes had a decreased GV (3.0% vs. 13.7%, p < 0.01) and increased telophase I incidence (34.6% vs. 17.6%, p < 0.05) after IVM, increased normal meiotic spindles (68.8% vs. 50.0%, p < 0.001), and an increased nuclear maturation rate (75.1% vs. 66.8%, p < 0.001). Moreover, in metaphase II oocytes, the percentage of FLI-treated oocytes with a diffuse mitochondrial distribution was higher (87.7% vs. 77.5%, p < 0.05) and with a cortical mitochondrial distribution was lower (11.6% vs. 17.4%, p < 0.05). Additionally, FLI-supplemented oocytes had more pattern I cortical granules (21.3% vs. 14.4%, p < 0.05). These data suggest that FLI supplementation in bovine in vitro maturation medium coordinates nuclear and cytoplasmic maturation to produce higher-quality oocytes.

A rare case of intra-ovarian oocyte maturation.

The intra-ovarian presence of ootids, i.e. female gametes that have completed meiosis, is considered exceptional in the animal kingdom. The present study explores the first such case to be reported in a sea cucumber (Echinodermata: Holothuroidea). In the overwhelming majority of animals, including holothuroids, oocytes (i.e. immature female gametes) that are developing in the ovary undergo a primary arrest at the prophase stage of meiosis, which may last from days to decades. In free-spawning taxa, this arrest is normally lifted only during or shortly before transit in the gonoduct, when gamete release (spawning) is imminent. However, oocytes of the holothuroid Chiridota laevis were discovered to have resumed the second meiotic division including the completion of germinal vesicle breakdown and polar-body expulsion inside the ovary, effectively reaching the ootid stage concomitantly with ovulation (i.e. escape from follicle cells) prior to spawning. The potential drivers and significance of this exceptionally rare case of full intra-ovarian oogenic maturation are discussed.

Optimal ICSI timing on immature oocytes for low prognosis patients under the POSEIDON classification.

BACKGROUND: The optimal timing of performing ICSI on immature oocytes for POSEIDON patients is still unknown to get better early embryonic development outcomes. The purpose of this study was to implore the most appropriate time to carry out ICSI on in vitro maturation GV and MI oocytes for POSEIDON patients. METHODS: Two hundred thirty-nine immature oocytes from 163 POSEIDON patients were prospectively performed ICSI at different timings: P-ICSI (ICSI was performed on in vitro matured oocytes 4-6 h after the first polar body extrusion, N = 81), R-ICSI (ICSI was performed on in vitro matured oocytes less than 4 h after the first polar body extrusion, N = 80), and E-ICSI (ICSI was performed on in vitro matured oocytes the next day after oocytes retrieval, N = 78). Fertilization and embryonic development outcomes were collected and statistically analyzed. Mitochondria distribution of cytoplasm of in vitro matured oocytes with different time cultures after the first polar body (PB1) extrusion was stained. RESULTS: Compared to the E-ICSI group, more day 3 embryos from P-ICSI became blastocysts after sequential culture though without statistical significance (OR = 3.71, 95% CI: 0.94-14.63, P = 0.061). Compared to the E-ICSI group, more embryos from both P-ICSI and R-ICSI groups were clinically used with statistical significance (OR = 5.67, 95% CI: 2.24-14.35, P = 0.000 for P-ICSI embryos; OR = 3.23, 95% CI: 1.23-8.45, P = 0.017 for R-ICSI embryos). Compared to the E-ICSI group, transferred embryos from P-ICSI and R-ICSI had a higher implantation rate though without statistical significance (35.3% for P-ICSI embryos; 9.1% or R-ICSI embryos and 0% for E-ICSI embryos, P = 0.050). Among the three group, there were most healthy babies delivered from the P-ICSI group (5, 1 and 0 for P-ICSI, R-ICSI and E-ICSI respectively). The mitochondria in the cytoplasm of in vitro matured oocytes with a less than 4 h and 4-6 h culture after PB1 extrusion presented semiperipheral and diffused distribution patterns, respectively. CONCLUSIONS: Our results revealed P-ICSI (ICSI was performed on in vitro matured oocytes 4-6 h after the first polar body extrusion) provided the most efficient method to utilize the immaturation oocytes basing on embryos utilization and live birth outcome for low prognosis patients under the POSEIDON classification. The mitochondria distribution of the in vitro matured oocytes' cytoplasm from P-ICSI varied that from R-ICSI.

In vitro fertilization and perinatal outcomes of patients with advanced maternal age after single frozen euploid embryo transfer: a propensity score-matched analysis of autologous and donor cycles.

OBJECTIVE: To evaluate in vitro fertilization (IVF) and perinatal outcomes of donor egg and autologous cycles in patients with advanced reproductive age after undergoing single frozen euploid embryo transfer. DESIGN: A multicenter, retrospective, cohort study. SETTING: University-affiliated and private IVF centers. PATIENT(S): Patients aged 39-46 years who underwent IVF with intracytoplasmic sperm injection and preimplantation genetic testing for aneuploidy using whole-chromosome sequencing with donor (n = 278) or autologous (n = 278) oocytes between October 2017 and October 2021. INTERVENTION(S): Single frozen euploid embryo transfer with donor or autologous euploid embryo. MAIN OUTCOME MEASURE(S): The main outcome measure was the live birth rate (LBR) after the first embryo transfer, calculated per embryo transfer. The secondary outcomes included the implantation rate, ectopic pregnancy rate, miscarriage rate, and gestational age and birth weight at the time of delivery. RESULT(S): Patients using donor or autologous oocytes had a similar likelihood of implantation (57.91% [51.87-63.78] vs. 57.19% [51.15-63.09]) and LBR (41.01% [95% confidence interval {CI}, 35.17-47.04] vs. 42.45% [95% CI, 36.56-48.49]). Furthermore, there were no significant differences in the ectopic pregnancy rate (0.72% [0.09-2.57] vs. 0.36% [0.01-1.99]), miscarriage rate (16.19% [12.06-21.05] vs. 14.39% [95% CI, 10.48-19.08]), gestational age (38.50 [38.08-38.92] vs. 39.16 [38.25-40.07] weeks), or birth weight of infants (2,982.25 [2,606.69-3,357.81] vs. 3,128.24 [2,962.30-3,294.17] kg). The univariate analysis showed no association between advanced maternal age and the LBR (relative risk, 1.03 [95% CI, 0.84-1.25]). Multivariate analysis using putative confounders for embryo competency found no associations with LBR (adjusted relative risk, 1.22 [95% CI, 0.75-1.98]). CONCLUSION(S): Patients with euploid blastocysts derived from donor or autologous oocytes did not reveal statistically significant differences in the LBR, implantation rate, ectopic pregnancy rate, miscarriage rate, duration of gestation, or infant birth weight. These findings suggest that age-related reproductive decline and/or poor IVF outcomes associated with women with advanced reproductive age undergoing IVF are heavily driven by embryonic aneuploidy.

Single-cell proteomics reveals decreased abundance of proteostasis and meiosis proteins in advanced maternal age oocytes.

Advanced maternal age is associated with a decline in oocyte quality, which often leads to reproductive failure in humans. However, the mechanisms behind this age-related decline remain unclear. To gain insights into this phenomenon, we applied plexDIA, a multiplexed data-independent acquisition, single-cell mass spectrometry method, to analyze the proteome of oocytes from both young women and women of advanced maternal age. Our findings primarily revealed distinct proteomic profiles between immature fully grown germinal vesicle and mature metaphase II oocytes. Importantly, we further show that a woman's age is associated with changes in her oocyte proteome. Specifically, when compared to oocytes obtained from young women, advanced maternal age oocytes exhibited lower levels of the proteasome and TRiC complex, as well as other key regulators of proteostasis and meiosis. This suggests that aging adversely affects the proteostasis and meiosis networks in human oocytes. The proteins identified in this study hold potential as targets for improving oocyte quality and may guide future studies into the molecular processes underlying oocyte aging.

Current practices in oncofertility counseling: updated evidence on fertility preservation and post-treatment pregnancies in young women affected by early breast cancer.

INTRODUCTION: Anticancer treatments have significantly contributed to increasing cure rates of breast cancer in the last years; however, they can also lead to short- and long-term side effects, including gonadotoxicity, and compromised fertility in young women. Oncofertility is a crucial issue for young patients who have not yet completed their family planning at the time of cancer diagnosis. AREAS COVERED: This review aims to cover all the latest available evidence in the field of oncofertility, including the gonadotoxicity of currently adopted anticancer therapies in the curative breast cancer setting, the available strategies for fertility preservation and the feasibility of achieving a pregnancy following anticancer treatment completion. EXPERT OPINION: Over the past years, a significant progress has been made in oncofertility care for young women with breast cancer. In the context of the currently available evidence, every young woman with newly diagnosed breast cancer should receive a proper and complete oncofertility counseling before starting any anticancer treatment to increase her chances of future pregnancies.

Use of membrane transport models to design cryopreservation procedures for oocytes.

Oocyte cryopreservation is increasingly being used in reproductive technologies for conservation and breeding purposes. Further development of oocyte cryopreservation techniques requires interdisciplinary insights in the underlying principles of cryopreservation. This review aims to serve this purpose by: (1) highlighting that preservation strategies can be rationally designed, (2) presenting mechanistic insights in volume and osmotic stress responses associated with CPA loading strategies and cooling, and (3) giving a comprehensive listing of oocyte specific biophysical membrane characteristics and commonly used permeation model equations. It is shown how transport models can be used to simulate the behavior of oocytes during cryopreservation processing steps, i.e., during loading of cryoprotective agents (CPAs), cooling with freezing as well as vitrification, warming and CPA unloading. More specifically, using defined cellular and membrane characteristics, the responses of oocytes during CPA (un)loading were simulated in terms of temperature- and CPA type-and-concentration-dependent changes in cell volume and intracellular solute concentration. In addition, in order to determine the optimal cooling rate for slow programmable cooling cryopreservation, the freezing-induced cell volume response was simulated at various cooling rates to estimate rates with tolerable limits. For vitrification, special emphasis was on prediction of the timing of reaching osmotic tolerance limits during CPA exposure, and the need to use step-wise CPA addition/removal protocols. In conclusion, we present simulations and schematic illustrations that explain the timing of events during slow cooling cryopreservation as well as vitrification, important for rationally designing protocols taking into account how different CPA types, concentrations and temperatures affect the oocyte.

In vitro matured oocytes have a higher developmental potential than in vivo matured oocytes after hormonal ovarian stimulation in Callithrix jacchus.

BACKGROUND: The common marmoset, Callithrix jacchus, is an invaluable model in biomedical research. Its use includes genetic engineering applications, which require manipulations of oocytes and production of embryos in vitro. To maximize the recovery of oocytes suitable for embryo production and to fulfil the requirements of the 3R principles to the highest degree possible, optimization of ovarian stimulation protocols is crucial. Here, we compared the efficacy of two hormonal ovarian stimulation approaches: 1) stimulation of follicular growth with hFSH followed by triggering of oocyte maturation with hCG (FSH + hCG) and 2) stimulation with hFSH only (FSH-priming). METHODS: In total, 14 female marmosets were used as oocyte donors in this study. Each animal underwent up to four surgical interventions, with the first three performed as ovum pick-up (OPU) procedures and the last one being an ovariohysterectomy (OvH). In total, 20 experiments were carried out with FSH + hCG stimulation and 18 with FSH-priming. Efficacy of each stimulation protocol was assessed through in vitro maturation (IVM), in vitro fertilization (IVF) and embryo production rates. RESULTS: Each study group consisted of two subgroups: the in vivo matured oocytes and the oocytes that underwent IVM. Surprisingly, in the absence of hCG triggering some of the oocytes recovered were at the MII stage, moreover, their number was not significantly lower compared to FSH + hCG stimulation (2.8 vs. 3.9, respectively (ns)). While the IVM and IVF rates did not differ between the two stimulation groups, the IVF rates of in vivo matured oocytes were significantly lower compared to in vitro matured ones in both FSH-priming and FSH + hCG groups. In total, 1.7 eight-cell embryos/experiment (OPU) and 2.1 eight-cell embryos/experiment (OvH) were obtained after FSH + hCG stimulation vs. 1.8 eight-cell embryos/experiment (OPU) and 5.0 eight-cell embryos/experiment (OvH) following FSH-priming. These numbers include embryos obtained from both in vivo and in vitro matured oocytes. CONCLUSION: A significantly lower developmental competence of the in vivo matured oocytes renders triggering of the in vivo maturation with hCG as a part of the currently used FSH-stimulation protocol unnecessary. In actual numbers, between 1 and 7 blastocysts were obtained following each FSH-priming. In the absence of further studies, FSH-priming appears superior to FSH + hCG stimulation in the common marmoset under current experimental settings.

Molecular Detection of Toxoplasma gondii Oocytes in Soil Samples from Guilan Province, Northern Iran.

Background: Soil is an appropriate substrate for the storage and transmission of oocytes of Toxoplasma gondii. Ingestion of soil contaminated with T. gondii oocysts is a major transmission route of human and animal toxoplasmosis. The present study was carried out to investigate soil contamination with T. gondii oocysts in urban and rural areas of Guilan Province, northern Iran. Methods: Overall, 208 soil samples were collected from 16 cities and villages in Guilan Province, northern Iran from Oct 2020 to Nov 2021. Soil samples were investigated using modified sucrose flotation technique. Real-time polymerase chain reaction was used to detect presence of T. gondii DNAs in the samples. Positive samples were further analyzed using nested polymerase chain reaction for GRA6 gene. Moreover, six selected positive samples were used for amplifying and sequencing of the GRA6 gene. Results: Overall, 31 samples were positive for T. gondii with frequency of 14.9% and ranging from 10.9% in rural areas to 16.3% in urban areas. Statistical analysis showed significant differences between the seasons (P=0.003). The phylogenetic analysis illustrated that our six sequences were similar and closely related to Type I strain of T. gondii. Conclusion: Results showed relatively high levels (14.9%) of T. gondii oocytes in soil samples of Guilan Province, northern Iran, which provided essential data for the effective prevention and control of toxoplasmosis in the region.

The impact of cumulus cell viability and pre-culture with the healthy cell mass on brilliant cresyl blue (BCB) staining assessment and meiotic competence of suboptimal porcine oocytes.

Objectives of the present study were to investigate the characteristics including glucose-6-phosphate dehydrogenase activity, as determined by Brilliant Cresyl Blue (BCB) staining, of suboptimal porcine oocytes and to enhance the meiotic competence of those through pre-culture with cumulus cell masses (CCMs). Percentage of oocyte-cumulus complexes (OCCs) derived from small follicles (SF; <3 mm in diameter) containing the oocytes that were assessed as BCB-negative (BCB-) was significantly higher than those derived from medium follicles (MF; 3-6 mm in diameter). Degrees of dead cumulus cells were significantly higher in OCCs containing BCB- oocytes, regardless of the origin of OCCs (MF vs. SF), than those containing BCB-positive (BCB+) ones. Exposing OCCs containing BCB+ oocytes to the apoptosis inducer, carbonyl cyanide m-chlorophenylhydrazone, for 20 h significantly induced the transition to BCB- and meiotic progression of exposed OCCs were significantly reduced in both SF and MF derived ones. Transit of BCB- oocytes to BCB+ was induced when OCCs were pre-cultured with CCMs of MF derived OCCs containing BCB+ oocytes for 20 h before IVM. This pre-culture also significantly increased the meiotic competence of BCB- oocytes, particularly in SF derived ones. However, reactive oxygen species levels were significantly higher in BCB+ oocytes as compared with BCB- ones, regardless of pre-culture with CCMs, whereas no significant differences were found in the ATP contents among the treatment groups. In conclusion, the BCB result of oocytes could be regulated by the healthy status and content of surrounding cumulus cells and the meiotic competence of suboptimal BCB- porcine oocytes is improved by pre-culture with healthy CCMs.

Astaxanthin Added during Post-Warm Recovery Mitigated Oxidative Stress in Bovine Vitrified Oocytes and Improved Quality of Resulting Blastocysts.

Various antioxidants are tested to improve the viability and development of cryopreserved oocytes, due to their known positive health effects. The aim of this study was to find whether astaxanthin (AX), a xanthophyll carotenoid, could mitigate deteriorations that occurred during the vitrification/warming process in bovine oocytes. Astaxanthin (2.5 µM) was added to the maturation medium during the post-warm recovery period of vitrified oocytes for 3 h. Afterward, the oocytes were fertilized in vitro using frozen bull semen and presumptive zygotes were cultured in the B2 Menezo medium in a co-culture with BRL-1 cells at 38.5 °C and 5% CO2 until the blastocyst stage. AX addition significantly reduced ROS formation, lipid peroxidation, and lysosomal activity, while increasing mitochondrial activity in vitrified oocytes. Although the effect of AX on embryo development was not observed, it stimulated cell proliferation in the blastocysts derived from vitrified oocytes and improved their quality by upregulation or downregulation of some genes related to apoptosis (BCL2, CAS9), oxidative stress (GPX4, CDX2), and development (GJB5) compared to the vitrified group without AX. Therefore, the antioxidant properties of astaxanthin even during short exposure to bovine vitrified/warmed oocytes resulted in improved blastocyst quality comparable to those from fresh oocytes.

The Identification of RPL4 as a Hub Gene Associated with Goat Litter Size via Weighted Gene Co-Expression Network Analysis.

Reproduction in goats is a highly complex and dynamic process of life regulation, involving coordinated regulation from various aspects such as central nervous system regulation, reproductive system development, oocyte maturation, and fertilized egg development. In recent years, researchers have identified numerous genes associated with goat reproductive performance through high-throughput sequencing, single-cell sequencing, gene knockout, and other techniques. However, there is still an urgent need to explore marker genes related to goat reproductive performance. In this study, a single-cell RNA sequencing dataset of oocytes (GSE136005) was obtained from the Gene Expression Omnibus (GEO) database. Weighted Gene Co-expression Network Analysis (WGCNA) was utilized to identify modules highly correlated with goat litter size. Through gene function enrichment analysis, it was found that genes within the modules were mainly enriched in adhesive junctions, cell cycle, and other signaling pathways. Additionally, the top 30 hub genes with the highest connectivity in WGCNA were identified. Subsequently, using Protein-Protein Interaction (PPI) network analysis, the top 30 genes with the highest connectivity within the modules were identified. The intersection of hub genes, key genes in the PPI network, and differentially expressed genes (DEGs) led to the identification of the RPL4 gene as a key marker gene associated with reproductive capacity in goat oocytes. Overall, our study reveals that the RPL4 gene in oocytes holds promise as a biological marker for assessing goat litter size, deepening our understanding of the regulatory mechanisms underlying goat reproductive performance.