Proteome and metabolomic profile of Mongolian horse follicular fluid during follicle development.

During follicular development, changes in the composition of the follicular fluid are synchronized with the development of oocytes. Our aim was to screen the key factors affecting oocyte maturation and optimize the in vitro culture protocol by understanding the changes of proteins and metabolites in follicular fluid. Follicles are divided into three groups according to their diameter (small follicle fluid (SFF): 10 mm < d < 20 mm; medium follicle fluid (MFF): 20 mm < d < 30 mm; large follicle fluid (LFF): 30 mm < d). Proteins and metabolites from the follicular fluid were analyzed by mass spectrometry. The results showed that: in LFF vs MFF, 20 differential abundant protein (DAP) and 88 differential abundant metabolites (DAM) were screened out; In SFF vs MFF, 3 DAPs and 65 DAMs were screened out; In MFF vs SFF, 24 DAPs and 35 DAMs were screened out. The analysis of differential proteins and metabolites showed that glycerophosphate hydrolysis decreased during follicular development, and proteins played a major role in metabolism and binding. In addition, DAMs and DAPs are co-enriched in the "linoleic acid metabolism" pathway. Combinatorial analysis reveals the dynamic profile of follicular fluid during follicular development and provides fundation for further exploring the function of follicular fluid in Mongolian horse.

3D culture of ovarian follicles in granular and nanofibrillar hydrogels.

3D culture of ovarian follicles in hydrogel matrices is an important emerging tool for basic scientific studies as well as clinical applications such as fertility preservation. For optimizing and scaling 3D culture of preantral follicles, there is a need for identifying biomaterial matrices that simplifies and improves the current culture procedures. At present, microencapsulation of follicles in alginate beads is the most commonly used approach. However, this technique involves notable manual handling and is best suited for encapsulation of single or several follicles. As a potential alternative, we here explore the suitability of different particle-based hydrogel matrices, where follicles can easily be introduced in tunable 3D environments, in large numbers. Specifically, we study the growth of secondary murine follicles in microgranular alginate and nanofibrillar cellulose matrices, with and without cell-binding cues, and map follicle growth against the viscoelastic properties of the matrices. We cultured follicles within the particle-based hydrogels for 10 days and continuously monitored their size, survival, and tendency to extrude oocytes. Interestingly, we observed that the diameter of the growing follicles increased significantly in the particle-based matrices, as compared to state-of-the-art alginate micro-encapsulation. On the other hand, the follicles displayed an increased tendency for early oocyte extrusion in the granular matrices, leading to a notable reduction in the number of intact follicles. We propose that this may be caused by impaired diffusion of nutrients and oxygen through thicker matrices, attributable to our experimental setup. Still, our findings suggest that viscoelastic, granular hydrogels represent promising matrices for 3D culture of early-stage ovarian follicles. In particular, these materials may easily be implemented in advanced culturing devices such as micro-perfusion systems.

Transcriptome Dynamics and Cell Dialogs Between Oocytes and Granulosa Cells in Mouse Follicle Development.

The development and maturation of follicles is a sophisticated and multistage process. The dynamic gene expression of oocytes and their surrounding somatic cells and the dialogs between these cells are critical to this process. In this study, we accurately classified the oocyte and follicle development into nine stages and profiled the gene expression of mouse oocytes and their surrounding granulosa cells and cumulus cells. The clustering of the transcriptomes showed the trajectories of two distinct development courses of oocytes and their surrounding somatic cells. Gene expression changes precipitously increased at Type 4 stage and drastically dropped afterward within both oocytes and granulosa cells. Moreover, the number of differentially expressed genes between oocytes and granulosa cells dramatically increased at Type 4 stage, most of which persistently passed on to the later stages. Strikingly, cell communications within and between oocytes and granulosa cells became active from Type 4 stage onward. Cell dialogs connected oocytes and granulosa cells in both unidirectional and bidirectional manners. TGFB2/3, TGFBR2/3, INHBA/B, and ACVR1/1B/2B of TGF-ß signaling pathway functioned in the follicle development. NOTCH signaling pathway regulated the development of granulosa cells. Additionally, many maternally DNA methylation- or H3K27me3-imprinted genes remained active in granulosa cells but silent in oocytes during oogenesis. Collectively, Type 4 stage is the key turning point when significant transcription changes diverge the fate of oocytes and granulosa cells, and the cell dialogs become active to assure follicle development. These findings shed new insights on the transcriptome dynamics and cell dialogs facilitating the development and maturation of oocytes and follicles.

The usefulness of peri-trigger female reproductive hormones (delta-FRH) in predicting oocyte maturation in normal ovarian reserve patients who received in vitro fertilization-embryo transfer: a retrospective study.

Objectives: To evaluate the efficacy of peri-trigger female reproductive hormones (FRHs) in the prediction of oocyte maturation in normal ovarian reserve patients during the in vitro fertilization-embryo transfer (IVF-ET) procedure. Materials and Methods: A hospital database was used to extract data on IVF-ET cases from January 2020 to September 2021. The levels of female reproductive hormones, including estradiol (E2), luteinizing hormone (LH), progesterone (P), and follicle-stimulating hormone (FSH), were initially evaluated at baseline, the day of the trigger, the day after the trigger, and the day of oocyte retrieval. The relative change in E2, LH, P, FSH between time point 1 (the day of trigger and baseline) and time point 2 (the day after the trigger and day on the trigger) was defined as E2_RoV1/2, LH_RoV1/2, P_RoV1/2, and FSH_RoV1/2, respectively. Univariable and multivariable regression were performed to screen the peri-trigger FRHs for the prediction of oocyte maturation. Results: A total of 118 patients were enrolled in our study. Univariable analysis revealed significant associations between E2_RoV1 and the rate of MII oocytes in the GnRH-agonist protocol group (p < 0.05), but not in the GnRH-antagonist protocol group. Conversely, P_RoV2 emerged as a potential predictor for the rate of MII oocytes in both protocol groups (p < 0.05). Multivariable analysis confirmed the significance of P_RoV2 in predicting oocyte maturation rate in both groups (p < 0.05), while the association of E2_RoV1 was not significant in either group. However, within the subgroup of high P_RoV2 in the GnRH-agonist protocol group, association was not observed to be significant. The C-index was 0.83 (95% CI [0.73-0.92]) for the GnRH-agonist protocol group and 0.77 (95% CI [0.63-0.90]) for the GnRH-antagonist protocol group. The ROC curve analysis further supported the satisfactory performance of the models, with area under the curve (AUC) values of 0.79 for the GnRH-agonist protocol group and 0.81 for the GnRH-antagonist protocol group. Conclusions: P_RoV2 showed significant predictive value for oocyte maturation in both GnRH-agonist and GnRH-antagonist protocol groups, which enhances the understanding of evaluating oocyte maturation and inform individualized treatment protocols in controlled ovarian hyperstimulation during IVF-ET for normal ovarian reserve patients.

Meiotic maturation failure in primary ovarian insufficiency: insights from a bovine model.

PURPOSE: Oocytes from women presenting primary ovarian insufficiency (POI) generate viable embryos at a lower rate than non-POI women, but the mechanisms responsible for the lower oocyte quality remain elusive. Due to the scarcity of human oocytes for research, animal models provide a promising way forward. We aimed at investigating the molecular events characterizing final maturation in POI oocytes in a well-defined POI-like bovine model. METHODS: Single-cell RNA-sequencing of bovine control and POI-like, GV, and MII oocytes (n = 5 per group) was performed. DEseq2 was used to identify differentially expressed genes. Further, a Gene set enrichment analysis and a transcriptomic meta-analysis between bovine and human oocytes were performed. RESULTS: In control cows, we found 2223 differentially expressed genes between the GV and MII stages. Specifically, the affected genes were related to RNA processing and transport, protein synthesis, organelle remodeling and reorganization, and metabolism. The meta-analysis with a set of young human oocytes at different maturation stages revealed 315 conserved genes through the GV-MII transition in cows and humans, mostly related to meiotic progression and cell cycle. Gene expression analysis between GV and MII of POI-like oocytes showed no differences in terms of differentially expressed genes, pointing towards a substantial failure to properly remodel the transcriptome in the POI model, and with the clustering analysis indicating that the cow's genetic background had a higher impact than the oocyte's maturation stage. CONCLUSION: Overall, we have identified and characterized a valuable animal model of POI, paving the way to identifying new molecular mechanisms involved in POI.

Novel PATL2 variants cause female infertility with oocyte maturation defect.

PURPOSE: Oocyte maturation defect (OOMD) is a rare cause of in vitro fertilization failure characterized by the production of immature oocytes. Compound heterozygous or homozygous PATL2 mutations have been associated with oocyte arrest at the germinal vesicle (GV), metaphase I (MI), and metaphase II (MII) stages, as well as morphological changes. METHODS: In this study, we recruited three OOMD cases and conducted a comprehensive multiplatform laboratory investigation. RESULTS: Whole exome sequence (WES) revealed four diagnostic variants in PATL2, nonsense mutation c.709C > T (p.R237*) and frameshift mutation c.1486_1487delinsT (p.A496Sfs*4) were novel mutations that have not been reported previously. Furthermore, the pathogenicity of these variants was predicted using in silico analysis, which indicated detrimental effects. Molecular dynamic analysis suggested that the A496S variant disrupted the hydrophobic segment, leading to structural changes that affected the overall protein folding and stability. Additionally, biochemical and molecular experiments were conducted on cells transfected with wild-type (WT) or mutant PATL2 (p.R237* and p.A496Sfs*4) plasmid vectors. CONCLUSIONS: The results demonstrated that PATL2A496Sfs*4 and PATL2R237* had impacts on protein size and expression level. Interestingly, expression levels of specific genes involved in oocyte maturation and early embryonic development were found to be simultaneously deregulated. The findings in our study expand the variation spectrum of the PATL2 gene, provide solid evidence for counseling on future pregnancies in affected families, strongly support the application of in the diagnosis of OOMD, and contribute to the understanding of PATL2 function.

Genetic factors of oocyte maturation arrest: an important cause for recurrent IVF/ICSI failures.

Oocyte maturation arrest (OMA) is a common phenotype observed in IVF/ICSI cycles, characterized by the production of immature oocytes which lead to infertility. Previous studies have demonstrated that genetic factors play an important role in OMA, but the genetic mechanisms underlying a group of patients remain to be elucidated. In the recent issue of Journal of Assisted Reproduction and Genetics, Hu et al. and Wan et al. identified novel PATL2 or ZFP36L2 variants in OMA patients, respectively. By conducting in vitro experiments, they demonstrated the destructive effect of the variants on protein function. These findings expand the mutational spectrum of PATL2 and ZFP36L2, and provide precise reference for genetic counseling of OMA patients.

Live birth after vitrification of oocytes from capacitation in vitro maturation.

Female fertility preservation is a rapidly growing field in medicine. Oocyte cryopreservation and assisted reproductive technique with vitrified-warmed oocytes have been successful with in vivo matured oocytes after conventional ovarian stimulation protocols. The use of in vitro matured oocytes after vitrification and warming has been limited. Capacitation in vitro maturation (CAPA-IVM) represents the latest refinement of IVM protocols and provides in vitro matured oocytes with improved competence. This case report describes the first successful live birth following oocyte vitrification from a CAPA-IVM cycle. This milestone achievement holds a significant promise to expand fertility preservation options and improve accessibility for women wishing to cryopreserve their eggs for future use.

Assessing the developmental competence of oocytes matured following rescue in vitro maturation: a systematic review and meta-analysis.

PURPOSE: To assess the developmental competence of oocytes matured following rescue in vitro maturation (IVM). METHODS: PubMed, EmBASE, and SCOPUS were systematically searched for peer-reviewed original papers using relevant keywords and Medical Subject Heading terms. Study quality was assessed using the Newcastle-Ottawa Scale. Odds ratios with a 95% confidence interval were calculated by applying a random effects model. The primary outcomes were fertilization and blastulation rates. Secondary outcomes included abnormal fertilization, cleavage, euploidy, clinical pregnancy, and live-birth rates. RESULT: Twenty-four studies were included in the meta-analysis. The oocytes matured following rescue IVM showed significantly reduced fertilization, cleavage, blastulation, and clinical pregnancy rates compared to sibling in vivo-matured oocytes. No significant differences were found for the euploidy and live-birth rates in euploid blastocyst transfer. In poor responders, a reduced fertilization rate was observed using in vitro-matured GV but not with in vitro-matured MI. A reduced cleavage rate in MI matured overnight compared to < 6 incubation hours was found. CONCLUSION: Our results showed compromised developmental competence in oocytes matured following rescue IVM. However, in poor responders, rescue IVM could maximize the efficiency of the treatment. Notably, our data suggests using in vitro MI matured within 6 incubation hours. CLINICAL TRIAL REGISTRATION NUMBER: CRD42023467232.

Spironolactone use and oocyte maturation in patients undergoing controlled ovarian hyperstimulation.

PURPOSE: To evaluate the association between spironolactone use and controlled ovarian hyperstimulation (COH) outcomes. METHODS: Retrospective study, including patients who underwent COH. Oocyte yield and maturation rates were compared by categories of spironolactone use at the start of their cycle. RESULTS: 402 patients were included. 83 patients continued spironolactone, 44 patients discontinued spironolactone, and 275 matched control patients were spironolactone-naïve. No difference was observed in the number of oocytes retrieved (17 ± 14 vs. 15 ± 13, p = 0.4) or mature oocytes vitrified (15 ± 9.5 vs. 12 ± 11, p = 0.4) in patients who continued spironolactone use and spironolactone naïve patients, respectively. When comparing patients who continued spironolactone use and patients who discontinued spironolactone use, no difference was seen in the number of oocytes retrieved (17 ± 14 vs. 17.5 ± 7.8, p = 0.9) or mature oocytes vitrified (15 ± 9.5 vs. 13.5 ± 6.5, p = 0.5), respectively. There was no observed relationship between total daily spironolactone dose (< 100mg/day, 100mg/day, 150mg/day and > 200 mg/day) and the total number of mature oocytes vitrified (respectively, 14.0 ± 13.0, 16.0 ± 7.8, 14.0 ± 4.5, 11.0 ± 7.0 oocytes, p = 0.4). CONCLUSIONS: This is the first study to evaluate the association between spironolactone and oocyte yield and maturation rates during COH cycles. These findings can assist in counseling patients on the implications of continuing spironolactone during COH cycle.

Maternal regulation of the vertebrate oocyte-to-embryo transition.

Maternally-loaded factors in the egg accumulate during oogenesis and are essential for the acquisition of oocyte and egg developmental competence to ensure the production of viable embryos. However, their molecular nature and functional importance remain poorly understood. Here, we present a collection of 9 recessive maternal-effect mutants identified in a zebrafish forward genetic screen that reveal unique molecular insights into the mechanisms controlling the vertebrate oocyte-to-embryo transition. Four genes, over easy, p33bjta, poached and black caviar, were found to control initial steps in yolk globule sizing and protein cleavage during oocyte maturation that act independently of nuclear maturation. The krang, kazukuram, p28tabj, and spotty genes play distinct roles in egg activation, including cortical granule biology, cytoplasmic segregation, the regulation of microtubule organizing center assembly and microtubule nucleation, and establishing the basic body plan. Furthermore, we cloned two of the mutant genes, identifying the over easy gene as a subunit of the Adaptor Protein complex 5, Ap5m1, which implicates it in regulating intracellular trafficking and yolk vesicle formation. The novel maternal protein Krang/Kiaa0513, highly conserved in metazoans, was discovered and linked to the function of cortical granules during egg activation. These mutant genes represent novel genetic entry points to decipher the molecular mechanisms functioning in the oocyte-to-embryo transition, fertility, and human disease. Additionally, our genetic adult screen not only contributes to the existing knowledge in the field but also sets the basis for future investigations. Thus, the identified maternal genes represent key players in the coordination and execution of events prior to fertilization.

Temporal metabolomics analysis reveals the metabolic patterns in goat cumulus cells during oocyte maturation.

Cumulus cells play a crucial role in the oocyte growth and maturation processes through providing necessary nutrients and growth signals by gap junction communication. However, a global overview of metabolic events in goat cumulus cells is still lacking. In the present study, we collected cumulus cells from goat cumulus-oocyte complexes (COCs) at different developmental stages. Metabolomics analysis was performed to investigate the global metabolic patterns in cumulus cells during oocyte in vitro maturation. In particular, we revealed the several significantly altered metabolic pathways and metaboliccharacteristics in goat cumulus cells, including the accumulation of fatty acids, steroid hormones metabolism, active catabolism of arginine during meiotic resumption, and a progressive decline in nucleotide metabolism. In conclusion, the dataset generated by our metabolomic profiling will provide valuable information to understand the key metabolic pathways and metabolites involved in COCs development.

Chemical reversion of age-related oocyte dysfunction fails to enhance embryo development in a bovine model of postovulatory aging.

PURPOSE: There are no clinical treatments to prevent/revert age-related alterations associated with oocyte competence decline in the context of advanced maternal age. Those alterations have been attributed to oxidative stress and mitochondrial dysfunction. Our study aimed to test the hypothesis that in vitro maturation (IVM) medium supplementation with antioxidants (resveratrol or phloretin) may revert age-related oocyte competence decline. METHODS: Bovine immature oocytes were matured in vitro for 23 h (young) and 30 h (aged). Postovulatory aged oocytes (control group) and embryos obtained after fertilization were examined and compared with oocytes supplemented with either 2 µM of resveratrol or 6 µM phloretin (treatment groups) during IVM. RESULTS: Aged oocytes had a significantly lower mitochondrial mass and proportion of mitochondrial clustered pattern, lower ooplasmic volume, higher ROS, lower sirtuin-1 protein level, and a lower blastocyst rate in comparison to young oocytes, indicating that postovulatory oocytes have a lower quality and developmental competence, thus validating our experimental model. Supplementation of IVM medium with antioxidants prevented the generation of ROS and restored the active mitochondrial mass and pattern characteristic of younger oocytes. Moreover, sirtuin-1 protein levels were also restored but only following incubation with resveratrol. Despite these findings, the blastocyst rate of treatment groups was not significantly different from the control group, indicating that resveratrol and phloretin could not restore the oocyte competence of postovulatory aged oocytes. CONCLUSION: Resveratrol and phloretin can both revert the age-related oxidative stress and mitochondrial dysfunction during postovulatory aging but were insufficient to enhance embryo developmental rates under our experimental conditions.

Identification of novel compound heterozygous ZFP36L2 variants implicated in oocyte maturation defects and female infertility.

PURPOSE: To explore the pathogenesis of oocyte maturation defects. METHODS: Whole exome sequencing was conducted to identify potential variants, which were then confirmed within the pedigree through Sanger sequencing. The functional characterization of the identified variants responsible for the disease, including their subcellular localization, protein levels, and interactions with other proteins, was verified through transient transfection in HeLa cells in vitro. Additionally, we employed real-time RT-PCR and single-cell RNA sequencing to examine the impact of ZFP36L2 pathogenic variants on mRNA metabolism in both HeLa cells and mouse or human oocytes. RESULTS: A novel compound heterozygous variant in ZFP36L2 (c.186T > G, p.His62Gln and c.869 C > T, p.Pro290Leu) was discovered in a patient with oocyte maturation defects. Our findings indicate that these variants lead to compromised binding capacity of the ZFP36L2-CONT6L complex and impaired mRNA degradation in HeLa cells and mouse oocytes. Furthermore, we characterized the changes in the human oocyte transcriptome associated with ZFP36L2 variants, with a particular emphasis on cell division, mitochondrial function, and ribosome metabolism. CONCLUSIONS: This study broadens the mutation spectrum of ZFP36L2 and constitutes the first report of human oocyte transcriptome alterations linked to ZFP36L2 variants. In conjunction with existing knowledge of ZFP36L2, our research lays the groundwork for genetic counseling aimed at addressing female infertility.

Maternal age at transfer following autologous oocyte cryopreservation is not associated with live birth rates.

PURPOSE: Our aim was to evaluate if maternal age at transfer following autologous oocyte cryopreservation is associated with live birth rate (LBR). METHODS: We performed a retrospective cohort study of all patients who thawed autologous oocytes and then underwent a single frozen euploid embryo transfer between 2011 and 2021 at a large urban university-affiliated fertility center. Each oocyte thaw patient was matched 2:1 to in vitro fertilization (IVF) patients who underwent single embryo transfer < 1 year after retrieval. Primary outcome was LBR. Secondary outcomes included implantation rates (IR) and spontaneous abortion rates (SABR). RESULTS: A total of 169 oocyte thaw patients were matched to 338 IVF patients. As expected, oocyte thaw patients were older (median age 42.5 vs. 37.6 years, p < 0.001) and waited longer between retrieval and transfer than in vitro fertilization patients (median time 59 vs. 1 month, p < 0.001). In univariate analysis, implantation and LBR differed among oocyte thaw and IVF patients (p < 0.05), but SABR did not (p = 0.57). Transfer outcomes in oocyte thaw patients did not differ based on transfer age group (IR: p = 0.18; SABR: p = 0.12; LBR: p = 0.24). In a multiple logistic regression model, age at transfer was not predictive of live birth when controlling for age at retrieval, embryo morphology, and day of blastulation. CONCLUSIONS: Maternal age at transfer after oocyte cryopreservation is not predictive of LBR; this suggests that "an aging womb" does not impair LBR after oocyte thaw and empowers patients to return for transfer when ready for childbearing.

Outcomes of oocyte retrievals completed by reproductive endocrinology and infertility fellows versus faculty: a 7-year retrospective review.

PURPOSE: To examine outcomes of oocyte retrievals completed by Reproductive Endocrinology and Infertility (REI) fellows versus faculty physicians. METHODS: This retrospective cohort study examined patients who underwent oocyte retrievals at Mayo Clinic from July 15, 2009, to December 15, 2016. The primary outcome was the oocyte retrieval rate (ORR) calculated per retrieval as the number of oocytes retrieved per follicles aspirated. The Wilcoxon signed-rank test was used to compare follicle and oocyte counts and ORR between fellows and faculty during the same bilateral retrieval. RESULTS: The study cohort included the first bilateral retrieval from 845 unique patients completed by 11 fellows and seven faculty. The median ORR was not statistically different for fellows and faculty (0.79 versus 0.80, p = 0.46). To assess for a learning curve, the outcomes of seven fellows who completed at least 80 retrievals in their first year were examined as four chronologically ordered sets of 20. When these sets were compared to the faculty physician mean ORR, no significant differences were found (p-values of 0.69, 0.69, 0.81, and 0.81, respectively). CONCLUSION: There were no significant differences in oocyte retrieval rates between fellows versus faculty over a 7-year period, with no significant learning curve observed. These findings suggest that fellows possess the requisite skills for successful oocyte retrieval upon entering REI fellowship following their OB/GYN residency. However, this does not diminish the critical role of comprehensive fellowship training and close supervision, especially in initial and complex cases.

Histological and Transcriptomic Insights into the Ovary Development of Hemibarbus labeo Injected with Spawn-Inducing Hormones.

Naturally, the ovaries of many farmed fish can only develop to stage IV (mainly including stage IV oocytes, known as full-grown postvitellogenic oocytes). Therefore, spawn-inducing hormone injections are used to promote ovary development and oocyte maturation, facilitating reproduction in the aquaculture industry. The study of spawn-inducing hormones and their underlying neuroendocrine mechanisms has been a recent focus in fish reproductive biology. However, the intra-ovarian regulatory mechanisms of ovary development and oocyte maturation after hormone injection require further investigation. In this study, we explored the histological and transcriptomic map of the ovary of Hemibarbus labeo after hormone injection to reveal changes in the ovary. The gonad index significantly increased after hormone injection for 5.5 h, after which no significant change was observed. Histological analysis showed that the nuclei had moved to one side of the oocytes at 5.5 h after hormone injection. Moreover, the volume of the oocytes increased and their yolk membranes thickened. Oocytes then underwent their first meiotic division at 5.5-11 h after hormone injection. Subsequently, the follicular membrane was ruptured, and ovulation was completed at 11-16.5 h after hormone injection. In addition, we identified 3189 differentially expressed genes (DEGs) on comparing the transcriptomes at different time points after hormone injection. These DEGs were significantly enriched in the GO terms of nervous system process, molecular transducer activity, and extracellular region, and the KEGG pathways of TNF signaling and cytokine-cytokine receptor interaction; these may play important roles in ovary development and oocyte maturation. Within these pathways, genes such as apoe, creb3, jun, junb, il11, and il8 may play important roles in steroid hormone synthesis and ovulation. Conclusively, our results show detailed sequential dynamics of oocyte development and provide new insights into the intra-ovarian regulatory mechanisms of ovarian development and oocyte maturation in H. labeo. These findings may be important for research on improving egg quality and reproduction in aquaculture.

Too many embryos: a critical perspective on a global challenge.

Over the past decade, significant endeavors have been directed toward establishing an optimal oocyte number to maximize the chances for successful in vitro fertilization outcomes. The effectiveness of assisted reproductive technologies has greatly improved, and more good-quality embryos are being created in each cycle. However, many of these embryos remain unused. Notably, in Europe, approximately one-third of couples did not use their surplus cryopreserved embryos. Surplus embryos pose a challenge for patients and clinics. Embryo disposal practices are not the same all over the continent, with embryo donation and embryo discharge not allowed in several countries. In this scenario, limiting the number of surplus embryos by reducing the number of inseminated oocytes, according to couple clinical history, could be a strategy.