Sperm cryopreservation is widely used for male fertility preservation and assisted reproduction technologies; however, the freezing and thawing process can induce irreversible structural and functional damage to human spermatozoa. This review summarizes the main mechanisms through which cryopreservation disrupts redox homeostasis, with particular emphasis on the generation of reactive oxygen species, mitochondrial dysfunction, lipid peroxidation, DNA fragmentation, and the consequent impairment of sperm motility and viability. Human spermatozoa are particularly vulnerable to oxidative damage because they possess limited cytoplasmic antioxidant defenses and a plasma membrane rich in polyunsaturated fatty acids (PFA), which are highly susceptible to peroxidative injury. In addition, the dilution or removal of seminal plasma during cryopreservation further compromise extracellular antioxidant protection. Experimental studies have shown that supplementation of freezing and/or thawing media with enzymatic and non-enzymatic antioxidants, including catalase, glutathione, ascorbic acid, tocopherols, melatonin, resveratrol, quercetin, MitoTEMPO, myo-inositol, elamipretide, brain-derived neurotrophic factor, and various natural extracts, may improve selected post-thaw sperm parameters and reduce biomarkers of oxidative stress. Nevertheless, no antioxidant strategy currently available is capable of completely preventing cryodamage, and the market heterogeneity of existing protocols limits their clinical translation. Further well-designed human studies are therefore required to determine the safety, efficacy, optimal concentration, and clinical utility of antioxidant supplementation in sperm cryopreservation media.

Nanomaterials are increasingly used in manufacturing, medicine, consumer products, and environmental technologies due to their unique physicochemical properties. Although these materials offer substantial technological and societal benefits, their widespread use has raised concerns about potential health risks. Of particular importance is exposure during pregnancy, as certain nanoparticles can cross the placental barrier and reach the developing embryo. Fetal tissues are highly sensitive to environmental insults, so maternal exposure to nanoparticles may disrupt normal development and increase the risk of abnormal pregnancy outcomes. This review examines the current understanding of nanoparticle-induced developmental toxicity, with a focus on the vulnerability of the maternal–fetal unit. We discuss the structure and function of the placental barrier and the mechanisms that enable nanoparticle transfer from mother to fetus. Particular attention is given to how nanoparticle characteristics, including size, shape, composition, and surface chemistry, influence biodistribution, placental transport, tissue accumulation, and toxicity. We summarize the major molecular and cellular mechanisms implicated in fetotoxicity, highlighting oxidative stress, apoptosis, autophagy, and DNA damage as recurring pathways identified across experimental studies. These interconnected processes contribute to placental dysfunction, impaired fetal growth, developmental abnormalities, and adverse pregnancy outcomes. We also compare findings across different classes of nanoparticles, including metal, metal oxide, carbon-based, and polymeric nanomaterials, identifying both shared toxicological mechanisms and material-specific effects. Evidence from animal models demonstrates that susceptibility varies according to nanoparticle properties, exposure conditions, and species, underscoring the complexity of nanoparticle–biological interactions and the limitations of extrapolating experimental findings directly to humans. Overall, the available evidence indicates that nanoparticle exposure during pregnancy represents a potential risk to fetal health, although important knowledge gaps remain regarding human exposure and long-term developmental outcomes. A better understanding of the mechanisms underlying nanoparticle-induced fetotoxicity is essential for improving human health risk assessment, refining experimental models, informing regulatory policies, and supporting the safe-by-design development of nanomaterials. Such knowledge will help ensure the responsible application of nanotechnology while minimizing potential risks during pregnancy. Finally, this review is distinguished by its integrated analysis of how the chemical characteristics of nanoparticles govern placental transfer and the mechanistic pathways of fetotoxicity across multiple nanomaterial classes, providing a unified framework that connects material properties with their potential for abnormal fetal development and adverse pregnancy outcomes.

Objective: To provide a rigorous, multidisciplinary synthesis of the epidemiological, genetic, immunological, and environmental factors contributing to Premature Ovarian Failure (POF), with particular attention to regional disparities, occupational exposures, the impact of chemotherapy, the occurrence and recurrence of endometrosis, and emerging fertility preservation strategies. Design: A structured literature review with an emphasis on recent advances in genetic and immunological understanding. Setting: Academic research and clinical insights from multidisciplinary contributors. Patients: Individuals diagnosed with POF as reported in the literature. Interventions: Review of literature concerning epidemiology, genetic mutations, immunological disorders, and surgical outcomes linked to POF. Main outcome measures: Identification of both established and emerging risk factors, validation of genetic and immunological markers, and clarification of diagnostic and preventive clinical approaches. Results: The prevalence of POF varies globally, affecting 1% of women under 40. Genetic factors, particularly mutations in the FMR1 and BMP15 genes, play a significant role, alongside autoimmune diseases. Chemotherapy is a leading iatrogenic cause, while endometriosis and ovarian cyst surgeries significantly contribute to diminished ovarian reserve. Conclusion: POF is a multifactorial condition with rising incidence in specific subgroups. Improved early detection, standardized biomarker use, and expanded access to fertility preservation are essential. Targeted genomic and occupational risk screening may enable personalized interventions. Further genomic studies are needed to elucidate rare mutations and their impact.

Background/Objectives: Male infertility is a complex, multifactorial condition influenced by infectious, inflammatory, and autoimmune components. Immunological factors, though implicated in reproduction, remain poorly understood. This study aims to deepen the understanding of infections, inflammation, and autoimmune factors in male infertility, with a focus on immune-related disorders affecting the testes and epididymis—immunologically privileged but vulnerable sites. These factors can impair sperm quality through oxidative stress (ROS) and antisperm antibodies (ASA), further compromising fertility. Methods: A narrative review was conducted by analyzing scientific literature from the past 10 years conducted on PubMed using keywords such as “male infertility”, “autoimmunity”, and “inflammatory disease”. Studies focusing on testicular and epididymal disorders, immunological impacts, and therapeutic approaches were included. Results: Our research highlights that conditions like epididymitis, vasectomy, testicular trauma, and previous surgeries can trigger inflammatory responses, leading to ASA formation and oxidative stress. ASA, particularly sperm-immobilizing antibodies, inhibits sperm motility and migration in the female reproductive tract. Infections caused by sexually transmitted bacteria or urinary pathogens frequently induce epididymo-orchitis, a primary contributor to male infertility. While standardized methodologies for ASA testing remain elusive, assisted reproductive treatments such as intracytoplasmic sperm injection (ICSI), in vitro fertilization (IVF), and intrauterine insemination (IUI) show promise in overcoming immune-mediated infertility. Conclusions: This review underscores the critical role of infection, inflammation, and autoimmune responses in male infertility. It highlights the necessity of improving diagnostic methods, understanding immune-pathological mechanisms, and addressing medicolegal issues associated with male infertility. This knowledge could pave the way for innovative therapies, ultimately enhancing fertility outcomes, and mitigating the societal and legal repercussions of infertility.

Semen quality is a key factor in male fertility, but defining normal reference values for semen parameters remain challenging. Over the past four decades, several authors have reported a noticeable decline in sperm parameters, raising concerns about male reproductive health. While the exact causes remain unclear, potential contributors include environmental pollution, endocrine disruptor chemicals (EDCs) and oxidative stress, with the latter becoming a growing concern. Environmental changes and increased exposure to EDCs, such as pesticides, herbicides, bisphenol A (BPA), phthalates, polychlorinated biphenyls (PCBs), and heavy metals, are believed to contribute significantly to the decline in sperm quality. These chemicals impact individuals from prenatal life through adulthood, potentially leading to long-term reproductive consequences. Overall, this review explores the relationship between environmental toxicants, including volatile organic compounds, EDCs, as well as oxidative stress and reduced male fertility. While a substantial body of research has found associations between these exposures and adverse fertility outcomes, some studies have reported no significant associations. The primary objective of this review is to provide a deeper understanding of the potential mechanisms between these environmental chemicals on testicular function and spermatogenesis. It also examines the broader evidence on the decline in sperm quality and explores its potential implications for overall fertility outcomes in humans. By doing so, the review will shed light on the broader public health implications of environmental pollutants and their impact on male reproductive health, emphasizing the need for further research in this critical area.

OBJECTIVE:Frozen embryo transfer in humans, especially at the blastocyst stage, provides a valid alternative to fresh embryo transfer. However, protocols for blastocyst vitrification are not yet standardized; for example, exposure to the first equilibration solution before vitrification commonly ranges from 2-3 minutes at 37°C or 2-15 minutes at room temperature. This study compared the clinical and neonatal outcomes involving vitrified-warmed blastocysts. METHODS:The main aim of this prospective study was to compare the clinical and neonatal outcomes for 831 warmed blastocysts, returned in 585 frozen embryo transfers with two exposure times to the equilibration solution at room temperature: (A) 7-8 minutes and (B) 9-10 minutes. RESULTS:The patients' characteristics were comparable between the two groups with no significant difference in their mean age, the average number of blastocysts transferred, basal Follicle Stimulating Hormone, body mass index (BMI), infertility duration, primary infertility, or endometrial thickness. The survival and clinical pregnancy rates of the vitrified-warmed laser collapsed blastocysts were not different between the two groups. The overall miscarriage rate was significantly lower in the 7-8 minute compared to the 9-10-minute equilibration group (A: 7.6% versus B: 14.2%, p<0.05). Live birth, multiple gestation and neonatal outcomes were similar between the two groups. CONCLUSIONS:Our results indicate that the equilibration time can affect the efficiency of the cryopreservation process of human blastocysts.

Assisted reproduction technology (ART) has advanced significantly over the past four decades, leading to improved pregnancy outcomes and a reduction in complications, particularly those associated with multiple pregnancies. These improvements largely stem from advances in understanding embryonic physiology, which has enabled better culture conditions. As a result, embryologists can now efficiently culture embryos to the blastocyst stage and successfully cryopreserve them for future use. However, while incubators aim to replicate the maternal environment of the oviduct and uterus, embryos in vitro are cultured in static conditions, unlike the dynamic, constantly changing environment they experience in vivo. Key factors such as pH, temperature, osmolality, and gas concentrations are crucial for establishing optimal embryo development and implantation potential. Moreover, the vitrification procedure for gametes or embryos can introduce oxidative stress, as well as osmotic shock and cryoprotectant toxicity, which may affect embryo viability and increase the risk of birth defects. Since the first successful ART birth in 1978, over 10 million babies have been conceived through these techniques. Although most of these children are healthy, concerns exist about potential birth defects or changes linked to the handling of gametes and embryos. The preimplantation period is marked by significant epigenetic reprogramming, which can be influenced by ART procedures such as ovarian stimulation, in vitro fertilization, embryo culture, and cryopreservation. However, the long-term health implications for offspring remain uncertain. Epigenetic reprogramming during early embryogenesis is essential for proper embryo development and can be changed by ART-related conditions. These concerns have raised questions about the possible connection between ART and a higher risk of birth defects or other changes in children born through these methods. Therefore, we conducted a scoping review following PRISMA-ScR guidelines to map evidence on ART-related risks, including epigenetic and birth defect outcomes.

During human in-vitro culture, morphological microscope analysis is routinely used to select embryos with the highest implantation potential for transfer, aiming for successful pregnancy and healthy live birth. This evaluation includes blastomere number, size, fragmentation, multinucleation, blastocyst (BL) expansion, and the inner-cell mass and trophectoderm appearance. However, this method requires removing embryos from the incubator, exposing them to non-physiological conditions such as fluctuations in pH, temperature, gases concentrations, as well as significant inter-observer variability. Continuous embryo culture using time-lapse monitoring (TLM) has revolutionized embryo evaluation by allowing continuous, real-time tracking of embryo development from fertilisation to blastocyst formation. This reduces the need to remove embryos from the incubator and helps maintain stable culture conditions. The monitoring system typically includes a standard incubator with an integrated microscope coupled to a digital camera, capturing images at regular intervals that are processed into a video for analysis. Despite its advantages, accurately predicting implantation rates in humans remains challenging. Recently, artificial intelligence (AI) has emerged as promising tool to objectively evaluate human embryos. AI can analyse large datasets, including embryological, clinical, and genetic information, and assist in individualizing treatment protocols. Integrating AI with TLM could improve embryo selection and enhance overall success rates. This paper explores the potential benefits of combining TLM and AI in reproductive and embryology laboratories, highlighting their potential to improve the outcomes of human ART.

Objective: To provide a rigorous, multidisciplinary synthesis of the epidemiological, genetic, immunological, and environmental factors contributing to Premature Ovarian Failure (POF), with particular attention to regional disparities, occupational exposures, the impact of chemotherapy, the occurrence and recurrence of endometrosis, and emerging fertility preservation strategies. Design: A structured literature review with an emphasis on recent advances in genetic and immunological understanding. Setting: Academic research and clinical insights from multidisciplinary contributors. Patients: Individuals diagnosed with POF as reported in the literature. Interventions: Review of literature concerning epidemiology, genetic mutations, immunological disorders, and surgical outcomes linked to POF. Main outcome measures: Identification of both established and emerging risk factors, validation of genetic and immunological markers, and clarification of diagnostic and preventive clinical approaches. Results: The prevalence of POF varies globally, affecting 1% of women under 40. Genetic factors, particularly mutations in the FMR1 and BMP15 genes, play a significant role, alongside autoimmune diseases. Chemotherapy is a leading iatrogenic cause, while endometriosis and ovarian cyst surgeries significantly contribute to diminished ovarian reserve. Conclusion: POF is a multifactorial condition with rising incidence in specific subgroups. Improved early detection, standardized biomarker use, and expanded access to fertility preservation are essential. Targeted genomic and occupational risk screening may enable personalized interventions. Further genomic studies are needed to elucidate rare mutations and their impact.

Background:Infertility is a multifactorial condition that affects both men and women and is influenced by various factors, including overweight and obesity. These conditions, especially in women with polycystic ovary syndrome (PCOS), are strongly associated with hormonal and metabolic imbalances that can impair fertility. Targeted nutritional interventions, such as nutraceutical supplementation, may offer support in improving reproductive outcomes. Methods:A narrative review was conducted using PubMed, focusing on publications from the past 12 years with the keywords "nutraceutical," "overweight," and "infertility." The review aimed to identify the main nutraceuticals used in managing infertility and to highlight the importance of a personalized approach tailored to individual patient characteristics. Results:Nutraceuticals may represent a safe and cost-effective adjunctive strategy to support fertility in overweight patients, particularly in those with PCOS. Evidence suggests that their effectiveness increases when integrated into a personalized treatment plan based on individual needs and clinical profiles. Conclusions:This review offers an updated overview of nutraceutical use in overweight individuals with infertility, outlining both benefits and limitations. It also addresses the often-overlooked medico-legal aspects of prescribing nutraceuticals, emphasizing the need for ethical and legal awareness when incorporating these interventions into clinical practice.

Objective: In assisted reproductive technology (ART), achieving a successful pregnancy requires optimizing an embryo culture and selecting the single embryo with the highest implantation potential, capable of resulting in a healthy pregnancy. The primary goal of this study was to determine the correlation between the blastocyst area and diameter and pregnancy outcomes in ART treatments. Methods: In this study, the blastocyst diameter and area were measured to determine whether these morphometric features could predict pregnancy outcomes in couples undergoing ART with ICSI. This is a retrospective trial analyzing 665 patients who underwent an ART cycle with the transfer of a single blastocyst on day 5. Results: Both morphometric features assessed were significantly associated with implantation and ongoing pregnancy outcomes. Our results showed that the implantation rate (IR) and ongoing clinical pregnancy rate (CPR) were significantly higher with a blastocyst area ≥ 25,000 µm2 compared to <25,000 µm2 (IR: 69.8% versus 47.9%, p < 0.001; CPR: 65.5% versus 45.9% p < 0.001). Additionally, a blastocyst diameter ≥ 170 µm resulted in a significantly higher IR and CPR compared to embryos with a diameter < 170 µm (IR: 68.8% versus 36.6%, p < 0.001; CPR: 66.3 versus 35.7%, p < 0.001). Conclusions: Blastocyst morphometric variables, being objective and measurable, are not subject to intra-operator variability and may serve as promising predictors of embryo viability and ongoing pregnancy success. These morphometric assessments could assist embryologists in selecting the embryo with the highest implantation potential from a cohort, as well as identifying those with a reduced chance of generating a successful pregnancy.

IntroductionInfertility is characterized by the failure to conceive after 12 months of unprotected sexual intercourse. In assisted reproduction technologies (ARTs), in-vitro fertilization and embryo transfer (IVF-ET) are pivotal, with the quality of embryo quality essential for successful implantation.ObjectiveThis systematic review with meta-analysis aimed to explore the prevalence of embryonic factors involved in the implantation process, concentrating on the following research inquiries: 1) the implantation rates of euploid versus untested embryo transfers; 2) the efficiency of transferring good embryos in different age groups; 3) the impact of age on good embryo transfers to gestational carriers; and 4) the transfer of donated gametes/embryos. The goal is to identify critical points in implantation to improve therapies.MethodsA comprehensive literature search identified 1474 relevant papers, 11 of which met the inclusion criteria. The information was gathered using a standardized form, and the risk of bias was evaluated. A meta-analysis of subgroups to determine euploid embryo transfer efficiency was conducted to synthesize and explore the results. Furthermore, data extracted from registries document the persistent secondary role of extraembryonic determinants in successful implantation.ResultsThe meta-analysis demonstrated that preimplantation genetic testing for aneuploidy (PGT-A) significantly increased the odds of implantation. Age was found to influence extraembryonic factors, with older women experiencing reduced embryo implantation as gestational carriers. However, the overall incidence of extraembryonic factors was low. This review highlights the need to focus on PGT-A, diagnostic hysteroscopy, and endometrial receptivity for improving implantation rates.ConclusionImplantation success in ARTs largely depends on embryo euploidy. While achieving three euploid embryos greatly increases success rates, it is challenging in older women. Extraembryonic factors, although present, have a marginal impact. Subsequent studies ought to concentrate on modulating endometrial responses immunologically and developing algorithms to improve the precision of predicting implantation success; as well as the timing of endometrial receptivity and the occurrence of dormant embryo phenomena also warrants further investigation.

In the last decades, to enhance success rates in assisted reproductive technology (ART) cycles, scientists have continually tried to optimize embryo culture and selection to increase clinical outcomes. In this scenario, the application of laser technology has increased considerably worldwide and is currently applied across ART in several ways: for assisted hatching (AH) or thinning of the zona pellucida (ZP), embryo biopsy, to immobilize and select the sperm during intracytoplasmic sperm injection, as well as to induce artificial blastocyst shrinkage before cryopreservation. Laser-AH has been suggested as a procedure to improve embryo implantation: the concept is that drilling holes through or thinning of the ZP could improve the hatching process and implantation. The artificial disruption of the ZP can be performed by different approaches: mechanically, chemically and with the laser, which is one of the most favourable and easy methods to remove part of the ZP and to augment the possibilities of implantation in patients defined as having a poor prognosis of success, or when the ZP is too thick. However, in the current literature, there is not sufficient evidence about the potential risk or impairment that laser utilization might induce on embryo development; therefore, the main aim of the current review is to provide an overview of the existing knowledge on the ZP and the mechanisms of manipulating it to improve the effectiveness of ART. Also, it emphasizes the positive aspect of laser application as a powerful tool that might increase the chance of pregnancy for infertile couples undergoing ART cycles.

Male infertility accounts for approximately 40% of infertility cases. There are many causes of male infertility, including environmental factors, age, lifestyle, infections, varicocele, and cancerous pathologies. Severe oligozoospermia, cryptozoospermia, and azoospermia (obstructive and non-obstructive) are identified as severe male factor infertility, once considered conditions of sterility. Today, in vitro fertilization (IVF) techniques are the only treatment strategy in cases of male factor infertility for which new methodologies have been developed in the manipulation of spermatozoa to achieve fertilization and increase success rates. This review is an update of in vitro manipulation techniques, in particular sperm selection, emphasizing clinical case-specific methodology. The success of an IVF process is related to infertility diagnosis, appropriate choice of treatment, and effective sperm preparation and selection. In fact, selecting the best spermatozoa to guarantee an optimal paternal heritage means increasing the blastulation, implantation, ongoing pregnancy and live birth rates, resulting in the greater success of IVF techniques.

Semen quality represents a compelling factor for fertility, and delineating the normal values has proven difficult. In the last four decades, several authors have reported a noticeable decline in sperm parameters. Also, studies investigating ‘time to pregnancy’ have shown that fecundity begins to be reduced when sperm numbers decrease below 30 million, even though according to the 6th edition of the WHO manual, the normal value is currently 16 million/mL or 39 million per ejaculate. There exists sufficient data to suggest a decline in sperm counts over time, even though the clear reason for this adverse trend is not well established, but some associations have been hypothesised, such as maternal smoking during pregnancy. Additional potential factors have yet to be fully illustrated but involve poor diet, increased obesity, and exposure to environmental toxins. Moreover, the change in environmental conditions and more common exposure to endocrine-disrupting chemicals (EDCs), such as pesticides and herbicides, as well as bisphenol A, phthalates, polychlorinated biphenyls, and heavy metals, starting from prenatal life and continuing into adulthood, may exhibit probable features explaining the reduction in sperm parameters. Therefore, the main goal of this narrative review is to furnish an overview of the possible effects of exposure to EDCs on testicular function and spermatogenesis and, also, to summarise the evidence regarding a decrease in sperm quality and examine its potential consequences.

The cryopreservation of gametes and embryos has increased notably over the past 20 years and is now an essential part of assisted reproductive technologies (ARTs). However, because the cryopreservation process is un-physiological for human cells, gametes, and embryos, cryobiologists have suggested diverse methods to successfully cryopreserve human gametes and embryos in order to maintain their viability and assure successful pregnancy. During the first period of early development, major waves of epigenetic reprogramming—crucial for the fate of the embryo—occur. Recently, concerns relating to the increased incidence of epigenetic anomalies and genomic-imprinting disorders have been reported after ARTs and cryopreservation. Epigenetic reprogramming is particularly susceptible to environmental and un-physiological conditions such as ovarian stimulation, embryo culture, and cryopreservation that might collectively affect epigenetics dysregulation. Additionally, recent literature suggests that epigenetic and transcriptomic profiles are sensitive to the stress induced by vitrification, osmotic shock, oxidative stress, rapid temperature and pH changes, and cryoprotectants; it is therefore critical to have a more comprehensive understanding of the potential induced perturbations of epigenetic modifications that may be associated with vitrification. The aim of this paper is to present a critical evaluation of the association of gamete and embryo cryopreservation, use of cryoprotectants, and epigenetic dysregulations with potential long-term consequences for offspring health.

Following the advancement of medically assisted reproduction (MAR) technology, and the rationale to extend the culture to the blastocyst stage, performing elective single embryo transfer (eSET), gamete quality and assessment have acquired large relevance in ART. Embryo quality is strictly correlated with gametes quality and culture conditions. Oocyte maturity assessment is therefore imperative for fertilization and embryo evolution. Mature oocytes at the metaphase II stage result in a higher fertilization rate compared to immature oocytes. Indeed, oocyte morphology evaluation represents an important and challenging task that may serve as a valuable prognostic tool for future embryo development and implantation potential. Different grading systems have been reported to assess human embryos, however, in many cases, it is still a major challenge to select the single embryo to transfer with the highest implantation potential. Further, eSET has conferred a challenge to embryologists, who must try to enhance embryo culture and selection to provide an adequate success rate, whilst reducing the overall number of embryos transferred. Above the standard morphological assessment, there are several invasive or non-invasive approaches for embryo selection such as preimplantation genetic testing, time-lapse technology, proteomics and metabolomics, as well as oxygen utilization and analysis of oxidative stress in culture medium. This short review is not designed to be a comprehensive review of all possible features that may influence oocyte quality. It does give, however, a brief overview and describes the prognostic value of the morphological characteristics of human oocytes on their developmental capacity following ART treatments.

Abstract Study question To explore the relationship between equilibration time pre-vitrification of human collapse blastocyst and clinical pregnancy and miscarriage rate in patients undergoing ART treatments Summary answer The miscarriage rate was significantly lower in the group with a short equilibration time of 7-8 minutes compared to the group of 9-10 minutes What is known already The introduction of vitrification represents one of the most important advancement in ART. However, protocols currently applied to cryopreserve human embryos still have some weak points that might be improved. A critical aspect is represented by the high concentration of cryoprotectants (CPAs) used during the vitrification and some of this CPAs might impact negatively cellular metabolism and function. Thus, this study investigated whether a shorter time in the equilibration solution before vitrification of artificially collapsed blastocysts might have an impact on survival and pregnancy outcomes, as well as live birth rate and risk of miscarriage in patients undergoing ART. Study design, size, duration This prospective study was performed at the Centre for Reproductive Medicine, Haikou Mary Hospital, China from March 2018 to May 2022. Informed consent for experimentation with human subjects was obtained before the patients start the ovarian stimulation. Female age <35 years, and causes of infertility included male factors, female infertility, and unexplained infertility. Following the fresh embryo transfer, supernumerary good quality blastocysts ≥ 2, according to Gardner’s score were vitrified and allocated as discussed below Participants/materials, setting, methods The study included a total of 831 expanded blastocysts, which were divided into two groups according to the equilibration time before vitrification: group (A) 7-8 minutes (413); and group (B) 9-10 minutes (418). Expanded blastocysts (grade 3 or more) were artificially shrunk by applying one or two laser pulses. Patients were included in the study only when at least two blastocysts were available for vitrification, in order to be allocated one in each group Main results and the role of chance Results: A total of 831 vitrified-warmed blastocysts were analysed in this study, of which 825 survived at the warming step (99.3%: 825/831). All surviving blastocysts were transferred in 585 embryo transfers. Overall, the clinical pregnancy rate per transfer was CPR: 68.5% (401/585), and live birth and miscarriage rates: 61.0% (357/585) and 11.0% (44/401). No significant differences were observed between the two groups regarding the mean age of patients, the average number of blastocysts transferred, the basal FSH, BMI, and infertility duration. Results show the same survival rate after warming for group A (99.3%) and group B (99.3%), as well as similar CPR (A: 69.1% versus B: 68.0%).The live birth (A: 63.8% versus B: 58.3%) and multiple gestation rates (A: 20.8% versus B: 23.5%) were comparable in the two groups. When analysing the overall miscarriage rate, data displayed a statistically significant difference (P < 0.05) in favour of group A (7.6%) compared to group B (14.2%). There were no differences between the two groups concerning the prevalence of male babies (A: 57.1% and B: 55.4%), average gestational length (A: 38.67±1.37 versus B: 38.33±1.21), preterm birth rate (A: 19.2% versus B: 20.6%), and birth weight (A: 2.95±0.58 versus B: 3.05±0.63 kg). Limitations, reasons for caution This is not a randomized controlled trial, it is a prospective observational cohort study aiming to calculate the effect of a shorted ES time on the risk of miscarriage. Also, potential confounding factors due to the heterogeneous nature of the sample investigated may impair the validity of our conclusions. Wider implications of the findings Results demonstrate that a shorter equilibration time resulted in optimal survival, clinical pregnancy, and live birth rates compared to exposure to ES for 9-10. Thus, suggesting that a longer exposure to the ES is not needed, and might negatively effect cellular metabolism and function, increasing the risk of miscarriage. Trial registration number Not applicatione