Reliable evaluation of blastocyst quality is critical for the success of in vitro fertilization (IVF) treatments. Current embryo grading practices primarily rely on visual assessment of morphological features, which introduces subjectivity, inter-embryologist variability, and challenges in standardizing quality assurance. In this study, we propose a multitask embedding-based approach for the automated analysis and prediction of key blastocyst components, including the trophectoderm (TE), inner cell mass (ICM), and blastocyst expansion (EXP). The method leverages biological and physical characteristics extracted from images of day-5 human embryos. A pretrained ResNet-18 architecture, enhanced with an embedding layer, is employed to learn discriminative representations from a limited dataset and to automatically identify TE and ICM regions along with their corresponding grades, structures that are visually similar and inherently difficult to distinguish. Experimental results demonstrate the promise of the multitask embedding approach and potential for robust and consistent blastocyst quality assessment.

Mitochondrial function can be affected by mutations in mitochondrial DNA (mtDNA). However, detecting de novo mutations in mtDNA has been challenging due to its high copy number, particularly in germline cells, and the low accuracy of conventional next-generation sequencing technologies. Using highly accurate duplex sequencing, we study the frequency of de novo insertion and deletion (indel) mtDNA mutations across multiple age groups in somatic and germline tissues of three mammalian species-mouse, macaque, and human. We demonstrate that, similar to de novo nucleotide substitutions, indels accumulate rapidly with age in somatic tissues with high energetic demand (brain and skeletal muscle) or high proliferation (liver). However, in oocytes, indels accumulate slower with age than nucleotide substitutions (or do not accumulate at all). The increases in indel frequency with age are driven mostly by deletions. Short tandem repeats are highly enriched for indels, implicating DNA replication slippage as a major driver of indel formation in mtDNA. For some species and tissues, indels are depleted at protein-coding sequences; however, indels that are multiples of 3 bp are not overrepresented. Ours is the most detailed study of de novo small indels in mtDNA to date. It provides parameters for models of mtDNA evolution, informs molecular mechanisms for a multitude of human genetic diseases, and illuminates the accumulation of indel mutations with age. Such accumulation may have functional consequences, as it affects reproduction later in life and drives the decline of mitochondrial function during aging.

STUDY QUESTION:What is the current good practice in the IVF laboratory, based on the best available evidence in the literature, if available, and the expertise of the working group? SUMMARY ANSWER:The updated ESHRE Recommendations on Good Practice in the IVF laboratory provides recommendations on all activities in the IVF laboratory. WHAT IS KNOWN ALREADY?:A previous version of the Good Practice in the IVF laboratory guideline was published in 2015. The adoption of new techniques, the increasing complexity of procedures, and the introduction of a revised European legislation on the quality and safety of tissues and cells for human application pertinent to assisted reproduction together necessitated an update of the document. STUDY DESIGN, SIZE, DURATION:This document was developed according to a predefined methodology for ESHRE Good Practice recommendations. The working group reviewed the document of 2015, and based on this assessment, each group member updated one or more sections. Recommendations are supported by data from the literature, if available, and the expertise of the working group and were discussed until consensus was reached within the working group. PARTICIPANTS/MATERIALS, SETTING, METHODS:The working group included 10 members representing the ESHRE Special Interest Groups for Embryology, Safety and Quality, and Andrology, with different areas of expertise and representing different European countries and settings. Based on the available evidence and the expertise of the working group, recommendations were formulated. Following stakeholder review of the initial draft, the final version was approved by the working group and ultimately by the ESHRE Executive Committee. MAIN RESULTS AND THE ROLE OF CHANCE:The ESHRE IVF labs working group updated the recommendations on the general organization of an IVF laboratory (staffing and direction, quality management, and laboratory safety) and on the specific aspects of the procedures performed in IVF laboratories (identification of patients and traceability of their reproductive cells, consumables, handling of biological material, oocyte retrieval, sperm preparation, insemination of oocytes, scoring for fertilization, embryo culture and transfer, embryo biopsy for pre-implantation genetic testing, cryopreservation, and emergency procedures). The section on embryo culture and transfer was split up, the section on sperm preparation was expanded to cover all general andrological procedures, and a new section on biopsy procedure was introduced. LIMITATIONS, REASONS FOR CAUTION:Not all recommendations are supported by evidence. Other recommendations, published in legal documents, relevant and recent documents, manuals, and consensus papers, were taken into account when formulating the recommendations. WIDER IMPLICATIONS OF THE FINDINGS:The guideline group is confident that this document will be helpful to directors and managers involved in the management and organization of IVF laboratories and also to embryologists and laboratory technicians performing daily tasks. STUDY FUNDING/COMPETING INTEREST(S):The guideline was developed by ESHRE, which funded the guideline meetings, literature searches, and dissemination of the guideline. The guideline group members did not receive any financial incentives; all work was provided voluntarily. G.C. reports consulting fees from Gedeon Richter and Cooper Surgical and was part of the working group of the 'Guide to the quality and safety of tissues and cells for human application' of the European Directorate for the Quality of Medicines and Healthcare, on behalf of the Council of Europe. T.E. reports consulting fees from Nexpring Health and Esco Medical and speaker's fees from Nexpring Health and Esco Medical. J.K.-B. reports research grants from Gates Foundation and NIHR, consulting fees from Bayer, speakers fees from Merck, IBSA, Ferring and Cooper Surgical, and travel support from Merck, IBSA, Ferring, and Cooper Surgical. I.S. reports speaker's fees from Vitrolife and Cooper Surgical. I.S. also declares being a member of ARCS Scientific Committee. The other authors disclosed no conflicts of interest. DISCLAIMER:This guideline represents the views of ESHRE, which were achieved after careful consideration of the scientific evidence available at the time of preparation. In the absence of scientific evidence on certain aspects, a consensus between the relevant ESHRE stakeholders has been obtained. Adherence to these clinical practice guidelines does not guarantee a successful or specific outcome nor does it establish a standard of care. Clinical practice guidelines do not replace the need for application of clinical judgment to each individual presentation nor variations based on locality and facility type. ESHRE makes no warranty, express or implied, regarding the clinical practice guidelines and specifically excludes any warranties of merchantability and fitness for a particular use or purpose (full disclaimer available at www.eshre.eu/guidelines).

Embryo selection is one of multiple crucial steps in in-vitro fertilization, commonly based on morphological assessment by clinical embryologists. Although artificial intelligence methods have demonstrated their potential to support embryo selection by automated embryo ranking or grading methods, the overall impact of AI-based solutions is still limited. This is mainly due to the required adaptation of automated solutions to custom clinical data, reliance on time lapse incubators and a lack of interpretability to understand AI reasoning. The modern, informed patient is questioning expert decisions, particularly if the treatment is not successful. Thus, evidence-based decision justification in tasks like embryo selection would support transparent decision making and respectful patient communication. To support this aim, we hereby present an expert-annotated dataset consisting of embryo images and corresponding morphological description using natural language. The description contains relevant information on embryonic cell cycle, developmental stage and morphological features. This dataset enables the finetuning of modern foundational vision-language models to learn and improve over time with high accuracy. Predicted embryo descriptions can then be leveraged to automatically extract scientific evidence from literature, facilitating well-informed, evidence-based decision-making and transparent communication with patients. Our proposed dataset supports research in language-based, interpretable, and transparent automated embryo assessment and has the potential to enhance the decision-making process and improve patient outcomes significantly over time.

Mitochondrial dysfunction can cause a multitude of systemic diseases, and thus, the origin of such dysfunction is of great interest to many clinicians and researchers. Part of mitochondrial dysfunction can be explained by mutations in mitochondrial DNA (mtDNA), which can be measured both in somatic and germline cells. As the average maternal age continues to increase, determining if mutations in mtDNA increase with age is an important aspect of reproductive health care. This study was designed to assess the frequency of de novo mutations in mtDNA in oocytes based on age, compared with the frequency of mutations in somatic cells. This study used high-quality, full-length mtDNA sequences for 22 individuals between the ages of 20 and 42, obtaining samples from blood, saliva, and oocytes. Duplex sequencing was used to identify variants in mtDNA; median mtDNA enrichment was 85.4%, 0.7%, and 4.0% and a median duplex consensus sequence (DCS) depth of 1440X, 78X, and 158X for oocytes, blood, and saliva, respectively. There were 3525 high-confidence variants identified across all samples. Across age groups and geographic regions, there were significantly fewer mutations in oocytes compared with blood and saliva. When predicting the probability of a mutation in mtDNA as a function of age, there were significant increases in mutation frequency in blood and saliva, but not in oocytes (P<0.001, P<0.001, P=0.688, respectively). Comparing different age groups, mutation frequency was higher in blood and saliva in older women compared with younger women, but there was no significant difference in oocyte mutation frequency. Within oocyte mtDNA, mutations were more frequent in the D-loop than in the "coding region" for each age group, consistent with previous literature. This remained true in several different analyses of regions of mtDNA, though no significant differences were found related to age. A total of 109 de novo mutations were located at confirmed disease-associated sites, with 32 variants in 29 positions. Lower proportions of disease-related mutations were present in oocytes compared with blood or saliva, suggesting preferential selection for oocytes against mutations. Hotspots for variants were identified in each type of cell, showing 58 sites overlapping between cell types; 40 of these were in the D-loop, 4 in rRNA, 4 in tRNA, and 10 in protein-coding regions. This supports the previous finding of high mutation frequency in the D-loop for oocytes. These results indicate that there is no increase in mutation frequency in oocytes as women age, though there was an increase in mutation frequency in both blood and saliva. This contrasts with previous literature in animal models that showed either continuous increases in mutation frequency with age or increases up to a certain age after which there was a plateau. The age group of this study was limited to 20-42, meaning that there could be an increase in mutation frequency that occurs either before or after this time period in humans. This is the first study to use duplex sequencing, and thus the most accurate to date in terms of pinpointing variants and mutation sites. Future research should focus on validating these results with similar methods as well as implementing duplex sequencing of pedigrees to form a more complete picture.

Oocytes are densely packed with mitochondria, the energy-producing organelles that contain their own genome, mitochondrial DNA (mtDNA). Each cell contains multiple copies of mtDNA, with copy number varying among tissue types. Oocytes possess the highest mtDNA copy number, containing hundreds of thousands of mtDNA molecules per cell. Because mitochondria are inherited exclusively through the maternal lineage, accurate detection of mtDNA variants is essential for studies of inheritance, aging, and disease. The presence of multiple mtDNA copies allows wild-type and mutant molecules to coexist within the same cell, a condition known as heteroplasmy, in which low-frequency and de novo variants may occur at frequencies below 1%. Conventional next-generation sequencing (NGS) lacks sufficient accuracy to reliably distinguish these rare variants from errors introduced during library preparation and sequencing. Here, we present a protocol for enriching mtDNA from single human oocytes using Exonuclease V to remove linear DNA, followed by duplex sequencing library preparation for highly accurate mtDNA analysis. This workflow enables error-corrected sequencing of individual oocytes, facilitating reliable detection of low-frequency mtDNA variants and analysis of heteroplasmy and de novo mutagenesis. The protocol provides a reproducible approach for investigating mitochondrial genome variation in single oocytes using Illumina-compatible sequencing platforms.

The application of artificial intelligence (AI) in IVF has shown promise in improving consistency and standardization of decisions, but often relies on annotated data and does not make use of the multimodal nature of IVF data. We investigated whether foundational vision-language models can be fine-tuned to predict natural language descriptions of embryo morphology and development. Using a publicly available embryo time-lapse dataset, we fine-tuned PaliGemma-2, a multi-modal vision-language model, with only 1,000 images and corresponding captions, describing embryo morphology, embryonic cell cycle and developmental stage. Our results show that the fine-tuned model, InVitroVision, outperformed a commercial model, ChatGPT 5.2, and base models in overall metrics, with performance improving with larger training datasets. This study demonstrates the potential of foundational vision-language models to generalize to IVF tasks with limited data, enabling the prediction of natural language descriptions of embryo morphology and development. This approach may facilitate the use of large language models to retrieve information and scientific evidence from relevant publications and guidelines, and has implications for few-shot adaptation to multiple downstream tasks in IVF.

The selection of the optimal embryo for transfer is a critical yet challenging step in in vitro fertilization (IVF), primarily due to its reliance on the manual inspection of extensive time-lapse imaging data. A key obstacle in this process is predicting blastocyst formation from the limited number of daily images available. Many clinics also lack complete time-lapse systems, so full videos are often unavailable. In this study, we aimed to predict which embryos will develop into blastocysts using limited daily images from time-lapse recordings. We propose a novel hybrid model that combines DINOv2, a transformer-based vision model, with an enhanced long short-term memory (LSTM) network featuring a multi-head attention layer. DINOv2 extracts meaningful features from embryo images, and the LSTM model then uses these features to analyze embryo development over time and generate final predictions. We tested our model on a real dataset of 704 embryo videos. The model achieved 96.4

This study introduces Blasto-Net, a multi-task deep learning model for comprehensive blastocyst analysis. The proposed model performs three tasks simultaneously in a single forward pass: segmentation of the ZP, TE, and ICM compartments, morphological grading, and implantation outcome prediction. Accurate blastocyst analysis in in vitro fertilization (IVF) is challenging. The compartments often have similar textures but very different structures. To address these challenges, Blasto-Net employs an EfficientNet-B3 encoder with a UNet-style decoder enhanced by the Convolutional Block Attention Module (CBAM) and a novel Edge-Aware Attention Module (EAAM) to effectively capture both semantic and boundary information. To handle distinct compartment topologies, the network employs specialized segmentation heads and a composite region- and boundary-based loss. Additionally, Grad-CAM++ visualizations are used to verify the anatomical consistency of the model's predictions. Evaluated on a public HMC blastocyst dataset, Blasto-Net achieves Dice scores of 94.93

Chronic endometritis (CE) is frequently diagnosed in women with repeated implantation failure (RIF) and recurrent pregnancy loss (RPL), yet the lack of standardized diagnostic criteria and uncertainty about the timing of assessment and optimal treatment lead to open questions regarding its clinical relevance. This study aims to identify clinical risk factors that may guide targeted CE testing and to evaluate an ideal time point in the diagnostic pathway to offer CE assessment in women with RIF and RPL. In this retrospective cohort study, 392 women with RIF (no pregnancy after two or more transfers of good quality blastocysts) and 119 women with RPL (two or more subsequent miscarriages) who underwent endometrial biopsy with CD138 immunohistochemistry between 2016 and 2024 were analyzed. Odds ratios for presumed CE risk factors were calculated and CE prevalence and reproductive outcomes were assessed. Women in the RPL group had a higher prevalence of CE compared to the RIF group (39.5 vs. 25.0

Research question: What are the effects of pipette- versus laser-assisted artificial blastocyst collapse (ABC) on the morphokinetics of warmed blastocyst re-expansion, and what is the potential effect on treatment outcomes? Design: Surplus blastocysts were extracted from 203 patients. These were divided into three groups: study group A, artificial collapsed by the aspiration of blastocoel fluid with a pipette; study group B, trophectoderm opened with a laser pulse; control group, no manipulation before vitrification was performed. During the 5-year study period, 257 associated single-warm blastocyst transfers were scheduled. The start and duration of the re-expansion process before transfer were annotated. Pregnancy and live birth data were also collected for the transfers. Results: The overall blastocyst survival rate was 96.9%, with no effect observed as a result of the two ABC methods. The re- expansion of blastocysts in study group B was initiated significantly sooner after warming (0.50 f 0.37 h) than in group A (0.79 f 0.56 h) or the control group (1.22 f 1.00 h). The duration of the re-expansion process was significantly reduced in study groups A (P = 0.021) and B (P = 0.004) compared with the control group. The embryos of participants who achieved a live birth had a significantly (P < 0.001) faster start of re-expansion (0.60 f 0.42 h) than the embryos in those who did not produce an ongoing pregnancy (1.05 f 0.92 h). Conclusions: Laser-treated blastocysts exhibited substantially shorter re-expansion times. Because faster re-expansion of the blastocyst is associated with positive treatment outcomes, the laser technique should be prioritized over the pipetting technique if ABC is considered.

What are the current recommended criteria for morphological assessment of oocytes, zygotes and embryos? The present ESHRE/Alpha Scientists in Reproductive Medicine consensus document provides several novel recommendations to assess oocyte and embryo morphology and rank embryos for transfer. A previous Alpha Scientists in Reproductive Medicine/ESHRE consensus on oocyte and embryo morphological assessment was published in 2011. After more than a decade, and the integration of time-lapse technology into embryo culture and assessment, a thorough review and update was needed. A working group consisting of Alpha Scientists in Reproductive Medicine executive committee members and ESHRE Special interest group of Embryology members formulated recommendations on oocyte and embryo assessment. The working group included 17 internationally recognized experts with extensive experience in clinical embryology. Seven members represented Alpha Scientists in Reproductive Medicine and eight members represented ESHRE, along with to two methodological experts from the ESHRE central office. Based on a systematic literature search and discussion of existing evidence, the recommendations of the Istanbul Consensus (2011) were reassessed and, where appropriate, updated based on consensus within the working group. A stakeholder review was organized after the updated draft was finalized. The final version was approved by the working group, the Alpha executive committee and the ESHRE Executive Committee. This updated consensus paper provides 20 recommendations focused on the timeline of preimplantation developmental events and morphological criteria for oocyte, zygote, and embryo assessment. Based on duration of embryo culture, recommendations are given on the frequency and timing of assessments to ensure consistency and effectiveness. Several criteria relevant to oocyte and embryo morphology have not been well studied, leading to either a recommendation against their use for grading or for their use in ranking rather than grading. Future updates may require further revision of these recommendations. This document provides embryologists with advice on best practices when assessing oocyte and embryo quality based on the most recent evidence.

In vitro maturation (IVM) of immature oocytes has been explored for research and clinical purposes since the dawn of assisted reproductive technologies. Oocyte maturation is a highly specific process, on the basis of complex mutual relationships between the germ and somatic cell compartments. The complexity of this relationship has made the quest for achieving oocyte maturation in vitro arduous. In its classical form, in which intact cumulus-enclosed oocytes are collected after very mild or no ovarian stimulation, oocyte IVM is nonexperimental and has been proposed as a more friendly treatment for patients with polycystic ovary or polycystic ovary syndrome. By contrast, rescue IVM (r-IVM), which is the maturation in vitro of immature oocytes collected in standard ovarian stimulation cycles, is an experimental procedure, proposed to mitigate the impact of low oocyte maturation rates in certain patients. Achieving effective r-IVM has turned out to be an even more daunting task because oocytes are cultured only after cumulus cell removal and, therefore, without the crucial somatic metabolic and regulative support. Immature oocyte arrested at the germinal vesicle or metaphase I stage require different management for their maturation in vitro and exhibit different developmental and chromosomal competence. Therefore, their possible use for treatment suggests a dedicated approach. Overall, r-IVM has limited clinical efficacy due to suboptimal maturation and developmental competence of immature oocytes. This raises a cost/benefit question: that is, the definition of appropriate clinical indications. Rescue IVM is probably irrelevant to treatment cycles in which the absolute number of mature oocytes is high. Conversely, specific poor prognosis cases, involving low maturation rates, low oocyte yield, and/or low oocyte quality, could benefit from the contribution of even a single embryo generated from an in vitro matured oocyte. Future progress in this field will depend on our ability to mimic in vitro the support provided by cumulus cells to oocyte nuclear and cytoplasmic maturation.

This European Society of Human Reproduction and Embryology (ESHRE)/Alpha Scientists in Reproductive Medicine (ALPHA) consensus document provides several novel recommendations to assess oocyte and embryo morphology and rank embryos for transfer. A previous ALPHA/ESHRE consensus on oocyte and embryo morphological assessment was published in 2011. After more than a decade, and the integration of time-lapse technology into embryo culture and assessment, a thorough review and update was needed. A working group consisting of ALPHA members and ESHRE Special interest group of Embryology members formulated recommendations on oocyte and embryo assessment. The working group included 17 internationally recognized experts with extensive experience in clinical embryology. Seven members represented ALPHA and eight members represented ESHRE, along with two methodological experts from the ESHRE central office. Based on a systematic literature search and discussion of existing evidence, the recommendations of the Istanbul Consensus (2011) were reassessed and, where appropriate, updated based on consensus within the working group. A stakeholder review was organized after the updated draft was finalized. The final version was approved by the working group, the ALPHA Executive Committee and the ESHRE Executive Committee. This updated consensus paper provides 20 recommendations focused on the timeline of preimplantation developmental events and morphological criteria for oocyte, zygote and embryo assessment. Based on the duration of embryo culture, recommendations are given on the frequency and timing of assessments to ensure consistency and effectiveness. Several criteria relevant to oocyte and embryo morphology have not been well studied, leading to either a recommendation against their use for grading or for their use in ranking rather than grading. Future updates may require further revision of these recommendations. This document provides embryologists with advice on best practices when assessing oocyte and embryo quality based on the most recent evidence.

Mitochondria, cellular powerhouses, harbor DNA [mitochondrial DNA (mtDNA)] inherited from the mothers. mtDNA mutations can cause diseases, yet whether they increase with age in human oocytes remains understudied. Here, using highly accurate duplex sequencing, we detected de novo mutations in single oocytes, blood, and saliva in women 20 to 42 years of age. We found that, with age, mutations increased in blood and saliva but not in oocytes. In oocytes, mutations with high allele frequencies were less prevalent in coding than noncoding regions, whereas mutations with low allele frequencies were more uniformly distributed along the mtDNA, suggesting frequency-dependent purifying selection. Thus, mtDNA in human oocytes is protected against accumulation of mutations with aging and having functional consequences. These findings are particularly timely as humans tend to reproduce later in life.

Abstract Study question What are the current recommended criteria for morphological assessment of oocytes, zygotes and embryos? Summary answer The present ESHRE/Alpha Scientists in Reproductive Medicine consensus document provides several novel recommendations to assess oocyte and embryo morphology and rank embryos for transfer. What is known already A previous Alpha Scientists in Reproductive Medicine / ESHRE consensus on oocyte and embryo morphological assessment was published in 2011. After more than a decade, a thorough review and update was needed. Study design, size, duration A working group consisting of Alpha Scientists in Reproductive Medicine executive board members and ESHRE Special interest group of Embryology members formulated recommendations on oocyte and embryo morphological assessment. Participants/materials, setting, methods The working group included 17 internationally recognized experts with extensive experience in clinical embryology. Seven members represented Alpha Scientists in Reproductive Medicine and 8 members represented ESHRE, in addition to two methodological experts from the ESHRE central office. Based on a systematic literature search and discussion of existing evidence, the recommendations of the Istanbul Consensus (2011) were reassessed and, where appropriate, updated based on consensus within the working group. After stakeholder review, the final version will be approved by the working group, the Alpha executive board and the ESHRE Executive Committee. Main results and the role of chance This updated consensus paper provides 20 recommendations focused on the timeline of preimplantation developmental events and morphological criteria for oocyte, zygote, and embryo assessment. Based on duration of embryo culture, recommendations are given on the frequency and timing of assessments to ensure safety and effectiveness. Limitations, reasons for caution Several criteria relevant to oocyte and embryo morphology have not been well studied, leading to either a recommendation against their use for grading or for use in ranking rather than grading. Future updates may require further revision of these recommendations. Wider implications of the findings This document provides embryologists with clear advice on best practices when assessing oocyte and embryo quality based on the most recent evidence.

Delayed fatherhood results in a higher risk of inheriting a new germline mutation that might result in a congenital disorder in the offspring. In particular, some FGFR3 mutations increase in frequency with age, but there are still a large number of uncharacterized FGFR3 mutations that could be expanding in the male germline with potentially early- or late-onset effects in the offspring. Here, we used digital polymerase chain reaction to assess the frequency and spatial distribution of 10 different FGFR3 missense substitutions in the sexually mature male germline. Our functional assessment of the receptor signaling of the variants with biophysical methods showed that 9 of these variants resulted in a higher activation of the receptor´s downstream signaling, resulting in 2 different expansion behaviors. Variants that form larger subclonal expansions in a dissected postmortem testis also showed a positive correlation of the substitution frequency with the sperm donor's age, and a high and ligand-independent FGFR3 activation. In contrast, variants that measured high FGFR3 signaling and elevated substitution frequencies independent of the donor's age did not result in measurable subclonal expansions in the testis. This suggests that promiscuous signal activation might also result in an accumulation of mutations before the sexual maturation of the male gonad with clones staying relatively constant in size throughout time. Collectively, these results provide novel insights into our understanding of the mutagenesis of driver mutations and their resulting mosaicism in the male germline with important consequences for the transmission and recurrence of associated disorders.

Taking into consideration earlier attempts at assisted reproduction and previous pregnancies allows a more differentiated approach when counseling couples regarding their chances in assisted reproductive treatments. The aim of this study was to investigate whether the outcome of fresh embryo transfers affects subsequent same-cohort vitrified–warmed cycles. This retrospective cohort study analyzes the outcome of all fresh and frozen embryo transfers (n = 8118) between January 1, 2011, and December 31, 2022. All women who received fresh or frozen embryo transfers at Kepler University Hospital Linz were included. The use of donor eggs, previously cryopreserved oocytes, and freeze-all cycles were excluded from the analysis. Positive serum βhCG after a fresh cycle has a significant impact on the chances of positive βhCG in a subsequent vitrified–warmed cycle (36.3

Mitochondria, cellular powerhouses, harbor DNA (mtDNA) inherited from the mothers. MtDNA mutations can cause diseases, yet whether they increase with age in human germline cells-oocytes-remains understudied. Here, using highly accurate duplex sequencing of full-length mtDNA, we detected de novo mutations in single oocytes, blood, and saliva in women between 20 and 42 years of age. We found that, with age, mutations increased in blood and saliva but not in oocytes. In oocytes, mutations with high allele frequencies (≥1%) were less prevalent in coding than non-coding regions, whereas mutations with low allele frequencies (<1%) were more uniformly distributed along mtDNA, suggesting frequency-dependent purifying selection. In somatic tissues, mutations caused elevated amino acid changes in protein-coding regions, suggesting positive or destructive selection. Thus, mtDNA in human oocytes is protected against accumulation of mutations having functional consequences and with aging. These findings are particularly timely as humans tend to reproduce later in life.