Search Results (169)

This study presents the first successful generation and comprehensive characterization of trophoblastic organoids (TOs) and the derivation of three-dimensional cavity- or sac-like structures—termed lacunoids/cystoids—from sheep intracytoplasmic sperm injection (ICSI) embryos. TOs were generated from sheep ICSI embryos for the first time and were shown to express trophoblastic markers at levels comparable to those in embryonic tissue. Detailed morphological characterization was conducted for both the TOs and the derived lacunoids/cystoids. Additionally, the TOs’ interactions with endometrial organoids (EOs), as well as those with preimplantation embryos, were investigated through co-culture experiments. The TOs expressed key trophoblastic markers, including CDX2, GATA3, syncytin-1, KRT18, KRT7, and Sox2, confirming their validity as a model for studying sheep trophoblast biology. The generation of lacunoids/cystoids from the TOs further revealed their structural and developmental characteristics, contributing valuable insights into early placental development and trophoblast-related pathologies. The TOs also supported extended embryonic development, and their co-culture with EOs induced dynamic changes in gene expression, particularly in angiogenesis-related genes, in both organoid types. This novel and reproducible in vitro model offers a reliable platform to study early placental development, effectively recapitulating the biological crosstalk between the trophectoderm and endometrium. The in-depth characterization of TOs and lacunoids/cystoids highlights their potential to advance our understanding of trophoblast differentiation and related developmental disorders. Full article

A retrospective cohort study was conducted comparing conventional intracytoplasmic sperm injection (ICSI) with a pre-catching sperm (PCS)-ICSI technique. Cases with at least 0.5 million motile sperm and 5 mature oocytes were included. Conventional ICSI involves simultaneous loading of sperm and oocytes onto the dish, followed by identification, immobilization, and loading of sperm into pipettes for oocyte injection. In the PCS-ICSI technique, suitable sperm were identified and immobilized prior to oocyte loading onto the dish, thus reducing the oocyte exposure time. Variables of interest included rate of fertilized and degenerated oocytes, abnormal fertilization, quality blastocyst formation, and pregnancy outcomes. Statistical analysis utilized Student’s t-test and Fisher’s Exact Test. Our study included 330 PCS-ICSI and 287 conventional ICSI cases. Female age, BMI, AMH, total number of collected oocytes, and rate of abnormal fertilization were similar between groups. The PCS-ICSI group demonstrated an increased rate of oocyte fertilization (84.0% vs. 79.3%, p < 0.001), good quality blastocyst formation (54.9% vs. 48.0%, p < 0.001), and a lower rate of oocyte degeneration (1.4% vs. 3.5%, p < 0.001). Positive pregnancy rate, clinical pregnancy, and live birth rate were similar between groups. The expansion of this technique resulted in increased oocyte fertilization and good quality blastocyst formation, and decreased oocyte degeneration. Further studies will evaluate the effectiveness of this technique in broader patient populations. Full article

The increasing use of assisted reproductive technologies (ARTs) globally, such as intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF), has highlighted the pressing need to determine the modifiable factors affecting the success of implantation and the outcomes of early pregnancy. Scientific interest in the role of nutrition in fertility is growing, but outside of celiac disease, little is known about gluten, a dietary protein with immunogenic and inflammatory properties. With an emphasis on ART results, this narrative review summarizes the most recent data regarding the possible effects of gluten consumption on reproductive health, focusing primarily on individuals with celiac disease (CD) and non-celiac gluten sensitivity (NCGS). In addition to discussing potential molecular processes connecting gluten-induced inflammation, increased gut permeability, autoimmune, and decreased endometrial receptivity, we further explore the documented link between CD and infertility and investigate new information on NCGS. These findings are tentative and based on scant low-quality evidence, although some case reports and small clinical studies have indicated that avoiding gluten may help some people undergoing ART, especially those with immune-mediated diseases or infertility that cannot be explained. There is currently no robust prospective evidence confirming that gluten restriction improves infertility outcomes. Therefore, before gluten elimination is advised in this situation, more carefully planned extensive research is required to generate reliable scientific proof. Beyond traditional celiac disease, we suggest that gluten sensitivity might be an underappreciated factor in ART failure that merits more research. A gluten-free diet may serve as a low-risk supplementary option for appropriately selected patients, pending the results of more extensive controlled studies. Full article

The significant decline in amphibians worldwide is demanding the development of reliable techniques to save species and their genetic diversity. Considerable efforts are currently in progress to develop assisted reproductive technologies (ARTs), focusing mainly on sperm cryopreservation and in vitro fertilization (IVF). In Xenopus, a simple and efficient transgenesis method based on the intracytoplasmic injection (ICSI) of cryoconserved sperm was developed several decades ago, allowing for quick generation of large numbers of transgenic animals, for biological research. Such a methodology could be critical for the recovery of species and their genetic diversity, contributing to amphibian conservation. However, this approach raised the question of whether the sperm preservation method used with ICSI is compatible with long-term storage. To address this question, animals were generated by ICSI using a twenty-year-old cryopreserved sperm preparation. Their development, behavior, and reproduction ability were compared with those of animals obtained using a recently frozen sperm preparation and those of animals obtained via IVF using fresh semen. Although lower than with IVF, we showed that fertilization rates using ICSI after 20 years of cryopreservation are similar to those of a recent preparation, with viable offspring leading to normal F2 generation. This pioneering achievement is proof of concept for long-term sperm cryopreservation using simple and readily available technologies for the conservation of endangered amphibians. Full article

Objective: Most studies concentrate on comparisons between the cleavage stage and blastocyst stage of embryos during in vitro stimulation treatment. We aimed, in this study, to compare the pregnancy rates of day 4 or day 5 blastocyst transfers, all derived from fresh, antagonist-regulated in vitro fertilization (IVF) cycles, and to evaluate the factors affecting pregnancy success. Methods: This retrospective cohort study evaluated 3681 fresh embryo transfer cycles conducted at a private IVF center between 2019 and 2021. Patients were divided into two groups based on the day of embryo transfer: day 4 (Group 1) and day 5 (Group 2). Subgroup analyses were performed according to age (≤40 vs. >40 years) and the number of oocytes retrieved (≤4 vs. >4). All patients underwent ovarian stimulation with FSH alone or in combination with hMG, and GnRH antagonist protocols were used for pituitary suppression. Final oocyte maturation was triggered with recombinant hCG, and fertilization was achieved via intracytoplasmic sperm injection (ICSI) for all cases. Embryos were cultured in sequential media and assessed daily until transfer on day 4 or day 5, based on embryo morphology and clinic logistics. Results: Pregnancy was more likely among women under 40 than among women over 40. There were a total of 1217 women who underwent day 4 transfer and 2464 women who underwent day 5 transfer. A total of 660 (54.2%) of the women transferred on day 4 developed pregnancy. Among those transferred on day 5, 1610 (65.3%) developed pregnancy. When compared to the 4th day, a single embryo transfer on the 5th day enhances pregnancy success by 1.8 times, while two embryo transfers raise it by 1.6 times. Furthermore, when the number of oocytes is greater than four and the number of embryos transferred is two, the pregnancy success rate is 2.5 times higher when embryo transfer is performed on the fifth day versus the fourth day. Regardless of age, oocyte count, or number of embryos transferred, 5th-day fresh embryo transfers enhanced pregnancy success by 1.9 times compared to 4th-day transfer. Conclusions: Transfers of fresh embryos on day 5 are superior to those on day 4 and should be favored, especially for people over the age of 40, regardless of the number of embryos transferred, even if that individual has fewer than four oocytes. Full article

In addition to the male genome, the fertilizing spermatozoon delivers to the oocyte several factors whose deficiency can cause embryo dysfunction. Sperm oocyte-activating factor, identified as phoshoplipase C zeta (PLCζ), drives oocyte exit from meiotic arrest through a signaling pathway initiated by periodic rises of free cytosolic Ca²⁺ concentration (calcium oscillations). Sperm centrioles, together with oocyte proteins, form centrosomes that are responsible for aster formation, pronuclear migration, and DNA polarization before nuclear syngamy and subsequent mitotic divisions. Sperm DNA fragmentation can be at the origin of aneuploidies, while epigenetic issues, mainly abnormal methylation of DNA-associated histones, cause asynchronies of zygotic gene activation among embryonic cells. Sperm long and short non-coding RNAs are important epigenetic regulators affecting critical developmental processes. Dysfunction of sperm PLCζ, centrioles, DNA, and RNA mostly converge to aneuploidy, developmental arrest, implantation failure, miscarriage, abortion, or offspring disease. With the exception of DNA fragmentation, the other sperm issues are more difficult to diagnose. Specific tests, including heterologous human intracytoplasmic sperm injection (ICSI) into animal oocytes, genetic testing for mutations in PLCZ1 (the gene coding for PLCζ in humans) and associated genes, and next-generation sequencing of sperm transcriptome, are currently available. Oral antioxidant treatment and in vitro selection of healthy spermatozoa can be used in cases of sperm DNA fragmentation, while ICSI with assisted oocyte activation is useful to overcome oocyte-activation defects. No clinically confirmed therapy is yet available for sperm RNA issues. Full article

Background: Thyroid disorders, particularly thyroid autoimmunity, are increasingly prevalent among women of reproductive age and have been linked to fertility outcomes. While current endocrinology guidelines define distinct thyroid-stimulating hormone (TSH) target values for women undergoing assisted reproductive technology (ART), the optimal preconception TSH range for in vitro fertilization (IVF) success remains a topic of debate. Objectives: This study aimed to assess the impact of baseline TSH levels within the recommended normal range on IVF outcomes, specifically clinical pregnancy and live birth rates. Additionally, we assessed the predictive value of procedural and preprocedural factors, including maternal body mass index (BMI) and TSH, using machine learning models. Methods: We conducted a retrospective, single-center cohort study at the Institute of Reproductive Medicine, University of Szeged, involving 996 women who underwent IVF, with or without intracytoplasmic sperm injection. Biometric, medical history, laboratory, and procedural factors were analyzed. Pregnancy and live birth predictions were modeled using support vector machine (SVM), random forest (RF), and extreme gradient boosting (XGBoost) algorithms. The significance of features in the RF and XGBoost models was assessed. Results: SVM models achieved a mean accuracy of 72.26% in predicting pregnancy but were less effective for live birth classification. RF and XGBoost models demonstrated an area under the receiver operating characteristic curve of 0.76 and 0.74 for pregnancy and 0.67 and 0.61, respectively, for live birth. Key predictors included embryo score, maternal age, BMI, and specific hormone levels. Notably, male factors also contributed to outcome prediction. Analysis suggested that variations in maternal TSH within the normal range (0.3–4.0 mIU/L) had no significant impact on IVF success. Conclusions: Our study suggests that preconception TSH levels within the reference range do not significantly influence IVF success, which indirectly supports the validity of the current recommendations on this matter. While machine learning models demonstrated promising predictive performance, larger prospective studies are needed to refine thyroid function targets in ART, with a separate analysis of women with thyroid autoimmunity. Full article

Introduction: Fertility is fundamental to human well-being, significantly impacting both individual lives and societal development. In particular, sperm morphology—referring to the shape, size, and structural integrity of sperm cells—is a key indicator in diagnosing male infertility and selecting viable sperm in assisted reproductive technologies such as in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI). However, traditional manual evaluation methods are highly subjective and inconsistent, creating a need for standardized, automated systems. Objectives: This study aims to develop a robust and fully automated sperm morphology classification framework capable of accurately identifying a wide range of morphological abnormalities, thereby minimizing observer variability and improving diagnostic support in reproductive healthcare. Methods: We propose a novel ensemble-based classification approach that combines convolutional neural network (CNN)-derived features using both feature-level and decision-level fusion techniques. Features extracted from multiple EfficientNetV2 variants are fused and classified using Support Vector Machines (SVM), Random Forest (RF), and Multi-Layer Perceptron with Attention (MLP-Attention). Decision-level fusion is achieved via soft voting to enhance robustness and accuracy. Results: The proposed ensemble framework was evaluated using the Hi-LabSpermMorpho dataset, which contains 18 distinct sperm morphology classes. The fusion-based model achieved an accuracy of 67.70%, significantly outperforming individual classifiers. The integration of multiple CNN architectures and ensemble techniques effectively mitigated class imbalance and enhanced the generalizability of the model. Conclusions: The presented methodology demonstrates a substantial improvement over traditional and single-model approaches in automated sperm morphology classification. By leveraging ensemble learning and multi-level fusion, the model provides a reliable and scalable solution for clinical decision-making in male fertility assessment. Full article

Hashimoto’s thyroiditis is the most prevalent autoimmune thyroid disorder, and it disproportionately affects women of reproductive age. Its impact on fertility and assisted reproductive technologies [ART] has become an area of growing clinical interest. Thyroid autoimmunity can influence female reproductive health through multiple interconnected mechanisms, including subtle thyroid hormone imbalances, reduced ovarian reserve, altered endometrial receptivity, and dysregulated immune responses. Subclinical hypothyroidism and the presence of anti-thyroid antibodies have been linked to increased miscarriage risk and reduced success rates in ART, particularly in intracytoplasmic sperm injection (ICSI) cycles. Although levothyroxine supplementation is widely used, its benefits in euthyroid women remain uncertain. Recent studies suggest that gut microbiota may modulate immune function and affect fertility outcomes among women with autoimmune thyroid conditions. This narrative review synthesizes findings from a broad literature base of over 40 peer-reviewed publications published between 2010 and 2025, with 30 of the most relevant and methodologically robust studies selected for detailed analysis. The review integrates clinical, endocrine, immunological, and microbiome-related perspectives. The evidence supports the need for personalized fertility management in women with Hashimoto’s thyroiditis and highlights directions for future research into immune and microbiota-targeted therapies. Full article

Background/Objectives: Preimplantation genetic screening improves embryo selection in intracytoplasmic sperm injection cycles, especially for women of advanced maternal age. As chromosomal normality declines with age, high-dose gonadotropins are commonly used to enhance follicular response. This study compares minimal and high-dose stimulation protocols in terms of euploidy, pregnancy, and live birth rates following single embryo transfer. Methods: In this prospective study, 198 women aged 38–45 years were enrolled and divided into two groups: minimal stimulation (100 mg clomiphene citrate and 75 IU human menopausal gonadotropin) and high stimulation (300–450 IU gonadotropins). Women with severe male factor infertility, endometriosis, or absolute tubal factor were excluded. Clinical outcomes were compared using a t-test or Mann–Whitney U test. Results: Baseline characteristics were similar between groups. The high-dose group had a significantly higher number of retrieved oocytes (p = 0.009) and metaphase II oocytes (p = 0.003). However, there were no significant differences in euploid embryo rates (35.4% vs. 37.4%, p = 0.768), clinical pregnancy rates (67.6% vs. 69.4%, p > 0.999), gestational sac rates (58.8% vs. 58.3%, p > 0.999), or live birth rates (47.1% vs. 50.0%, p = 0.995). Conclusions: This is the first prospective study to compare euploid embryo rates, pregnancy rates, and live birth rates between minimal stimulation protocol and high stimulation protocol in AMA patients. Although there has been no difference in euploid and pregnancy rates, minimal stimulation protocol has advantages in cost and comfort. Full article

We investigated whether FMR1 allelic complexity—integrating CGG repeat length with the number and pattern of AGG interspersions—can be used as a predictor of ovarian reserve and in vitro fertilization (IVF) success. This cohort study included 124 females with infertility attributed to female factors undergoing intracytoplasmic sperm injection (ICSI). The total CGG repeat lengths and AGG interspersion patterns of the FMR1 gene were determined by conventional polymerase chain reaction (PCR) and triplet-primed PCR. The allelic complexity (allelic score) was calculated using a previously described formula by combining the allelic scores, allowing for the stratification of samples into equivalent and dissimilar groups. No statistically significant differences were observed in ovarian reserve markers or overall IVF outcomes between the two groups. However, within the dissimilar group, the allelic score of allele 1 was significantly correlated with the number of both injected metaphase II and two-pronuclei oocytes. These findings suggest that FMR1 allelic complexity may contribute to predicting IVF success, particularly in females classified in the dissimilar group, who appear more susceptible to IVF failure than those in the equivalent group. Further research into the predictive utility of FMR1 could provide valuable insights for fertility assessment and enhance assisted reproductive technologies. Full article

Background and Objectives: Pregnancies resulting from assisted reproductive technology (ART) have been associated with placenta-related adverse outcomes. Uterine artery Doppler pulsatility index (UtA-PI) reflects placental function. This study aimed to examine whether second-trimester UtA-PI differs according to the conception method after adjusting for potential confounding factors. Materials and Methods: In this retrospective cohort study, we included data from February 2015 to August 2024, at the third Department of Obstetrics and Gynecology, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, Greece, on singleton pregnancies presenting for their routine antenatal care, including a second-trimester anomaly scan. Pregnancies conceived via ART, including those conceived via ovulation induction/intrauterine insemination (OI/IUI) or in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI), were compared to those conceived spontaneously. Multiple linear regression was employed to investigate the association between the mode of conception and log₁₀ UtA-PI values, adjusting for various confounders, including gestational age at the time of the scan, maternal weight, height, age, parity, mode of delivery, smoking status, pre-existing diabetes mellitus (type I or II), and pre-existing thyroid disease. Results: The study included 15,552 singleton pregnancies, of which 82 (0.5%) were conceived via OI/IUI and 690 (4.4%) were conceived via IVF/ICSI. The median UtA-PI values were 0.99 (IQR: 0.85–1.17) for spontaneous conception (SC), 1.00 (IQR: 0.86–1.16) for OI/IUI, and 0.90 (IQR: 0.76–1.12) for IVF/ICSI. The Kruskal–Wallis test indicated a statistically significant difference among these groups (p < 0.001). Pairwise comparisons using the Wilcoxon rank-sum test with Bonferroni correction revealed that UtA-PI values in IVF/ICSI pregnancies were significantly lower compared to both SC and OI/IUI pregnancies (p < 0.001 for both). No significant difference was observed between the SC and OI/IUI groups. In the multivariable linear regression model, IVF/ICSI conception was independently associated with lower log₁₀ UtA-PI values (estimate = −0.076, 95% CI: −0.096, −0.056) while no association was found for OI/IUI conception. Conclusions: Although ART has been associated with placental-related complications, mid-trimester UtA flow was found to be lower in IVF/ICSI pregnancies, suggesting better utero-placental flow in ART pregnancies and other possible mechanisms in the maternal–placental interplay for the development of pregnancy complications. Full article

In vitro maturation (IVM) of oocytes retrieved from ovum pick-up (OPU) or ovarian tissue (OT) is a standard approach for patients with specific conditions where prior hormonal stimulation is contraindicated. However, the developmental competence of oocytes matured in vitro is still inferior to that of oocytes matured in vivo. Capacitation IVM (CAPA-IVM) includes an extra step of pre-maturation culture (PMC) with c-type natriuretic peptide (CNP) as a meiotic arrestor to better synchronize cytoplasmic and nuclear maturity in oocytes by allowing the cytoplasm additional time to acquire essential components critical for optimal competency. This study aims to evaluate the effect of CAPA-IVM on equine oocyte quality and developmental competence. Immature cumulus–oocyte complexes (COCs) were retrieved from slaughterhouse ovaries and matured in vitro either in CAPA-IVM (short 6 h, long 24 h pre-maturation) or standard IVM. Mature oocytes from each group were analyzed for calcium-releasing potential (n = 52) and single-oocyte proteomics (n = 44), and embryo development (n = 229) was assessed after fertilization with piezo-drilled intracytoplasmic sperm injection (ICSI). Genetic analysis of developed blastocysts (n = 41) was performed to detect chromosomal aberrations. Our findings demonstrate that CAPA-IVM of equine COCs yields significantly higher maturation rates than controls. Moreover, short CAPA-IVM with six hours pre-maturation culture showed substantially higher embryo development potential than the control group (20/69 vs. 9/63, respectively). Genetic analysis revealed a high euploidy rate in equine blastocysts regardless of the maturation conditions. Live calcium imaging of the fertilized oocytes demonstrated that the majority of oocytes displayed non-continuous calcium oscillation patterns, irrespective of maturation conditions. Single-oocyte proteomics reveals a comparable proteomic landscape between mature oocytes subjected to short CAPA-IVM and standard IVM. However, we identified four enriched gene sets with positive enrichment scores after short CAPA-IVM, related to cytoskeleton regulation, ribosomal function, and cytosolic components. Our findings indicate that CAPA-IVM holds the potential to improve oocyte quality and competence in horses. However, further fine-tuning of culture conditions would benefit the effective use of these IVM systems. Moreover, given that the mare serves as an excellent model for human reproduction, the molecular trends identified in this study could provide valuable insights for advancing human artificial reproductive technologies. Full article

The use of microfluidic sperm sorting (MFSS) systems in infertility treatment is increasing due to their practicality and ease of use. While often presented as highly effective, their efficacy in patients with varying sperm analysis results remains uncertain. In this study, we evaluated the effectiveness of MFSS compared with the swim-up (SU) technique in terms of oxygen radical levels and spermiogram parameters. Samples from each patient were processed using both methods, followed by assessments of sperm concentration, motility, morphology, DNA integrity, acrosomal status, and mitochondrial membrane potential. Participants were selected based on sperm analysis and categorized as normozoospermic (n = 40) or non-normozoospermic (n = 28). An analysis of separation techniques revealed no significant differences, except for a lower percentage of DNA-fragmented sperm in the MFSS group compared with SU within the non-normozoospermic cohort (SU: 10.0% vs. MFSS: 5.69%, p = 0.027). No differences were observed between SU and MFSS in normozoospermic men. The MFSS method is a simple technique, frequently used in laboratories, that yields good results but does not offer a substantial advantage over SU. The primary benefit of MFSS appears to be a significant reduction in the proportion of sperm with DNA fragmentation compared with SU in patients with abnormal sperm analysis results. Full article

Phospholipase C zeta (PLCζ) has emerged as a pivotal sperm-specific factor responsible for triggering oocyte activation, a process essential for successful fertilization and early embryogenesis. A narrative review was conducted to examine the molecular architecture and biochemical features of PLCζ, with particular emphasis on how its distinctive structural domains facilitate the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) and the induction of calcium (Ca²⁺) oscillations in the oocyte. Notably, PLCζ exhibits unique sensitivity to basal Ca²⁺ levels and the capacity to sustain repetitive intracellular Ca²⁺ transients that drive meiotic progression and block polyspermy. Clinically, PLCζ deficiency—whether caused by genetic mutations, reduced expression, or improper localization—represents a unifying explanation for certain forms of male infertility, including total fertilization failure (TFF) following intracytoplasmic sperm injection (ICSI). Globozoospermia is a prime example; this condition is characterized by round-headed sperm devoid of acrosomes and exhibiting significantly reduced or absent PLCζ and often results in fertilization failure. Diagnostic methods such as immunofluorescence, Western blotting, and the mouse oocyte-activation test collectively support the identification and characterization of PLCζ-related defects, while genetic testing for mutations in the PLCZ1 gene has proven valuable for identifying hereditary causes of sperm-borne oocyte-activation deficiency (OAD). Therapeutic approaches range from assisted oocyte activation (AOA) with calcium ionophores to emerging interventions that introduce functional PLCζ protein or mRNA directly into the oocyte. These advancements demonstrate the rapid translation of foundational discoveries into clinically actionable interventions. Future investigations are poised to refine diagnostic assays, standardize measurement protocols, and explore the potential of gene therapy or CRISPR/Cas9-mediated correction for heritable PLCζ abnormalities. By addressing both the molecular basis and translational applications of PLCζ, recent findings underscore its indispensable role in fertility care and lay out a path toward further innovation in assisted reproductive technologies. Full article