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Simple Summary: Heat stress is a major problem for the health and productivity of livestock, including cattle. Since the 1950s, researchers have studied how high environmental temperature affects cattle fertility through both laboratory and field studies. When a cow's rectal or vaginal temperature goes above 39.1 °C, it is considered heat-stressed, which can harm its ability to reproduce. However, there is not enough information about temperatures in the uterus, oviducts, and ovarian follicles, especially in lactating dairy cows, to ensure current laboratory models are accurate. These data are crucial for understanding how heat stress impacts oocyte (egg) development and early embryo survival in cattle. Additionally, it is important to improve live animal stress models to correctly identify when cows are truly experiencing heat stress, shown by signs like increased panting, body temperature, and heart rate, rather than just assuming they are stressed based on exposure to a high temperature–humidity index. Improving these models will make them more reliable and help identify cattle that can better tolerate heat. Rectal and vaginal temperatures are utilised in both in vivo and in vitro models to study the effects of heat stress on oocyte competence and embryo viability in cattle. However, uterine temperature increases by only 0.5 °C in heat-stressed cows, significantly lower than simulated increases in in vitro models. Temperature variations within oviducts and ovarian follicles during heat stress are poorly understood or unavailable, and evidence is lacking that oocytes and pre-implantation embryos experience mild (40 °C) or severe (41 °C) heat stress inside the ovarian follicle and the oviduct and uterus, respectively. Gathering detailed temperature data from the reproductive tract and follicles is crucial to accurately assess oocyte competence and embryo viability under realistic heat stress conditions. Potential harm from heat stress on oocytes and embryos may result from reduced nutrient availability (e.g., diminished blood flow to the reproductive tract) or other unidentified mechanisms affecting tissue function rather than direct thermal effects. Refining in vivo stress models in cattle is essential to accurately identify animals truly experiencing heat stress, rather than assuming heat stress exposure as done in most studies. This will improve model reliability and aid in the selection of heat-tolerant animals. [ABSTRACT FROM AUTHOR]

According to the society of assisted reproductive technologies (SART), the top fertility clinics in North America have reported overall pregnancy rates of up to 50% in good prognosis patients. Although new techniques in advanced reproductive technologies have begun to emerge in the twenty-first century, including time-lapse monitoring of embryo development, analysis of metabolites in embryo culture media, and the use of artificial intelligence in morphometric embryo grading, currently chromosomal status obtained via pre-implantation genetic testing for aneuploidy (PGT-A) appears to be the most definitive in evaluating embryonic potential. Yet still, in most clinics, approximately 30% of euploid or chromosomally normal embryos fail to achieve a viable pregnancy resulting in stress and anxiety for patients, repeat cycles, and increased cost of treatment. Recent literature suggests that blastocysts with an increased mitochondrial DNA (mtDNA) ratio have greatly reduced implantation potential, however, there exists controversial studies which do not report a relationship. This review summarizes the current literature on utilizing mitochondrial DNA as a possible biomarker for embryo implantation, highlights the reasons for inconsistencies in some studies and suggests possible improvements to the study design of future mtDNA ratio research.

The present study was designed to evaluate equine blastocyst re-expansion rate, quality, and sex following perforation of the blastocoel, collection of blastocoel fluid (BF), and PCR amplification of free DNA. Experiment 1 tested the feasibility of the BF sample collection with a hand-held, small-gauged needle (26g) and subsequent PCR amplification of the TSP-Y gene for males and AMEL-Y gene for males and AMEL-X gene for females. Experiment 2 tested the application of the technique. Equine embryos were collected via uterine flushes 8d after ovulation. Thereafter, embryos (n = 19) were initially assessed and transferred to a 50 μL droplet of holding medium in which the blastocoel was manually perforated as in Experiment 1. Within 1 min of detecting a diameter decrease or collapse, the entire volume of each droplet of medium was collected and stored at −20 °C until PCR. In Experiment 1, amplification of the TSP-Y gene was positive for males at 60% (9/15) and negative for females at 40% (6/15). In Experiment 2, a total of 42 embryos were randomly assigned to a collapsed embryo (CE) or intact embryo (IE) groups and stored at room temperature (RT, 25 °C) or cold temperature (CT, 5 °C) for 24h as follows: 1) CERT, n = 11; 2) CECT n = 11; 3) IERT, n = 10; and 4) IECT, n = 10. After 24h, embryo diameter and quality were reassessed. For all collapsed embryos (n = 19), blastocoel fluid was subjected to double PCR amplification of the TSPY gene with blood from adult male and female horses as controls. Positive gene amplification indicated 57.9% (11/19) of embryos were male and negative amplification indicated 31.6% (6/19) of embryos were female. Relative to the least diameter (0%) after perforation of collapsed embryos or fullest diameter (100%) of intact embryos at T0, percentage change in diameter and quality Grade 1 or 2 embryos after 24h of storage for all groups were, respectively: 31.2% and 54% for CERT group, 28.2% and 0% for CECT group, 25.9% and 100% for IERT group, 4.3% and 80% for IECT group, respectively. Thus, needle-induced leakage and collapse of the blastocoel at T0 resulted in a high rate of blastocyst re-expansion (69%) with many embryos (54%) achieving good quality at T24 with potential for transfer as either male or female embryos. For both collapsed and intact embryos, it was observed that storage for 24h at room temperature (25 °C) was associated with improved embryo growth and morphological quality compared to storage at cold temperature (5 °C). • It is possible to perform the sexing of equine embryos through blastocoels fluid samples collected by hand-held, small gauged needle and PCR based on positive amplification of the TSP-Y gene. • Collapsed embryos are capable of a partial re-expansion after 24h storage culture in a complete embryo medium and that recovery was greater when storage was maintained at room vs cold temperature. • Room temperature maintained intact embryo growth and morphological quality of the embryos that resulted in a greater percentage of embryos as potentially transferable for pregnancy success. [ABSTRACT FROM AUTHOR]

Simple Summary: The conservation of local chicken breeds is globally considered because of their interesting characteristics, such as their tolerance to heat stress, resistance to diseases and capacity to survive in severe environments. In recent years, conservation programs of native chicken breeds have been developed in Italy, and this work has focused on Bionda Piemontese, Bianca di Saluzzo and Pepoi, the first two of which are catalogued as endangered and the third as a 'critically maintained' breed. Semen cryopreservation is the only method that is currently feasible for the ex situ management of genetic diversity in birds, and poultry sperm shows a greater susceptibility during the freezing/thawing process due to its unique features. Therefore, the choice of the most suitable rooster sperm donor is fundamental in the establishment of a semen cryobank. The present study aims to analyze the efficiency of bird and semen management required for the implementation of the Italian Semen Cryobank of Autochthonous Chicken and Turkey Breeds created with the "TuBAvI" project. Our results suggest that male donors need to be selected based on the quality of fresh ejaculates, regardless of sperm outcome after freezing/thawing, in order to identify individuals who can ensure the survival of a breed with regard to increased fertilizing capacity. Native breed conservation is an important component of poultry biodiversity. The aim of this work is to describe different steps that lead to donor selection for the implementation of the Italian Semen Cryobank of Autochthonous Chicken and Turkey Breeds. The variability within and between breeds was evaluated, and the stored semen reproductive capacity was in vivo tested using artificial insemination. Semen from Bionda Piemontese, Bianca di Saluzzo and Pepoi roosters was collected and processed. Concentration, volume, sperm membrane integrity, total motile sperm, progressive motile sperm and kinetic parameters were analyzed; sperm parameters accounting for bird variability were used to select male donors. Fresh semen quality parameters measured in donor ejaculates showed significant differences between breeds; no differences were found after cryopreservation. Variability in the fertilizing ability of cryopreserved semen was found within a breed (5–16%) and between birds within a breed (BP = 3–7%; BS = 7–31%; PP = 6–22%); only sperm quality parameters measured in fresh ejaculates, not frozen/thawed, may be associated with in vivo fertility results. In conclusion, sperm concentration and progressive motility were successfully used as selection parameters to identify chicken male donors with improved sperm quality for sperm cryobanking. However, new reliable sperm markers to predict cryopreserved semen's fertilizing ability are required. [ABSTRACT FROM AUTHOR]

Rectal and vaginal temperatures are utilised in both in vivo and in vitro models to study the effects of heat stress on oocyte competence and embryo viability in cattle. However, uterine temperature increases by only 0.5 °C in heat-stressed cows, significantly lower than simulated increases in in vitro models. Temperature variations within oviducts and ovarian follicles during heat stress are poorly understood or unavailable, and evidence is lacking that oocytes and pre-implantation embryos experience mild (40 °C) or severe (41 °C) heat stress inside the ovarian follicle and the oviduct and uterus, respectively. Gathering detailed temperature data from the reproductive tract and follicles is crucial to accurately assess oocyte competence and embryo viability under realistic heat stress conditions. Potential harm from heat stress on oocytes and embryos may result from reduced nutrient availability (e.g., diminished blood flow to the reproductive tract) or other unidentified mechanisms affecting tissue function rather than direct thermal effects. Refining in vivo stress models in cattle is essential to accurately identify animals truly experiencing heat stress, rather than assuming heat stress exposure as done in most studies. This will improve model reliability and aid in the selection of heat-tolerant animals.

Abstract Purpose We aimed to study the association between adjusted mtDNA levels in human trophectoderm biopsy samples and the developmental potential of euploid and mosaic blastocysts. Methods We analyzed relative mtDNA levels in 2,814 blastocysts obtained from 576 couples undergoing preimplantation genetic testing for aneuploidy from June 2018 to June 2021. All patients underwent in vitro fertilization in a single clinic; the study was blinded—mtDNA content was unknown at the time of single embryo transfer. The fate of the euploid or mosaic embryos transferred was compared with mtDNA levels. Results Euploid embryos had lower mtDNA than aneuploid and mosaic embryos. Embryos biopsied on Day 5 had higher mtDNA than those biopsied on Day 6. No difference was detected in mtDNA scores between embryos derived from oocytes of different maternal ages. Linear mixed model suggested that blastulation rate was associated with mtDNA score. Moreover, the specific next-generation sequencing platform used have a significant effect on the observed mtDNA content. Euploid embryos with higher mtDNA content presented significantly higher miscarriage rates and lower live birth rates, while no significant difference was observed in the mosaic cohort. Conclusion Our results will aid in improving methods for analyzing the association between mtDNA level and blastocyst viability.

: To investigate the impact of recryopreservation on embryo viability and the outcomes of in vitro fertilization (IVF) by comparison with single cryopreservation. There is a lack of consensus and reliable evidence regarding the impact of recryopreservation techniques on human embryos, particularly with respect to embryo viability and IVF outcomes. Systematic review and meta-analysis. Not applicable. Various databases such as PubMed, Embase, Cochrane Library, and Scopus were searched until October 10, 2022. All comparative studies comparing embryonic and IVF outcomes between repeated and single cryopreservation of embryos were included. The random-effect and fixed-effect meta-analysis models were used to pool the odds ratio (OR) and corresponding 95% confidence intervals (CIs). A subgroup analysis was performed based on different methods of cryopreservation and different times of embryo cryopreservation or transfer. Outcomes referring to embryo surviva l, IVF outcomes (including clinical pregnancy rate, embryo implantation rate, miscarriage rate, and live birth rate), and neonatal outcomes (including low birth weight rate and preterm birth rate) were evaluated. Fourteen studies were eligible for the present meta-analysis, involving 4,525 embryo transfer cycles in total (3,270 cycles with single cryopreservation [control group] and 1,255 with recryopreservation [experimental group]). Decreased embryo survival (OR, 0.51; 95% CI, 0.27–0.96) and clinical pregnancy rates (OR, 0.47; 95% CI, 0.23–0.96) were found in embryos that were recryopreserved by slow freezing. The live birth rate of revitrified embryos was also notably affected (OR, 0.60; 95% CI, 0.38–0.94). Overall, recryopreservation resulted in a decreased live birth rate (OR, 0.67; 95% CI, 0.50–0.90) and an increased miscarriage rate (OR, 1.52; 95% CI, 1.16–1.98) compared with single cryopreservation. No significant difference was found in neonatal outcomes. When embryos were cryopreserved and transferred at the blastocyst stage, both the embryo implantation rate (OR, 0.59; 95% CI, 0.39–0.89) and live birth rate (OR, 0.60; 95% CI, 0.37–0.96) were significantly different between the 2 groups. The present meta-analysis suggested that recryopreservation, compared with single cryopreservation, can lead to impaired embryo viability and a lower rate of IVF success, with no affected neonatal outcomes. Clinicians and embryologists should retain a cautious attitude toward recryopreservation strategies. CRD42022359456 [ABSTRACT FROM AUTHOR]

Exogenous application of silicon (Si) provides benefits to various plant species, including alfalfa. However, knowledge about the effects of Si on seed germination in alfalfa under salt stress is still scarce. Therefore, this study aimed at exploring the effect of Si on Medicago sativa L. seed germination and seedling growth under salinity. Seeds of two Moroccan alfalfa varieties, Ouad Lmaleh (OL) and Demnate 201 (Dm), and the European variety NS Mediana ZMS V (NS Med) were germinated with 200 mM NaCl solution with or without 3 mM Si. The obtained results showed that salt stress significantly reduced germination traits, reserve mobilization and embryo viability. However, exogenous Si counteracted the negative impact of salinity on seed germination by enhancing reserve mobilization and embryo viability. Si alleviated salt-mediated oxidative stress in alfalfa by reducing hydrogen peroxide (H2O2), electrolyte leakage and malondialdehyde contents and by increasing proline accumulation. Besides, H2O2 staining revealed that Si reduced H2O2 accumulation induced by salt stress. Enzyme activity analyses revealed that Si triggered the activity of some critical enzymes related to oxidative stress alleviation, including superoxide dismutase (SOD) and catalase, together with an accumulation of SOD isoforms, as visualized by native gel PAGE. Furthermore, as compared to Si-untreated salt-stressed control, data showed a key role of Si in alleviating the devastating impact of salt by reducing Na+ content and increasing K+ content, leading to higher K+/Na+ ratio. Our findings suggest that 3 mM Si application can constitute a promising way to improve alfalfa seed germination and seedlings growth under salt stress. [ABSTRACT FROM AUTHOR]

Native breed conservation is an important component of poultry biodiversity. The aim of this work is to describe different steps that lead to donor selection for the implementation of the Italian Semen Cryobank of Autochthonous Chicken and Turkey Breeds. The variability within and between breeds was evaluated, and the stored semen reproductive capacity was in vivo tested using artificial insemination. Semen from Bionda Piemontese, Bianca di Saluzzo and Pepoi roosters was collected and processed. Concentration, volume, sperm membrane integrity, total motile sperm, progressive motile sperm and kinetic parameters were analyzed; sperm parameters accounting for bird variability were used to select male donors. Fresh semen quality parameters measured in donor ejaculates showed significant differences between breeds; no differences were found after cryopreservation. Variability in the fertilizing ability of cryopreserved semen was found within a breed (5–16%) and between birds within a breed (BP = 3–7%; BS = 7–31%; PP = 6–22%); only sperm quality parameters measured in fresh ejaculates, not frozen/thawed, may be associated with in vivo fertility results. In conclusion, sperm concentration and progressive motility were successfully used as selection parameters to identify chicken male donors with improved sperm quality for sperm cryobanking. However, new reliable sperm markers to predict cryopreserved semen’s fertilizing ability are required.

Purpose: What is the rate of euploidy and clinical viability of embryos resulting from micro 3 pronuclei zygotes? Methods: Retrospective cohort analysis in a single, academic in vitro fertilization (IVF) center from March 2018 to June 2021. Cohorts were separated by fertilization as either a 2 pronuclear zygote (2PN) or micro 3 pronuclear zygote (micro 3PN). PGT-A was performed to identify embryonic ploidy rates in embryos created from micro 3PN zygotes. The clinical outcomes of all transferred euploid micro 3PN zygotes were evaluated from frozen embryo transfer (FET) cycles. Results: During the designated study period, 75,903 mature oocytes were retrieved and underwent ICSI. Of these, 60,161 were fertilized as 2PN zygotes (79.3%) and 183 fertilized as micro 3PN zygotes (0.24%). Of the micro 3PN-derived embryos that underwent biopsy, 27.5% (n=11/42) were deemed euploid by PGT-A, compared to 51.4% (n=12,301/23,923) of 2PN-derived embryos, p=0.06. Four micro 3PN-derived embryos were transferred in subsequent single euploid FET cycles, which includes one live birth and one ongoing pregnancy. Conclusion: Micro 3PN zygotes that develop to the blastocyst stage and meet the criteria for embryo biopsy have the potential to be euploid by preimplantation genetic testing for aneuploidy (PGT-A) and if selected for transfer can achieve a live birth. Although there are a significantly lower number of micro 3PN embryos that make it to blastocyst biopsy, the potential to continue to culture abnormally fertilized oocytes may give these patients a chance at pregnancy that they previously did not have. [ABSTRACT FROM AUTHOR]

Purpose: We aimed to study the association between adjusted mtDNA levels in human trophectoderm biopsy samples and the developmental potential of euploid and mosaic blastocysts. Methods: We analyzed relative mtDNA levels in 2,814 blastocysts obtained from 576 couples undergoing preimplantation genetic testing for aneuploidy from June 2018 to June 2021. All patients underwent in vitro fertilization in a single clinic; the study was blinded—mtDNA content was unknown at the time of single embryo transfer. The fate of the euploid or mosaic embryos transferred was compared with mtDNA levels. Results: Euploid embryos had lower mtDNA than aneuploid and mosaic embryos. Embryos biopsied on Day 5 had higher mtDNA than those biopsied on Day 6. No difference was detected in mtDNA scores between embryos derived from oocytes of different maternal ages. Linear mixed model suggested that blastulation rate was associated with mtDNA score. Moreover, the specific next-generation sequencing platform used have a significant effect on the observed mtDNA content. Euploid embryos with higher mtDNA content presented significantly higher miscarriage rates and lower live birth rates, while no significant difference was observed in the mosaic cohort. Conclusion: Our results will aid in improving methods for analyzing the association between mtDNA level and blastocyst viability. [ABSTRACT FROM AUTHOR]

Purpose: Staff management is the most cited ART/IVF laboratory inspection deficiency. Small ART/IVF clinics may be challenged to perform these activities by low staff volume; similarly, large ART/IVF networks may be challenged by high staff volume and large datasets. Here, we sought to investigate the performance of an automated, digital platform solution to manage this necessary task. Methods: The ART Compass (ARTC) digital staff management platform was used to assess the clinical decision-making of ART laboratory staff. The survey modules presented standardized instructions to technologists and measured inter- and intra-technologist variability for subjective "clinical decision-making" type questions. Internal and external comparisons were achieved by providing technologists two answers: (1) a comparison to their own lab director and (2) to the most popular response collectively provided by all lab director level accounts. The platform is hosted on HIPAA compliant Amazon web servers, accessible via web browser and mobile applications for iOS (Apple) and Android mobile devices. Results: Here, we investigated the performance of a digital staff management platform for single embryologist IVF practices and for three IVF lab networks (sites A, B, C) from 2020 to 2022. Embryology dish preparation survey results show variance among respondents in the following: PPE use, media volume, timing of oil overlay, and timing of moving prepared dishes to incubators. Surveying the perceived Gardner score and terms in use for early blastocysts reveals a lack of standardization of terminology and fair to poor agreement. We observed moderate inter-technologist agreement for ICM and TE grade (0.47 and 0.52, respectively). Lastly, the clinical decision of choice to freeze or discard an embryo revealed that agreement to freeze was highest for the top-quality embryos, and that some embryos can be highly contested, evenly split between choice to freeze or discard. Conclusions: We conclude that a digital platform is a novel and effective tool to automate, routinely monitor, and assure quality for staff-related parameters in ART and IVF laboratories. Use of a digital platform can increase regulatory compliance and provide actionable insight for quality assurance in both single embryologist practices and for large networks. Furthermore, clinical decision-making can be augmented with artificial intelligence integration. [ABSTRACT FROM AUTHOR]

Cryopreservation is one of the most important strategies in reproductive medicine now with well-established fertility preservation treatments, successful pregnancy rates. Purpose This work investigates ICSI outcome between LASER Artificial Shrinkage (LAS) and Micro-Needle Artificial Shrinkage (MNAS) before vitrification. Patients and methods Four hundred and nine women were included in the study; which were randomly divided into two groups according to the technique used for artificial shrinkage step of the blastocyst before vitrification: in the first group, Laser beam was used while in the second group the micro-needle was used. Ovarian stimulation was done before the ICSI procedure either by long, short or antagonist protocol. Results The statistical analysis of our study revealed that there was no statistically significant difference between the two groups regarding age, number of cases, AMH, Basal FSH, BMI, male factor, usage ovarian stimulation protocol, high quality blastocysts, the mean number of transferred embryos. While, there was a statistically significant difference between two groups after thawing with p-value < .001 in favor of the LAS method regarding the morphology of originally high quality blastocysts, blastocysts healthiness (not degenerated), pregnancy rate, the implantation rates. Conclusion LASER artificial shrinkage of human blastocysts is a promising technology that could be implemented on a wider basis to improve ART practice, as our study revealed that the usage of LASER pulse for artificial shrinkage of blastocysts before vitrification has better implantation rate as well as better chemical and clinical pregnancy rate in comparison to the usage of micro-needle artificial shrinkage of blastocysts before vitrification. There is a statistically significant difference regarding live birth rate being more in the LASER group as compared to needle group, also the number of twins ether identical or non-identical are larger in laser group than in needle group but with no statistically significant difference. [ABSTRACT FROM AUTHOR]

Vitrification is an important tool to store surplus embryos in assisted reproductive technology (ART). However, vitrification increases oxidative damage and results in decreased viability. Studies have reported that L-glutathione (GSH) supplementation improves the preimplantation development of murine embryos. Glutathione constitutes the major non-protein sulphydryl compound in mammalian cells, which confers protection against oxidative damage. However, the effect of GSH supplementation on embryonic vitrification outcomes has yet to be reported. This study aims to determine whether GSH supplementation in culture media improves in vitro culture and vitrification outcomes, as observed through embryo morphology and preimplantation development. Female BALB/c mice aged 6–8 weeks were superovulated through an intraperitoneal injection of 10 IU of pregnant mare serum gonadotrophin (PMSG), followed by 10 IU of human chorionic gonadotrophin (hCG) 48 h later. The mated mice were euthanized by cervical dislocation 48 h after hCG to harvest embryos. Two-cell embryos were randomly assigned to be cultured in either Group 1 (GSH-free medium), Group 2 (GSH-free medium with vitrification), Group 3 (0.01 mM GSH-supplemented medium), or Group 4 (0.01 mM GSH-supplemented medium with vitrification). Non-vitrified (Groups 1 and 3) and vitrified (Groups 2 and 4) embryos were observed for morphological quality and preimplantation development at 24, 48, 72, and 96 h. In the non-vitrified groups, there were significant increases in the number of Grade-1 blastocysts in GSH cultures (p < 0.05). Similarly, in the vitrified groups, GSH supplementation was also seen to significantly increase blastocyst formation. Exogenous GSH supplementation resulted in a significant increase in intracellular GSH, a release of cytochrome c from mitochondria, and a parallel decrease in intracellular reactive oxygen species (ROS) levels in vitrified eight-cell embryos (p < 0.05). GSH supplementation was shown to upregulate Bcl2 expression and downregulate Bax expression in the vitrified preimplantation embryo group. The action of exogenous GSH was concomitant with an increase in the relative abundance of Gpx1 and Sod1. In conclusion, our study demonstrated the novel use and practical applicability of GSH supplementation for improving embryonic cryotolerance via a decrease in ROS levels and the inhibition of apoptotic events by improvement in oxidative status. [ABSTRACT FROM AUTHOR]

A retrospective case control study was undertaken at the molecular biology department of a private center for reproductive medicine in order to determine whether any correlation exists between mitochondrial DNA (mtDNA) content of cleavage-stage preimplantation embryos and their developmental potential. A total of 69 couples underwent IVF treatment (averaged women age: 36.5, SD 4.9) and produced a total of 314 embryos. A single blastomere was biopsied from each embryo at the cleavage stage (day-3 post-fertilization) subjected to low-pass next generation sequencing (NGS), for the purpose of detecting aneuploidy. For each sample, the number of mtDNA reads obtained after analysis using NGS was divided by the number of reads attributable to the nuclear genome. The mtDNA copy number amount was found to be higher in aneuploid embryos than in those that were euploid (mean mtDNA ratio ± SD: 6.3 ± 7.5 versus 7.1 ± 5.8, p < 0.004; U Mann–Whitney test), whereas no statistically significant differences in mtDNA content were seen in relation to embryo morphology (6.6 ± 4.8 vs. 8.5 ± 13.6, p 0.09), sex (6.6 ± 4.1 vs. 6.2 ± 6.8, p 0.16), maternal age (6.9 ± 7.8 vs. 6.7 ± 4.5, p 0.14) or its ability to implant (7.4 ± 6.6 vs. 5.1 ± 4.6, p 0.18). The mtDNA content cannot serve as a useful biomarker at this point in development. However, further studies investigating both quantitative and qualitative aspects of mtDNA are still required to fully evaluate the relationship between mitochondrial DNA and human reproduction.

Nutritional status and gene polymorphisms of one-carbon metabolism confer a well-known interaction that in pregnant women may affect embryo viability and the health of the newborn. Folate metabolism directly impacts nucleotide synthesis and methylation, which is of increasing interest in the reproductive medicine field. Studies assessing the genetic influence of folate metabolism on IVF treatments have currently been performed in women using their own oocytes. Most of these patients seeking to have a child or undergoing IVF treatments are advised to preventively intake folate supplies that restore known metabolic imbalances, but the treatments could lead to the promotion of specific enzymes in specific women, depending on their genetic variance. In the present study, we assess the influence of candidate gene variants related to folate metabolism, such as Serine Hydroxymethyltransferase 1 SHMT1 (rs1979276 and rs1979277), Betaine-Homocysteine S-Methyltransferase BHMT (rs3733890), Methionine synthase reductase MTRR (rs1801394), Methylenetetrahydrofolate reductase MTHFR (rs1801131 and rs1801133), methionine synthase MTR (rs12749581), ATP Binding Cassette Subfamily B Member 1 ABCB1 (rs1045642) and folate receptor alpha FOLR1 (rs2071010) on the success of IVF treatment performed in women being recipients of donated oocytes. The implication of such gene variants seems to have no direct impact on pregnancy consecution after IVF; however, several gene variants could influence pregnancy loss events or pregnancy maintenance, as consequence of folic acid fortification. [ABSTRACT FROM AUTHOR]

Superovulation treatments aim to stimulate multifollicular recruitment, maximizing the number of oocytes or transferable embryos produced. Factors associated with the superovulation protocol, female characteristics and many other factors are determinants in the number and quality of oocytes obtained. An accurate way to assess oocyte quality more precise than morphological appearance is genetic expression. The present study aims to compare the response of nulliparous and multiparous females to superovulatory stimulation, studying its effect on the expression of some genes associated with the activation, growth, development and oocyte-embryo transition of oocytes, as well as its impact on in vivo embryonic development and viability rate at birth. In a first experiment, the effect of stimulation treatment on the ovulation response and the expression of the MSY2, MATER, ITPR1, ITPR2, ITPR3, eIF4E, PAR1, PAPOL-A, PAPOL-G, ZAR1 and YY1 genes in nulliparous and multiparous females were determined. In a second experiment, the implantation and viability at birth of embryos from superovulated nulliparous and multiparous females were analysed. The ovulation rate was significantly higher in the superovulation groups than in the control groups. The ovulation rate was significantly increased in nulliparous females compared with multiparous does. From the eleven genes analysed, only the expression of MATER, PAPOL-A, PAPOL-G and ZAR-1 genes was shown to be different among experimental groups. Finally, in terms of implantation rate and viability at birth, the nulliparous control group showed better results than the rest of the groups. Both hyperstimulation treatment and reproductive female's history seem to alter the transcriptome of important genes related to oocyte maturation and competence acquisition, affecting in vivo embryo viability. • CTP + hCG elicits a greater response in nulliparous than in multiparous rabbit does. • The expression of MATER, PAPOL-A, PAPOL-G and ZAR1 is affected by superovulation. • Reproductive female history affects genes related to oocyte maturation. • Reproductive female history affects genes related to competence acquisition. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate animal and ovarian parameters that affected fertilization and embryo development up to the morula stage. The data were combined from four studies in which cows were inseminated between 46 and 60 days in milk, following a synchronization protocol, and flushed for embryo collection five or six days later. Parity and body condition score on the day of insemination were recorded. Cyclicity, ovarian structures, and circulating hormone concentrations before and on the day of insemination were also assessed. The recovered structures were graded on a 5-point scale (excellent-good quality, fair quality, poor quality, degenerated, and not fertilized). For recovered embryos, the total number of blastomeres, the number of nonviable blastomeres, and the number of accessory spermatozoa were assessed by epifluorescence microscopy. The risk factors for fertilization and embryo quality were identified using cumulative link mixed models. A total of 418 structures from 389 lactating Holstein cows (34% primiparous and 66% multiparous) were recovered. Thirty-five percent of the recovered structures were excellent-good quality embryos, 21% were fair quality embryos, 11% were poor quality embryos, 16% were degenerated embryos, and 17% were unfertilized oocytes. Structures from primiparous cows, from those with greater progesterone concentration at insemination, and from cows with seven or less accessory spermatozoa were less likely to be fertilized or of better quality than structures from multiparous cows, from those with lower progesterone concentration in plasma, and from those with more than seven accessory spermatozoa, respectively. Embryos with more blastomeres or without nonviable blastomeres were more likely to be of better quality than embryos with fewer blastomeres or with nonviable blastomeres. The results of this analysis highlight the importance of low circulating concentrations of progesterone near artificial insemination and potential positive association with number of accessory spermatozoa reaching the embryo and improvement of embryo development up to the morula stage. • Cumulative link models were used to identify risk factors for embryo quality as a categorical outcome. • Multiparous cows had higher probability to produce good quality embryo than primiparous cows. • Circulating progesterone on the day of estrus was associated with embryo quality. [ABSTRACT FROM AUTHOR]

In the present study, we investigated the effects of exogenous silicon (Si) treatment on seed germination, embryo viability, seedling growth, antioxidant molecules and osmotic regulation in fenugreek under salt stress. The seeds were germinated in Petri dishes at 25 ± 1 °C for 8 days under 0 mM (control) versus 200 mM NaCl (salt stress) with or without 3 mM Si treatment. The obtained results showed that salt stress significantly reduced final germination percentage, germination speed, velocity index, germination energy, peak value, germination value and vitality index, but increased the mean germination time of fenugreek seeds. Salinity also significantly impaired seedling length and fresh weight. However, Si supplementation contracted the negative impacts of salinity and significantly increased all of the studied germination traits except of mean germination time. Additionally, Si alleviated the negative effects of salt on embryo viability and improved its ability to mobilize sugar and protein reserves. Furthermore, 200 mM NaCl increased Na+ content and induced the oxidative stress reflected by high malonyldialdehyde content, reactive oxygen species accumulation and electrolyte leakage percentage. However, salt-mediated oxidative stress was alleviated by Si treatment through a significant decrease of Na+ content and an increase of endogenous Si content, which correlated with a significant induction of enzymatic and non-enzymatic molecules with antioxidant function. Altogether, these findings suggested that, the exogenous Si treatment could be a potential method to enhance salt tolerance of fenugreek during seed germination. [ABSTRACT FROM AUTHOR]

The aim of this study was to compare the effect of two permeant-cryoprotectants, dimethylacetamide (DMA) and N-methylacetamide (NMA) used at different concentrations (0%, 2%, 4%, 6%) on the quality and fertility of post-thaw rooster semen. Ejaculates were processed in 7 treatments: Lake pre-freezing+0.1 M trehalose (LPF-T) (control treatment), LPF-T+2% DMA, LPF-T+4% DMA, LPF-T+6% DMA, LPF-T+2% NMA, LPF-T+4% NMA, LPF-T+6% NMA. Sperm quality [sperm membrane integrity (SMI), motility and kinetic parameters] was assessed before and after cryopreservation. Fertility and embryo viability were recorded. Increasing both DMA and NMA concentration from 2 to 6% improved SMI, total motile sperm, progressive motile sperm (PMS), VCL, VSL and VAP values. PMS recovery rates were significantly the highest in 6% DMA, 4% NMA and 6% NMA treatments. Semen cryopreserved with DMA produced the best fertility and embryo viability at 6%; progressive lower values were recorded at lower concentrations, with no viable embryos at 2%. Semen cryopreserved with NMA showed the best fertility values at 2% and lower values were recorded at higher concentrations; live embryos were found in all NMA treatments. Finally, NMA and DMA showed a similar positive concentration dependent effect of the quality of cryopreserved semen. NMA, not DMA, provided the highest fertility and embryo viability values at the lowest 2%. Therefore, the use of NMA is recommended in order to reduce the cryoprotectant concentration, with a concomitant reduction in the risk of toxicity, providing at the same time the adequate cryoprotective action to obtain viable embryos after artificial insemination of cryopreserved chicken semen. • Lower cryoprotectant concentrations in chicken sperm cryopreservation. • Evaluation of chicken sperm motility parameters. • Dimethylacetamide and N-methylacetamide in freezing poultry semen. • N-methylacetamide in synergy with Trehalose in frozen chicken sperm. • Live embryos obtained using rooster semen cryopreserved with N-methylacetamide. [ABSTRACT FROM AUTHOR]

A retrospective case control study was undertaken at the molecular biology department of a private center for reproductive medicine in order to determine whether any correlation exists between the mitochondrial DNA (mtDNA) content of trophectoderm and embryo developmental potential. A total of 275 couples underwent IVF treatment, producing a total of 716 embryos. The trophectoderm was biopsied from each embryo at the blastocyst stage (day 5 or day 6 post-fertilization) subjected to low-pass next-generation sequencing (NGS), for the purpose of detecting aneuploidy. For each sample, the number of mtDNA reads obtained after analysis using NGS was divided by the number of reads attributable to the nuclear genome. The mtDNA copy number was found to be higher in aneuploid embryos than in those that were euploid (mean mtDNA ratio ± SD: 1.13 ± 1.37 versus 1.45 ± 1.78, p = 0.02) and in day 5 biopsies compared to day 6 biopsies (1.41 ± 1.66 vs. 1.19 ± 1.27, p = 0.001), whereas no statistically significant differences in mtDNA content were seen in relation to embryo morphology (1.58 ± 2.44 vs. 2.19 ± 2.89, p = 0.12), genetic sex (1.27 ± 1.29 vs. 1.27 ± 1.18, p = 0.99), maternal age (1.31 ± 1.41 vs. 1.33 ± 1.29, p = 0.43), or its ability to implant (1.14 ± 0.88 vs. 1.21 ± 1.16, p = 0.39). mtDNA has small potential to serve as an additional, independent biomarker for embryo selection. [ABSTRACT FROM AUTHOR]

An increasing number of studies have related the mitochondrial DNA (mtDNA) content to embryo viability and transfer outcomes. However, previous studies have focused more on the relationship between mtDNA and embryo implantation, few studies have studied the effect of the mtDNA content on live birth. In the study, we investigated whether mtDNA content is a reliable screening biomarker for live birth after single blastocyst transfer. A total of 233 couples with 316 blastocyst stage embryos undergoing in vitro fertilization treatment and pre-implantation genetic testing analysis were included in the study. All embryos were chromosomally normal and had undergone single-embryo transfers. There was no significant difference observed in the blastocyst mtDNA content among the live birth, miscarriage and non-implanted groups (p=0.999), and the mtDNA content in blastocysts from the miscarriage and live birth groups was similar [median (interquartile range), 1.00*108(7.59*107- 1.39*108) vs 1.01*108 (7.37*107- 1.32*108)]. Similarly, no significant association was observed between mtDNA content and embryo implantation potential (p=0.965). After adjusting for multiple confounders in a logistic regression analysis with generalized estimating equations, no associations between mtDNA content and live birth were observed in all blastocysts, Day-5 and Day-6 blastocysts (p=0.567, p=0.673, p=0.165, respectively). The live birth rate was not significantly different between blastocysts with an elevated mtDNA content and blastocysts with a normal mtDNA content (26.7% vs 33.6% p=0.780). Additionally, there was no linear correlation between the mtDNA content and maternal age (p=0.570). In conclusion, the mtDNA content does not seem to be a potential biomarker for embryo transfer outcomes (i.e., implantation and live birth) based on the existing testing tools. Embryos with an elevated mtDNA content also have development potential for successful live birth. [ABSTRACT FROM AUTHOR]

The main goal of assisted reproductive technology (ART) is to achieve a healthy singleton live birth after the transfer of one embryo. A major objective of IVF scientists has always been to use adequate criteria for selecting the embryo for transfer according to its implantation potential. Indeed, embryo quality is usually assessed by evaluating visual morphology, which relies on the removal of the embryo from the incubator and might include inter- and intra-evaluator variation among embryologists. Recently, an advancement in embryo culture has taken place with the introduction of a new type of incubator with an integrated time-lapse monitoring system, which enables embryologists to analyse the dynamic events of embryo development from fertilization to blastocyst formation. This novel practice is rapidly growing and has been used in many IVF centres worldwide. Therefore, the main aim of this review is to present the benefits of time-lapse monitoring in a modern embryology laboratory; in particular, we discuss blastocyst collapse and morphometric blastocyst assessment, and analyse their association with embryo viability and implantation potential. In addition, we highlight preliminary studies involving artificial intelligence and machine learning models as non-invasive markers of clinical pregnancy. [ABSTRACT FROM AUTHOR]

Microbiome formation and assemblage are essential processes influencing proper embryonal and early-life development in neonates. In birds, transmission of microbes from the outer environment into the egg’s interior has been found to shape embryo viability and hatchling phenotype. However, microbial transmission may be affected by egg-white antimicrobial proteins (AMPs), whose concentration and antimicrobial action are temperature-modulated. As both partial incubation and clutch covering with nest-lining feathers during the pre-incubation period can significantly alter temperature conditions acting on eggs, we experimentally investigated the effects of these behavioural mechanisms on concentrations of both the primary and most abundant egg-white AMPs (lysozyme and avidin) using mallard (Anas platyrhychos) eggs. In addition, we assessed whether concentrations of egg-white AMPs altered the probability and intensity of bacterial trans-shell penetration, thereby affecting hatchling morphological traits in vivo.We observed higher concentrations of lysozyme in partially incubated eggs. Clutch covering with nest-lining feathers had no effect on egg-white AMP concentration and we observed no association between concentration of eggwhite lysozyme and avidin with either the probability or intensity of bacterial transshell penetration. The higher egg-white lysozyme concentration was associated with decreased scaled body mass index of hatchlings. These outcomes demonstrate that incubation prior to clutch completion in precocial birds can alter concentrations of particular egg-white AMPs, though with no effect on bacterial transmission into the egg in vivo. Furthermore, a higher egg white lysozyme concentration compromised hatchling body condition, suggesting a potential growth-regulating role of lysozyme during embryogenesis in precocial birds. [ABSTRACT FROM AUTHOR]

Microbiome formation and assemblage are essential processes influencing proper embryonal and early-life development in neonates. In birds, transmission of microbes from the outer environment into the egg’s interior has been found to shape embryo viability and hatchling phenotype. However, microbial transmission may be affected by egg-white antimicrobial proteins (AMPs), whose concentration and antimicrobial action are temperature-modulated. As both partial incubation and clutch covering with nest-lining feathers during the pre-incubation period can significantly alter temperature conditions acting on eggs, we experimentally investigated the effects of these behavioural mechanisms on concentrations of both the primary and most abundant egg-white AMPs (lysozyme and avidin) using mallard (Anas platyrhychos) eggs. In addition, we assessed whether concentrations of egg-white AMPs altered the probability and intensity of bacterial trans-shell penetration, thereby affecting hatchling morphological traits in vivo. We observed higher concentrations of lysozyme in partially incubated eggs. Clutch covering with nest-lining feathers had no effect on egg-white AMP concentration and we observed no association between concentration of egg-white lysozyme and avidin with either the probability or intensity of bacterial trans-shell penetration. The higher egg-white lysozyme concentration was associated with decreased scaled body mass index of hatchlings. These outcomes demonstrate that incubation prior to clutch completion in precocial birds can alter concentrations of particular egg-white AMPs, though with no effect on bacterial transmission into the egg in vivo. Furthermore, a higher egg white lysozyme concentration compromised hatchling body condition, suggesting a potential growth-regulating role of lysozyme during embryogenesis in precocial birds.

An increasing number of studies have related the mitochondrial DNA (mtDNA) content to embryo viability and transfer outcomes. However, previous studies have focused more on the relationship between mtDNA and embryo implantation, few studies have studied the effect of the mtDNA content on live birth. In the study, we investigated whether mtDNA content is a reliable screening biomarker for live birth after single blastocyst transfer. A total of 233 couples with 316 blastocyst stage embryos undergoing in vitro fertilization treatment and pre-implantation genetic testing analysis were included in the study. All embryos were chromosomally normal and had undergone single-embryo transfers. There was no significant difference observed in the blastocyst mtDNA content among the live birth, miscarriage and non-implanted groups (p=0.999), and the mtDNA content in blastocysts from the miscarriage and live birth groups was similar [median (interquartile range), 1.00*108(7.59*107- 1.39*108) vs 1.01*108 (7.37*107- 1.32*108)]. Similarly, no significant association was observed between mtDNA content and embryo implantation potential (p=0.965). After adjusting for multiple confounders in a logistic regression analysis with generalized estimating equations, no associations between mtDNA content and live birth were observed in all blastocysts, Day-5 and Day-6 blastocysts (p=0.567, p=0.673, p=0.165, respectively). The live birth rate was not significantly different between blastocysts with an elevated mtDNA content and blastocysts with a normal mtDNA content (26.7% vs 33.6% p=0.780). Additionally, there was no linear correlation between the mtDNA content and maternal age (p=0.570). In conclusion, the mtDNA content does not seem to be a potential biomarker for embryo transfer outcomes (i.e., implantation and live birth) based on the existing testing tools. Embryos with an elevated mtDNA content also have development potential for successful live birth.

Mitochondria are commonly recognized as the powerhouses of the cell, primarily responsible for energy production through oxidative phosphorylation. Alongside this vital function, they also play crucial roles in regulating calcium signaling, maintaining membrane potential, and modulating apoptosis. Their involvement in various cellular pathways becomes particularly evident during oogenesis and embryogenesis, where mitochondrial quantity, morphology, and distribution are tightly controlled. The efficiency of the mitochondrial network is maintained through multiple quality control mechanisms that are essential for reproductive success. These include mitochondrial unfolded protein response, mitochondrial dynamics, and mitophagy. Not surprisingly, mitochondrial dysfunction has been implicated in infertility and ovarian aging, prompting investigation into mitochondria as diagnostic and therapeutic targets in assisted reproduction. To date, mitochondrial DNA copy number in oocytes, cumulus cells, and trophectoderm biopsies, and fluorescent lifetime imaging microscopy-based assessment of NADH and flavin adenine dinucleotide content have been explored as potential predictors of embryo competence, yielding limited success. Despite challenges in the clinical application of mitochondrial diagnostic strategies, these enigmatic organelles have a significant impact on reproduction, and their potential role as diagnostic targets in assisted reproduction is likely to remain an active area of investigation in the foreseeable future., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Vitrification is an important tool to store surplus embryos in assisted reproductive technology (ART). However, vitrification increases oxidative damage and results in decreased viability. Studies have reported that L-glutathione (GSH) supplementation improves the preimplantation development of murine embryos. Glutathione constitutes the major non-protein sulphydryl compound in mammalian cells, which confers protection against oxidative damage. However, the effect of GSH supplementation on embryonic vitrification outcomes has yet to be reported. This study aims to determine whether GSH supplementation in culture media improves in vitro culture and vitrification outcomes, as observed through embryo morphology and preimplantation development. Female BALB/c mice aged 6–8 weeks were superovulated through an intraperitoneal injection of 10 IU of pregnant mare serum gonadotrophin (PMSG), followed by 10 IU of human chorionic gonadotrophin (hCG) 48 h later. The mated mice were euthanized by cervical dislocation 48 h after hCG to harvest embryos. Two-cell embryos were randomly assigned to be cultured in either Group 1 (GSH-free medium), Group 2 (GSH-free medium with vitrification), Group 3 (0.01 mM GSH-supplemented medium), or Group 4 (0.01 mM GSH-supplemented medium with vitrification). Non-vitrified (Groups 1 and 3) and vitrified (Groups 2 and 4) embryos were observed for morphological quality and preimplantation development at 24, 48, 72, and 96 h. In the non-vitrified groups, there were significant increases in the number of Grade-1 blastocysts in GSH cultures (p < 0.05). Similarly, in the vitrified groups, GSH supplementation was also seen to significantly increase blastocyst formation. Exogenous GSH supplementation resulted in a significant increase in intracellular GSH, a release of cytochrome c from mitochondria, and a parallel decrease in intracellular reactive oxygen species (ROS) levels in vitrified eight-cell embryos (p < 0.05). GSH supplementation was shown to upregulate Bcl2 expression and downregulate Bax expression in the vitrified preimplantation embryo group. The action of exogenous GSH was concomitant with an increase in the relative abundance of Gpx1 and Sod1. In conclusion, our study demonstrated the novel use and practical applicability of GSH supplementation for improving embryonic cryotolerance via a decrease in ROS levels and the inhibition of apoptotic events by improvement in oxidative status.

Nutritional status and gene polymorphisms of one-carbon metabolism confer a well-known interaction that in pregnant women may affect embryo viability and the health of the newborn. Folate metabolism directly impacts nucleotide synthesis and methylation, which is of increasing interest in the reproductive medicine field. Studies assessing the genetic influence of folate metabolism on IVF treatments have currently been performed in women using their own oocytes. Most of these patients seeking to have a child or undergoing IVF treatments are advised to preventively intake folate supplies that restore known metabolic imbalances, but the treatments could lead to the promotion of specific enzymes in specific women, depending on their genetic variance. In the present study, we assess the influence of candidate gene variants related to folate metabolism, such as Serine Hydroxymethyltransferase 1 SHMT1 (rs1979276 and rs1979277), Betaine-Homocysteine S-Methyltransferase BHMT (rs3733890), Methionine synthase reductase MTRR (rs1801394), Methylenetetrahydrofolate reductase MTHFR (rs1801131 and rs1801133), methionine synthase MTR (rs12749581), ATP Binding Cassette Subfamily B Member 1 ABCB1 (rs1045642) and folate receptor alpha FOLR1 (rs2071010) on the success of IVF treatment performed in women being recipients of donated oocytes. The implication of such gene variants seems to have no direct impact on pregnancy consecution after IVF; however, several gene variants could influence pregnancy loss events or pregnancy maintenance, as consequence of folic acid fortification.

The Juniperus excelsa (Cupressaceae) forest in northern Greece has been facing a documented severe decline of natural regeneration over the last few years. In order to dissect the physiological components of the low regeneration, pollen originated from different sites as well as from trees of different height was investigated in terms of viability and in vitro germination capacity. In addition, embryo development and viability of the first year immature seeds were further assessed. Pollen was found to present high viability (61.5-93.2%) in all sites and tree height classes. However, pollen germination was quite low (maximum germination percentage recorded was 30.2%). The low pollen germination capacity recorded in trees above 6 m in height was reflected in the subsequent low percentages of successful embryo development and viability found in the produced seeds. These results suggest that low seed fertility in Juniperus excelsa is associated with reduced pollen germination, suggesting a complex regeneration mechanism and is considered an additional step in shedding light on the physiological basis of the low natural regeneration of Juniperus excelsa.

Abstract Mounting evidence suggests that nongenetic paternal effects on offspring may be widespread among animal taxa, but the mechanisms underlying this form of nongenetic inheritance are not yet fully understood. Here, we show that seminal fluids underlie paternal effects on early offspring survival in an insect, the cricket Teleogryllus oceanicus, and quantify the contribution of this paternal effect to the inheritance of this important fitness trait. We used castrated males within a full‐sib half‐sib experimental design to show that seminal fluid donors were responsible for variation in the survival of developing embryos to hatching, and in their subsequent survival to adulthood. Increased expression of two seminal fluid protein genes, previously found to be positively associated with sperm quality, was found to be negatively associated with embryo survival. These nongenetic paternal effects hold important implications for the evolution of adaptive maternal responses to sperm competition, and more broadly for the interpretation of sire effects from classic quantitative genetic breeding designs.

A retrospective case control study was undertaken at the molecular biology department of a private center for reproductive medicine in order to determine whether any correlation exists between the mitochondrial DNA (mtDNA) content of trophectoderm and embryo developmental potential. A total of 275 couples underwent IVF treatment, producing a total of 716 embryos. The trophectoderm was biopsied from each embryo at the blastocyst stage (day 5 or day 6 post-fertilization) subjected to low-pass next-generation sequencing (NGS), for the purpose of detecting aneuploidy. For each sample, the number of mtDNA reads obtained after analysis using NGS was divided by the number of reads attributable to the nuclear genome. The mtDNA copy number was found to be higher in aneuploid embryos than in those that were euploid (mean mtDNA ratio ± SD: 1.13 ± 1.37 versus 1.45 ± 1.78, p = 0.02) and in day 5 biopsies compared to day 6 biopsies (1.41 ± 1.66 vs. 1.19 ± 1.27, p = 0.001), whereas no statistically significant differences in mtDNA content were seen in relation to embryo morphology (1.58 ± 2.44 vs. 2.19 ± 2.89, p = 0.12), genetic sex (1.27 ± 1.29 vs. 1.27 ± 1.18, p = 0.99), maternal age (1.31 ± 1.41 vs. 1.33 ± 1.29, p = 0.43), or its ability to implant (1.14 ± 0.88 vs. 1.21 ± 1.16, p = 0.39). mtDNA has small potential to serve as an additional, independent biomarker for embryo selection.

Objective: To determine the effect of patient and treatment parameters on 19 embryo morphokinetic parameters using pronuclear fading as time zero.Design: Single-site, retrospective cohort analysis.Setting: Fertility treatment center.Patients(s): Patients undergoing treatment between September 2014 and January 2016 (n = 639) whose embryos were cultured in the EmbryoScope for 6 days (n = 2,376).Intervention(s): None.Main Outcome Measure(s): Multiple regression analysis of body mass index; maternal age; infertility diagnosis; treatment type; suppression protocol on time to each cellular division (tn): t2, t3, t4, t5, t6, t7, t8, t9, time to start of compaction (tM), start of blastulation (tSB), full blastocyst (tB); and interval measurements: s2, s3, cc2, cc3, cc4, t9-tM, tM-tSB, and tSB-tB. Beta coefficients were analyzed to quantify any significant effects.Result(s): Embryos appeared to be subtly affected by patient and treatment parameters, exhibiting complex relationships between various morphokinetic parameters and specific patient and treatment factors, rather than a systemic effect.Conclusion(s): These findings outline the need for the consideration of confounding factors when assessing an embryo's ability to achieve implantation. Although morphokinetic parameters have been related to embryo viability, it is likely that this will vary depending on the embryo's origin. [ABSTRACT FROM AUTHOR]

Summary: During human in vitro culture, a morphological microscope analysis is normally performed to select the best embryo to transfer, with the hope of obtaining a successful pregnancy. The morphological evaluation may combine number and size of blastomeres, fragmentation, multinucleation, blastocyst expansion, inner-cell mass and trophectoderm appearance. However, standard microscopy evaluation involves the removal of the embryos from the incubator, exposing them to changes in pH, temperature, and oxygen level. Additionally, morphological assessments might include high inter-observer variability. Recently, continuous embryo culture using time-lapse monitoring (TLM) has allowed embryologists to analyse the dynamic and morphokinetic events of embryo development and, based on that, the embryologist is able to scrutinize the complete sequence of embryonic evolution, from fertilization to the blastocyst formation. Therefore, TLM allows an uninterrupted culture condition, reducing the need to remove embryos from the incubator. The monitoring system is normally composed of a standard incubator with an integrated microscope coupled to a digital camera, which is able to collect images at regular times, and subsequently processed into video. These data can be annotated and analyzed using an integrated software, therefore this allows embryologists to facilitate the process of embryo selection for transfer. The main aim of this paper is to discuss the potential benefits and uses of the TLM in the embryology laboratory. [ABSTRACT FROM AUTHOR]

The present study aimed to elucidate the developmental kinetics, growth potential, and viability of bovine embryos produced in vitro with sexed semen. Bovine oocytes were fertilized in vitro using unsorted and X-sorted semen from the same Holstein bulls, and the kinetics of in vitro development were continuously monitored for 10 d through time-lapse cinematography. The blastocyst formation rate was lower in the X-sorted group than in the unsorted group (P < 0.01), whereas the normal fertilization rate did not differ between groups. Morphokinetic evaluation revealed that the incidence of blastomere fusion during the first cleavage division, termed reverse cleavage, was higher in the X-sorted group (P < 0.01). Furthermore, embryos produced with X-sorted semen showed slower growth throughout the developmental period than embryos produced with unsorted semen (P < 0.01). The cell number of the trophectoderm and inner cell mass of blastocysts was reduced in the X-sorted group (P < 0.01). In embryos that developed to the blastocyst stage, the hatchability (P < 0.05), chromosomal normality (P < 0.01), and survivability after the conventional frozen-thawing process (P < 0.05) were reduced in the X-sorted group compared to that in the unsorted group, indicating a compromised viability of embryos derived from X-sorted semen. Taken together, the first cleavage dysmorphism, delayed embryo growth, and impaired viability of embryos developed to the blastocyst stage may explain the mechanism of reduced fertility in embryos derived from sexed semen. The kinetic evaluation of early embryo development and de-selection of embryos presenting the aberrant first cleavage would be valid for clinical application to produce sexed embryos with high implantation potential. • Bovine embryos were produced in vitro with X-sorted semen. • Blastomere fusion incidence increased during the first cleavage division. • X-sperm-derived embryos had slow growth and reduced blastulation rate. • These embryos had reduced hatchability, chromosomal normality, and cryotolerance. [ABSTRACT FROM AUTHOR]

The recent clinical introduction of time-lapse videography into in vitro fertilization laboratories has offered a novel opportunity for embryologists to explore improved methods for embryo selection. While the concept of uninterrupted culture of embryos provided by such systems is welcomed, the current evidence does not support its full application in routine clinical practice. The issue of whether or not algorithms for embryo selection can be extrapolated between laboratories, which may represent a major hurdle to its wide application, is currently gaining increasing attention amongst embryologists worldwide. In this commentary issues identified in time-lapse embryo selection/de-selection algorithms, such as quantitative versus qualitative parameters, are discussed alongside the reference start point for the timing system, and types of datasets used for developing and validating time-lapse algorithms. Considering these factors, alternative future research directions which could potentially solve current issues are proposed. [ABSTRACT FROM AUTHOR]

Background: Successful embryo implantation is a complex process that requires the coordination of a series of events, involving both the embryo and the maternal endometrium. Key to this process is the intricate cascade of molecular mechanisms regulated by endocrine, paracrine and autocrine modulators of embryonic and maternal origin. Despite significant progress in ART, implantation failure still affects numerous infertile couples worldwide and fewer than 10% of embryos successfully implant. Improved selection of both the viable embryos and the optimal endometrial phenotype for transfer remains crucial to enhancing implantation chances. However, both classical morphological embryo selection and new strategies incorporated into clinical practice, such as embryonic genetic analysis, morphokinetics or ultrasound endometrial dating, remain insufficient to predict successful implantation. Additionally, no techniques are widely applied to analyse molecular signals involved in the embryo-uterine interaction. More reliable biological markers to predict embryo and uterine reproductive competence are needed to improve pregnancy outcomes. Recent years have seen a trend towards 'omics' methods, which enable the assessment of complete endometrial and embryonic molecular profiles during implantation. Omics have advanced our knowledge of the implantation process, identifying potential but rarely implemented biomarkers of successful implantation.Objective and Rationale: Differences between the findings of published omics studies, and perhaps because embryonic and endometrial molecular signatures were often not investigated jointly, have prevented firm conclusions being reached. A timely review summarizing omics studies on the molecular determinants of human implantation in both the embryo and the endometrium will help facilitate integrative and reliable omics approaches to enhance ART outcomes.Search Methods: In order to provide a comprehensive review of the literature published up to September 2019, Medline databases were searched using keywords pertaining to omics, including 'transcriptome', 'proteome', 'secretome', 'metabolome' and 'expression profiles', combined with terms related to implantation, such as 'endometrial receptivity', 'embryo viability' and 'embryo implantation'. No language restrictions were imposed. References from articles were also used for additional literature.Outcomes: Here we provide a complete summary of the major achievements in human implantation research supplied by omics approaches, highlighting their potential to improve reproductive outcomes while fully elucidating the implantation mechanism. The review highlights the existence of discrepancies among the postulated biomarkers from studies on embryo viability or endometrial receptivity, even using the same omic analysis.Wider Implications: Despite the huge amount of biomarker information provided by omics, we still do not have enough evidence to link data from all omics with an implantation outcome. However, in the foreseeable future, application of minimally or non-invasive omics tools, together with a more integrative interpretation of uniformly collected data, will help to overcome the difficulties for clinical implementation of omics tools. Omics assays of the embryo and endometrium are being proposed or already being used as diagnostic tools for personalised single-embryo transfer in the most favourable endometrial environment, avoiding the risk of multiple pregnancies and ensuring better pregnancy rates. [ABSTRACT FROM AUTHOR]

The goal of this retrospective cohort study was to compare survival, implantation, clinical and ongoing pregnancy rates between blastocysts that were vitrified on post-fertilization days 5, 6 and 7. Before vitrification, blastocysts were evaluated in terms of morphology and blastocyst expansion, inner cell mass and trophectoderm quality. They were thawed and transfered in a subsequent artificial cycle. Embryo implantation rates were 39%, 25% and 25% for blastocysts that were vitrified on days 5, 6, and 7, respectively (p = 0.006). Clinical and ongoing pregnancy rates were 19%, 12%, 13% (p = 0.100) and 9%, 7%, 12% (p = 0.99) for days 5, 6 and 7 blastocysts, respectively. Day 5 blastocysts had significantly higher full-collapsing score after assisted-hatching compared to days 6 and 7 blastocysts (p = 0.014). As blastocyst quality increased, implantation and clinical pregnancy rates increased in all groups and both parameters were statistically significantly higher on day 5 blastocysts than on days 6 or 7 (p = 0.001). It was clearly found that good quality blastocysts obtained on day 5 have higher implantation and clinical pregnancy rates than 6th and 7th day cryopreserved embryos. There were no statistically significant differences between the cryopreserved embryos on days 6 and 7 regarding the implantation, clinic and ongoing pregnancy rates. [ABSTRACT FROM AUTHOR]

This study investigated the efficacy of four published day 3 embryo time-lapse algorithms based on different types of datasets (known implantation data [KID] and single embryo transfer [SET]), and the confounding effect of female age and conventional embryo morphology. Four algorithms were retrospectively applied to three types of datasets generated at Fertility North between February 2013 and December 2014: (a) KID dataset (n = 270), (b) a subset of SET (n = 144, end-point = implantation), and (c) SET (n = 144, end-point = live birth), respectively. All four algorithms showed progressively reduced predictive power (expressed as area under the receiver operating characteristics curve and 95% confidence interval [CI]) after application to the three datasets (a–c): Liu (0.762 [0.701–0.824] vs. 0.724 [0.641–0.807] vs. 0.707 [0.620–0.793]), KIDScore (0.614 [0.539–0.688] vs. 0.548 [0.451–0.645] vs. 0.536 [0.434–0.637]), Meseguer (0.585 [0.508–0.663] vs. 0.56 [0.462–0.658] vs. 0.549 [0.445–0.652]), and Basile (0.582 [0.505–0.659] vs. 0.519 [0.421–0.618] vs. 0.509 [0.406–0.612]). Furthermore, using KID dataset, the association (expressed as odds ratio and 95% CI) between time-lapse algorithms and implantation outcomes lost statistical significance after adjusting for conventional embryo morphology and female age in 3 of the 4 algorithms (KIDScore 1.832 [1.118–3.004] vs. 1.063 [0.659–1.715], Meseguer 1.150 [1.021–1.295] vs. 1.122 [0.981–1.284] and Basile 1.122 [1.008–1.249] vs. 1.038 [0.919–1.172]). In conclusion, SET is a preferred dataset to KID when developing or validating time-lapse algorithms, and day 3 conventional embryo morphology and female age should be considered as confounding factors. [ABSTRACT FROM AUTHOR]

The Juniperus excelsa (Cupressaceae) forest in northern Greece has been facing a documented severe decline of natural regeneration over the last few years. In order to dissect the physiological components of the low regeneration, pollen originated from different sites as well as from trees of different height was investigated in terms of viability and in vitro germination capacity. In addition, embryo development and viability of the first year immature seeds were further assessed. Pollen was found to present high viability (61.5-93.2%) in all sites and tree height classes. However, pollen germination was quite low (maximum germination percentage recorded was 30.2%). The low pollen germination capacity recorded in trees above 6 m in height was reflected in the subsequent low percentages of successful embryo development and viability found in the produced seeds. These results suggest that low seed fertility in Juniperus excelsa is associated with reduced pollen germination, suggesting a complex regeneration mechanism and is considered an additional step in shedding light on the physiological basis of the low natural regeneration of Juniperus excelsa. [ABSTRACT FROM AUTHOR]

Mounting evidence suggests that nongenetic paternal effects on offspring may be widespread among animal taxa, but the mechanisms underlying this form of nongenetic inheritance are not yet fully understood. Here, we show that seminal fluids underlie paternal effects on early offspring survival in an insect, the cricket Teleogryllus oceanicus, and quantify the contribution of this paternal effect to the inheritance of this important fitness trait. We used castrated males within a full‐sib half‐sib experimental design to show that seminal fluid donors were responsible for variation in the survival of developing embryos to hatching, and in their subsequent survival to adulthood. Increased expression of two seminal fluid protein genes, previously found to be positively associated with sperm quality, was found to be negatively associated with embryo survival. These nongenetic paternal effects hold important implications for the evolution of adaptive maternal responses to sperm competition, and more broadly for the interpretation of sire effects from classic quantitative genetic breeding designs. [ABSTRACT FROM AUTHOR]

Contents: Superovulation protocols are designed to achieve maximum embryo yields. Nevertheless, ovarian response control and the quality of obtained embryos are still a challenge. On the other hand, to save the superovulated embryos until their subsequent use, it is usual to cryopreserve them, so it is also crucial to assess their cryotolerance. The aim of this study was to compare the efficacy of a single injection of corifollitropin alfa (FSH‐CTP) alone or supplemented with human chorionic gonadotropin (hCG) and to determine the impact of this stimulation on in vitro and in vivo development of fresh or devitrified embryos. Our outcomes showed that ovulation rate and recovered embryos were significantly increased when hCG was used. In vitro development of fresh and devitrified embryos and survival at birth were not significantly affected by superstimulation treatment. Results of this study suggest that a single injection of long‐acting FSH‐CTP supplemented with hCG can be effectively used in rabbits to elicit an increase in ovulation rate and number of recovered embryos. Furthermore, we demonstrated that hCG supplementation had no negative effects in embryo cryosurvival and development, showing similar survival rate at birth than FSH‐CTP alone group. [ABSTRACT FROM AUTHOR]

Only one third of the in vitro fertilization treatments result in successful delivery following morphological viability assessment worldwide. A paper by Montskó et al. (2015) describes the identification of the alpha-1 chain of human haptoglobin as a potential marker of embryo viability. Using mass spectrometry, the concentration of the haptoglobin alpha-1 chain was determined in spent culture media samples of in vitro fertilized embryos and correlation was found with the outcome of the respective transfer. In the present study we investigated, whether the concentration of haptoglobin alpha-1 chain shows any correlation with morphological scores to clarify whether levels of the alpha-1 chain provide additional information on embryo viability unnoticed by the morphological assessment. In the study, pregnancy and live birth rates were examined in 143 transferred samples of 86 patients, retrospectively. Two sample groups were created. The control group contained embryos classified as 'good' or 'fair' based on the Istanbul Consensus Criteria System, while the double-assay group contained embryos assessed as 'good' or 'fair' by the morphological evaluation and as 'viable' by the haptoglobin assay. Clinical pregnancy rate was 30.2% in the control group, while 47.6% in the group scored parallel with morphological criteria and proteomic analysis (p < 0.05). The increased clinical pregnancy rate observed in the double-assayed group can be attributed to decreased false-positivity of the double assay. Abbreviations: IVF: in vitro fertilization; SEC: spent embryo culture medium; HSA: human serum albumin; Hpt: haptoglobin; HptA1: haptoglobin alpha-1 chain; ICCS: Istanbul Consensus Criteria System; BMI: body mass index; ICSI: intracytoplasmic sperm injection [ABSTRACT FROM AUTHOR]

Contents: The sperm mobility assay measures the ability of sperm to swim through a dense layer of Accudenz®, and the sperm mobility phenotype has been shown to predict fertility and other sperm performance traits in roosters and turkeys. In this study, we examined turkey sperm morphometry and rates of early embryonic death associated with high‐ and low‐mobility semen. We also assessed whether the hypo‐osmotic stress test, which evaluates the structural integrity of the sperm plasma membrane, may be used as a faster and simpler assay for sperm mobility and viability. We confirmed previous work that found that high‐mobility sperm are faster and swim more linearly than low‐mobility sperm, and that mobility traits were repeatable within males. In contrast to previous studies, we did not find higher rates of fertility, but low‐mobility sperm was associated with higher rates of early embryonic death, though this trend was not significant. High‐mobility sperm had longer sperm heads, explained by longer nuclei, despite shorter acrosomes. Although these sperm were faster, midpiece length and flagellum length did not differ between high‐ and low‐mobility sperm. Finally, mobility was not found to be associated with sperm performance in the hypo‐osmotic stress test. [ABSTRACT FROM AUTHOR]

The evaluation of embryo viability is typically based on morphologic and morphometric grading systems, which are known to be heavily affected by differences in subjective judgement. A significant proportion of euploid embryos do not implant even when the endometrium is normal, despite the wide incorporation of preimplantation genetic testing for aneuploidy. The need to improve reproductive outcomes following assisted reproductive treatments is culminating in the emergence of new research and technology with the potential to enhance embryo selection. The assessment of mitochondrial DNA content as a predictor of embryo viability has gained increasing attention among numerous promising approaches. As is common with new tools, this attention has led to both inflated expectations and serious disillusionment. Here we gather evidence to establish the true clinical applicability of this method. Controversy continues to surround this topic, adding both interest and confusion. [ABSTRACT FROM AUTHOR]