Mitochondria as determinants of reproductive senescence and competence: implications for diagnosis of embryo competence in assisted reproduction.

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.

Mitochondrial DNA quantification correlates with the developmental potential of human euploid blastocysts but not with that of mosaic blastocysts.

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.

Assessing the clinical viability of micro 3 pronuclei zygotes.

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.

Assuring quality in assisted reproduction laboratories: assessing the performance of ART Compass - a digital art staff management platform.

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.

Mitochondrial DNA Copy Number in Cleavage Stage Human Embryos-Impact on Infertility Outcome.

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.

Concentration dependent effect of dimethylacetamide and N-methylacetamide on the quality and fertility of cryopreserved chicken semen.

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.

Profiling the Influence of Gene Variants Related to Folate-Mediated One-Carbon Metabolism on the Outcome of In Vitro Fertilization (IVF) with Donor Oocytes in Recipients Receiving Folic Acid Fortification.

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.

Does Trophectoderm Mitochondrial DNA Content Affect Embryo Developmental and Implantation Potential?

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.

Focus on time-lapse analysis: blastocyst collapse and morphometric assessment as new features of embryo viability.

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.

Use of time-lapse monitoring in medically assisted reproduction treatments: a mini-review.

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.

Can paternal effects via seminal fluid contribute to the evolution of polyandry?

Genetic benefits from mating with multiple males are thought to favour the evolution of polyandry. However, recent evidence suggests that non-genetic paternal effects via seminal fluid might contribute to the observed effects of polyandry on offspring performance. Here, we test this hypothesis using the field cricket Teleogryllus oceanicus. Using interference RNA, we first show that at least one seminal fluid protein is essential for embryo survival. We then show that polyandrous females mated to three different males produced embryos with higher pre-hatching viability than did monandrous females mated with the same male three times. Pseudo-polyandrous females that obtained sperm and seminal fluid from a single male and seminal fluid from two additional males had embryos with viabilities intermediate between monandrous and polyandrous females. Our results suggest either that ejaculate mediated paternal effects on embryo viability have both genetic and non-genetic components, or that seminal fluids transferred by castrated males provide only a subset of proteins contained within the normal ejaculate, and are unable to exert their full effect on embryo viability.

Analysis of specific mRNA gene expression profiles as markers of egg and embryo quality for hybrid catfish aquaculture.

Despite best efforts to optimize reproduction, egg incubation, and larval performance in captivity, inconsistencies in hatchery fish production are still created by high variations in egg quality from individual females. In some hatchery species, egg quality and generation of viable embryos are correlated to abundances of specific mRNAs. Channel catfish females show considerable extremes in egg quality, causing inconsistencies in channel catfish, Ictalurus punctatus, female × blue catfish, Ictalurus furcatus, male hybrid fry production. The objectives of this study were to examine relative transcripts linked to egg and embryo quality and determine expression between low-hatch and high-hatch egg batches through early development (0, 24, 48, and 96 h post-fertilization; HPF). RNA was extracted from eggs/embryos of nine females (n = 4 high-quality, n = 5 low-quality) and Real-Time PCR was used to quantify relative gene expression. The transcripts assessed in this study perform critical cellular functions, including tubulin ß (tubb), cathepsin D (ctsd), cathepsin Z (ctsz), cathepsin B (ctsb), cyclin B (ccnb1), exportin-1 (xpo1), ring finger protein 213 (rnf213), glucocorticoid receptor-1 (GR-1), and heat shock protein 70 (hsp70). Relative gene expression of all transcripts except GR-1 and hsp70 were up-regulated in the high-hatch group and peaked at 48 HPF (neurulation stage), indicating the importance of these gene products at this threshold to normally progress until hatch. Due to lack of expression during earlier stages, maternally derived mRNAs for these genes do not seem to impact early embryonic development. Using mRNA markers as a selection mechanism for hatchery broodstock may lead to more high-hatch egg batches by reducing problems associated with poor egg quality.

Different incubation patterns affect selective antimicrobial properties of the egg interior: experimental evidence from eggs of precocial and altricial birds.

Avian eggs contend with omnipresent microorganisms entering the egg interior, where they affect embryo viability and hatchling phenotype. The incubation behaviour and deposition of egg white antimicrobial proteins (AMPs) vary highly across the avian altricial-precocial spectrum. Experimental evidence of how these alterations in avian reproductive strategies affect the antimicrobial properties of the precocial and altricial egg interior is lacking, however. Here, we tested the egg white antimicrobial activity in eggs of two representative model species, from each end of the avian altricial-precocial spectrum, against potentially pathogenic and beneficial probiotic microorganisms. Eggs were experimentally treated to mimic un-incubated eggs in the nest, partial incubation during the egg-laying period, the onset of full incubation and the increased deposition of two main egg white AMPs, lysozyme and ovotransferrin. We moreover assessed to what extent egg antimicrobial components, egg white pH and AMP concentrations varied as a result of different incubation patterns. Fully incubated precocial and altricial eggs decreased their antimicrobial activity against a potentially pathogenic microorganism, whereas partial incubation significantly enhanced the persistence of a beneficial probiotic microorganism in precocial eggs. These effects were most probably conditioned by temperature-dependent alterations in egg white pH and AMP concentrations. While lysozyme concentration and pH decreased in fully incubated precocial but not altricial eggs, egg white ovotransferrin increased along with the intensity of incubation in both precocial and altricial eggs. This study is the first to experimentally demonstrate that different incubation patterns may have selective antimicrobial potential mediated by species-specific effects on antimicrobial components in the egg white.

A single injection of corifollitropin alfa supplemented with human chorionic gonadotropin increases follicular recruitment and transferable embryos in the rabbit.

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.

Low-mobility sperm phenotype in the domestic turkey: Impact on sperm morphometry and early embryonic death.

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.

Mitochondrial DNA quantification as a tool for embryo viability assessment: retrospective analysis of data from single euploid blastocyst transfers.

STUDY QUESTION: Does the amount of mitochondrial DNA (mtDNA) in blastocyst biopsy specimens have the potential to serve as a biomarker of euploid embryo implantation ability, independent of morphology? SUMMARY ANSWER: The results of this study strongly suggest that elevated mtDNA levels, above a previously defined threshold, are strongly associated with blastocyst implantation failure and represent an independent biomarker of embryo viability. WHAT IS KNOWN ALREADY: Improved methods of embryo selection are highly desirable in order to increase the efficiency of IVF treatment. At present, even the transfer of chromosomally normal embryos of high morphological grade cannot guarantee that a pregnancy will follow. Recently, it has been proposed that the quantity of mtDNA in embryonic cells may be an indicator of developmental potential, with higher levels of mtDNA associated with reduced implantation. However, thus far reported data sets have been relatively small and in some cases have lacked appropriate validation. STUDY DESIGN, SIZE, DURATION: This large, blinded, retrospective study involved the analysis of relative mtDNA levels in 1505 euploid blastocysts obtained from 490 couples undergoing preimplantation genetic testing for aneuploidy. Implantation outcomes were compared to mtDNA levels in order to determine the capacity of the method to predict viability and to assess the validity of previously established thresholds. PARTICIPANTS/MATERIALS, SETTING, METHODS: DNA from blastocyst biopsy samples was amplified and then subjected to aneuploidy analysis using next generation sequencing or array comparative genomic hybridization. Only those embryos classified as chromosomally normal had their mtDNA levels assessed. This analysis was undertaken retrospectively using quantitative real-time PCR, without knowledge of the outcome of embryo transfer. Predictions of implantation failure, based upon mtDNA levels were subsequently compared to the observed clinical results. All cycles involved the transfer of a single embryo. MAIN RESULTS AND THE ROLE OF CHANCE: Of all blastocysts analyzed, 9.2% (139/1505) contained mtDNA levels above a previously established viability threshold and were therefore predicted to have reduced chances of implantation. To the date of analysis, 282 euploid blastocysts had been transferred with an overall implantation rate of 65.6% (185/282). Of the transferred embryos, 249 contained levels of mtDNA in the normal range, 185 of which produced a pregnancy, giving an implantation rate of 74.3% for euploid embryos with 'normal' quantities of mtDNA. However, 33 of the transferred embryos were determined to have elevated mtDNA quantities. None of these led to a pregnancy. Therefore, the negative predictive value of mtDNA assessment in this cohort was 100% (33/33). The difference between the implantation rates for embryos with normal and elevated mtDNA levels was highly significant (P < 0.0001). The mtDNA thresholds, used for classification of embryos, were unaffected by female age or the clinic in which the IVF was undertaken. The probability of an embryo having elevated levels of mtDNA was not influenced by variation in embryo morphology. LIMITATIONS, REASONS FOR CAUTION: This study provides strong evidence that mtDNA quantification can serve as a valuable tool to assist the evaluation of blastocyst viability. However, to determine the true extent of any clinical benefits, other types of investigations, such as non-selection studies and randomized controlled trials, will also be necessary. WIDER IMPLICATIONS OF THE FINDINGS: The results of this study suggest that mtDNA quantity can serve as an independent biomarker for the prediction of euploid blastocyst implantation potential. Prospective studies should now be undertaken to confirm these results. Additionally, investigations into the underlying biological cause(s) of elevated mtDNA levels and an enhanced understanding of how they relate to diminished implantation potential would be invaluable. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by funding provided by Reprogenetics. None of the authors have any competing interests.

Protein repair L-isoaspartyl methyltransferase 1 (PIMT1) in rice improves seed longevity by preserving embryo vigor and viability.

Damaged proteins containing abnormal isoaspartyl (isoAsp) accumulate as seeds age and the abnormality is thought to undermine seed vigor. Protein-L-isoaspartyl methyltransferase (PIMT) is involved in isoAsp-containing protein repair. Two PIMT genes from rice (Oryza sativa L.), designated as OsPIMT1 and OsPIMT2, were isolated and investigated for their roles. The results indicated that OsPIMT2 was mainly present in green tissues, but OsPIMT1 largely accumulated in embryos. Confocal visualization of the transient expression of OsPIMTs showed that OsPIMT2 was localized in the chloroplast and nucleus, whereas OsPIMT1 was predominately found in the cytosol. Artificial aging results highlighted the sensitivity of the seeds of OsPIMT1 mutant line when subjected to accelerated aging. Overexpression of OsPIMT1 in transgenic seeds reduced the accumulation of isoAsp-containing protein in embryos, and increased embryo viability. The germination percentage of transgenic seeds overexpressing OsPIMT1 increased 9-15% compared to the WT seeds after 21-day of artificial aging, whereas seeds from the OsPIMT1 RNAi lines overaccumulated isoAsp in embryos and experienced rapid loss of seed germinability. Taken together, these data strongly indicated that OsPIMT1-related seed longevity improvement is probably due to the repair of detrimental isoAsp-containing proteins that over accumulate in embryos when subjected to accelerated aging.

Preliminary trials of a specific gravity technique in the determination of early embryo growth potential†.

STUDY QUESTION: Can a modified specific gravity technique be used to distinguish viable from nonviable embryos? SUMMARY ANSWER: Preliminary data suggests a modified specific gravity technique can be used to determine embryo viability and potential for future development. WHAT IS KNOWN ALREADY: Single embryo transfer (SET) is fast becoming the standard of practice. However, there is currently no reliable method to ensure development of the embryo transferred. STUDY DESIGN, SIZE, DURATION: A preliminary, animal-based in vitro study of specific gravity as a predictor of embryo development using a mouse model. PARTICIPANTS/MATERIALS, SETTING, METHODS: After a brief study to demonstrate embryo recovery, experiments were conducted to assess the ability of the specific gravity system (SGS) to distinguish between viable and nonviable embryos. In the first study, 1-cell mouse embryos were exposed to the SGS with or without previous exposure to an extreme heat source (60°C); measurements were repeated daily for 5 days. In the second experiment, larger pools of 1-cell embryos were either placed directly in culture or passed through the SGS and then placed in culture and monitored for 4 days. MAIN RESULTS AND THE ROLE OF CHANCE: In the first experiment, viable embryos demonstrated a predictable pattern of descent time over the first 48 h of development (similar to previous experience with the SGS), while embryos that were heat killed demonstrated significantly altered drop patterns (P < 0.001); first descending faster. In the second experiment, average descent times were different for embryos that stalled early versus those that developed to blastocyst (P < 0.001). Interestingly, more embryos dropped through the SGS developed to blastocyst than the culture control (P < 0.01). LIMITATIONS, REASONS FOR CAUTION: As this is a preliminary report of the SGS technology determining viability, a larger embryo population will be needed. Further, the current in vitro study will need to be followed by fecundity studies prior to application to a human population. WIDER IMPLICATIONS OF THE FINDINGS: If proven, the SGS would provide a noninvasive means of assessing embryos prior to transfer after assisted reproductive technologies procedures, thereby improving fecundity and allowing more reliable SET. STUDY FUNDING/COMPETING INTERESTS: The authors gratefully acknowledge the funding support of the U.S. Jersey Association, the Laura W. Bush Institute for Women's Health and a Howard Hughes Medical Institute grant through the Undergraduate Science Education Program to Texas Tech University. None of the authors have any conflict of interest regarding this work. TRIAL REGISTRATION NUMBER: none.

Follicular Fluid Metabolomics: Tool for Predicting IVF Outcomes of Different Infertility Causes.

Infertility affects approximately 15% of couples at child-bearing ages and assisted reproductive technologies (ART), especially in vitro fertilization and embryo transfer (IVF-ET), provided infertile patients with an effective solution. The current paradox is that multiple embryo transfer that may leads to severe obstetric and perinatal complications seems to be the most valid measure to secure high success rate in the majority of clinic centers. Therefore, to avoid multiple transfer of embryos, it is urgent to explore biomarkers for IVF prognosis to select high-quality oocytes and embryos. Follicular fluid (FF), a typical biofluid constituted of the plasma effusion and granulosa-cell secretion, provides essential intracellular substances for oocytes maturation and its variation in composition reflects oocyte developmental competence and embryo viability. With the advances in metabolomics methodology, metabolomics, as an accurate and sensitive analyzing method, has been utilized to explore predictors in FF for ART success. Although FF metabolomics has provided a great possibility for screening markers with diagnostic and predictive value, its effectiveness is still doubted by some researchers. This may be resulted from the ignorance of the impact of sterility causes on the FF metabolomic profiles and thus its predictive ability might not be rightly illustrated. Therefore, in this review, we categorically demonstrate the study of FF metabolomics according to specific infertility causes, expecting to reveal the predicting value of metabolomics for IVF outcomes.

Evaluation of blastocyst re-expansion, quality in relation to storage temperature, and sexing using blastocoel fluid after manual perforation with a hand-held needle involving in vivo produced equine embryos.

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).