Identifying and optimizing human endometrial gene expression signatures for endometrial dating.

STUDY QUESTION: What are the key considerations for developing an enhanced transcriptomic method for secretory endometrial tissue dating? SUMMARY ANSWER: Multiple gene expression signature combinations can serve as biomarkers for endometrial dating, but their predictive performance is variable and depends on the number and identity of the genes included in the prediction model, the dataset characteristics and the technology employed for measuring gene expression. WHAT IS KNOWN ALREADY: Among the new generation of transcriptomic endometrial dating (TED) tools developed in the last decade, there exists variation in the technology used for measuring gene expression, the gene makeup and the prediction model design. A detailed study, comparing prediction performance across signatures for understanding signature behaviour and discrepancies in gene content between them, is lacking. STUDY DESIGN, SIZE, DURATION: A multicentre prospective study was performed between July 2018 and October 2020 at five different centres from the same group of clinics (Spain). This study recruited 281 patients and finally included in the gene expression analysis 225 Caucasian patients who underwent IVF treatment. After preprocessing and batch effect filtering, gene expression measurements from 217 patients were combined with artificial intelligence algorithms (support vector machine, random forest and k-nearest neighbours) allowing evaluation of different prediction models. In addition, secretory-phase endometrial transcriptomes from gene expression omnibus (GEO) datasets were analysed for 137 women, to study the endometrial dating capacity of genes independently and grouped by signatures. This provided data on the consistency of prediction across different gene expression technologies and datasets. PARTICIPANTS/MATERIALS, SETTING, METHODS: Endometrial biopsies were analysed using a targeted TruSeq (Illumina) custom RNA expression panel called the endometrial dating panel (ED panel). This panel included 301 genes previously considered relevant for endometrial dating as well as new genes selected for their anticipated value in detecting the secretory phase. Final samples (n = 217) were divided into a training set for signature discovery and an independent testing set for evaluation of predictive performance of the new signature. In addition, secretory-phase endometrial transcriptomes from GEO were analysed for 137 women to study endometrial dating capacity of genes independently and grouped by signatures. Predictive performance among these signatures was compared according to signature gene set size. MAIN RESULTS AND THE ROLE OF CHANCE: Testing of the ED panel allowed development of a model based on a new signature of 73 genes, which we termed 'TED' and delivers an enhanced tool for the consistent dating of the secretory phase progression, especially during the mid-secretory endometrium (3-8 days after progesterone (P) administration (P + 3-P + 8) in a hormone replacement therapy cycle). This new model showed the best predictive capacity in an independent test set for staging the endometrial tissue in the secretory phase, especially in the expected window of implantation (average of 114.5 ± 7.2 h of progesterone administered; range in our patient population of 82-172 h). Published sets of genes, in current use for endometrial dating and the new TED genes, were evaluated in parallel in whole-transcriptome datasets and in the ED panel dataset. TED signature performance was consistently excellent for all datasets assessed, frequently outperforming previously published sets of genes with a smaller number of genes for dating the endometrium in the secretory phase. Thus, this optimized set exhibited prediction consistency across datasets. LARGE SCALE DATA: The data used in this study is partially available at GEO database. GEO identifiers GSE4888, GSE29981, GSE58144, GSE98386. LIMITATIONS, REASONS FOR CAUTION: Although dating the endometrial biopsy is crucial for investigating endometrial progression and the receptivity process, further studies are needed to confirm whether or not endometrial dating methods in general are clinically useful and to guide the specific use of TED in the clinical setting. WIDER IMPLICATIONS OF THE FINDINGS: Multiple gene signature combinations provide adequate endometrial dating, but their predictive performance depends on the identity of the genes included, the gene expression platform, the algorithms used and dataset characteristics. TED is a next-generation endometrial assessment tool based on gene expression for accurate endometrial progression dating especially during the mid-secretory. STUDY FUNDING/COMPETING INTEREST(S): Research funded by IVI Foundation (1810-FIVI-066-PD). P.D.-G. visiting scientist fellowship at Oxford University (BEFPI/2010/032) and Josefa Maria Sanchez-Reyes' predoctoral fellowship (ACIF/2018/072) were supported by a program from the Generalitat Valenciana funded by the Spanish government. A.D.-P. is supported by the FPU/15/01398 predoctoral fellowship from the Ministry of Science, Innovation and Universities (Spanish Government). D.W. received support from the NIHR Oxford Biomedical Research Centre. The authors do not have any competing interests to declare.

PGDIS position statement on the transfer of mosaic embryos 2021.

Chromosome testing strategies, such as preimplantation genetic testing for aneuploidy (PGT-A), improve initial IVF outcomes by avoiding unwitting transfer of aneuploid embryos in morphology-based selection practices. Newer technologies have revealed that some embryos may appear to have intermediate whole chromosome (or parts of a chromosome termed segmental) copy number results suggesting trophectoderm mosaicism. An embryo with a trophectoderm mosaic-range result may be the only option for transfer for some patients. Recent data suggest that such mosaic embryos can be transferred without added risk of abnormal birth outcomes but may be associated with increased implantation failure and miscarriage rates, with higher values of mosaicism appearing to be less favourable for producing good outcomes. In this Position Statement, we provide guidance to laboratories, clinics, clinicians and counsellors to assist in discussions on the utility and transfer of mosaic embryos.

Holstein Friesian dairy cattle edited for diluted coat color as a potential adaptation to climate change.

BACKGROUND: High-producing Holstein Friesian dairy cattle have a characteristic black and white coat, often with large proportions of black. Compared to a light coat color, black absorbs more solar radiation which is a contributing factor to heat stress in cattle. To better adapt dairy cattle to rapidly warming climates, we aimed to lighten their coat color by genome editing. RESULTS: Using gRNA/Cas9-mediated editing, we introduced a three bp deletion in the pre-melanosomal protein 17 gene (PMEL) proposed as causative variant for the semi-dominant color dilution phenotype observed in Galloway and Highland cattle. Calves generated from cells with homozygous edits revealed a strong color dilution effect. Instead of the characteristic black and white markings of control calves generated from unedited cells, the edited calves displayed a novel grey and white coat pattern. CONCLUSION: This, for the first time, verified the causative nature of the PMEL mutation for diluting the black coat color in cattle. Although only one of the calves was healthy at birth and later succumbed to a naval infection, the study showed the feasibility of generating such edited animals with the possibility to dissect the effects of the introgressed edit and other interfering allelic variants that might exist in individual cattle and accurately determine the impact of only the three bp change.

The incidence and origin of segmental aneuploidy in human oocytes and preimplantation embryos.

STUDY QUESTION: What is the incidence, origin and clinical significance of segmental aneuploidy in human oocytes and preimplantation embryos? SUMMARY ANSWER: Segmental aneuploidy occurs at a considerable frequency in preimplantation embryos with a majority being mitotic in origin. WHAT IS KNOWN ALREADY: In recent years, accurate techniques for the detection of aneuploidy in single cells have been developed. Research using such methods has confirmed that aneuploidy is a common feature of human oocytes and preimplantation embryos. However, thus far research has mainly focused on loss or gain of whole chromosomes. We utilized sensitive molecular methods to study another important form of cytogenetic abnormality at the earliest stages of human development, namely segmental aneuploidy. STUDY DESIGN, SIZE, DURATION: Chromosomal copy number data was obtained from oocytes and embryos of 635 IVF patients, who requested chromosome screening for various reasons, most commonly for advanced maternal age or previously unsuccessful IVF treatments. A total of 3541 samples comprising of 452 human oocytes, 1762 cleavage stage and 1327 blastocyst stage embryos were investigated in the present study. PARTICIPANTS/MATERIALS, SETTING, METHODS: Whole genome amplification (Sureplex, Illumina) was performed on cells biopsied from oocytes and embryos of IVF patients who requested chromosome screening. The samples were subsequently processed and analyzed for their chromosome complement using microarray comparative genomic hybridization (aCGH), (Illumina, Cambridge, UK). MAIN RESULTS AND THE ROLE OF CHANCE: Segmental abnormalities, involving loss or gain of chromosomal fragments in excess of 15 Mb, were found to occur at a high frequency. The incidence of such abnormalities was 10.4% in oocytes, but this increased dramatically during the first 3 days of embryonic development (24.3%), before starting to decline as embryos reached the final (blastocyst) stage of preimplantation development (15.6%). While some segmental errors were clearly of meiotic origin, most appear to arise during the first few mitoses following fertilization. The reduction in frequency at the blastocyst stage suggests that many cells/embryos affected by segmental abnormalities are eliminated (e.g. via arrest of the affected embryos or apoptosis of abnormal cells). Interestingly, sites of chromosome breakage associated with segmental aneuploidy were not entirely random but tended to occur within distinct chromosomal regions. Some of the identified hotspots correspond to known fragile sites while others may be considered novel and may be specific to gametogenesis and/or embryogenesis. LIMITATIONS REASONS FOR CAUTION: The cytogenetic analysis was performed on biopsies of embryos, which might not be representative of the true incidence of mosaic segmental aneuploidy of the entire embryo. WIDER IMPLICATIONS OF THE FINDINGS: The findings of this study are valuable for understanding the origin of subchromosomal duplications and deletions, a clinically important class of abnormalities that are a common cause of congenital abnormalities and miscarriage. Furthermore, the results provide additional evidence that control of the cell cycle is more relaxed during the first few mitotic divisions following fertilization, permitting DNA double-strand breaks to occur and persist through cell division. The data are also of great relevance for preimplantation genetic testing, where the detection of segmental aneuploidy is currently considered problematic for embryo diagnosis and patient counseling. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by institutional funding (Reprogenetics UK). Additionally, DW is supported by the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre Programme. DB was supported by the University of Oxford's Clarendon funding. No conflict of interests to declare.

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.

Embryos with morphokinetic abnormalities may develop into euploid blastocysts.

Irregular cleavage divisions are expected to produce chromosomally deviant embryos. We investigated whether embryos from irregular cleavages could develop into euploid blastocysts, and, if so, whether any evidence existed of a self-correction mechanism of the embryo. We also investigated the role of different dynamic aspects of morula compaction in this process. A total of 791 embryos from 141 patients undergoing pre-implantation genetic screening were retrospectively analysed using a time-lapse imaging system, and multiple cell divisions were evaluated. A total of 276 embryos developed into blastocysts suitable for biopsy and chromosome screening through array-comparative genomic hybridization. As well as testing trophectoderm biopsy specimens for aneuploidy, excluded cells of 18 blastocysts, which developed from partially compacted morulas, were also analysed. Unique data on the developmental fate of embryos with cleavage abnormalities are presented, and a potential mechanism of 'aneuploidy rescue' is postulated through which mosaic embryos may form partially compacted morulas to exclude aneuploid cells. In addition, this process seems to be less efficient in older women. The data obtained also provide further evidence that excluded cells should not be used to infer the cytogenetic status of the embryo.

Precise and Accurate Measurements of Strong-Field Photoionization and a Transferable Laser Intensity Calibration Standard.

Ionization of atoms and molecules in strong laser fields is a fundamental process in many fields of research, especially in the emerging field of attosecond science. So far, demonstrably accurate data have only been acquired for atomic hydrogen (H), a species that is accessible to few investigators. Here, we present measurements of the ionization yield for argon, krypton, and xenon with percent-level accuracy, calibrated using H, in a laser regime widely used in attosecond science. We derive a transferable calibration standard for laser peak intensity, accurate to 1.3%, that is based on a simple reference curve. In addition, our measurements provide a much needed benchmark for testing models of ionization in noble-gas atoms, such as the widely employed single-active electron approximation.

Morphological and cytogenetic assessment of cleavage and blastocyst stage embryos.

Morphological assessments are the main way in which fertility clinics select in vitro generated embryo(s) for transfer to the uterus. However, it is widely acknowledged that the microscopic appearance of an embryo is only weakly correlated with its viability. Furthermore, the extent to which morphology is affected by aneuploidy, a genetic defect common in human preimplantation embryos, remains unclear. Aneuploidy is of great relevance to embryo selection as it represents one of the most important causes of implantation failure and miscarriage. The current study aimed to examine whether morphological appearance can assist in identifying embryos at risk of aneuploidy. Additionally, the data produced sheds light on how chromosomal anomalies impact development from the cleavage to the blastocyst stage. A total of 1213 embryos were examined. Comprehensive chromosome analysis was combined with well-established criteria for the assessment of embryo morphology. At the cleavage stage, chromosome abnormalities were common even amongst embryos assigned the best morphological scores, indicating that aneuploidy has little effect on microscopic appearance at fixed time points up until Day 3 of development. However, at the blastocyst stage aneuploidies were found to be significantly less common among embryos of optimal morphological quality, while such abnormalities were overrepresented amongst embryos considered to be of poor morphology. Despite the link between aneuploidy and blastocyst appearance, many chromosomally abnormal embryos were able to achieve the highest morphological scores. In particular, blastocysts affected by forms of aneuploidy with the greatest capacity to produce clinical pregnancies (e.g. trisomy 21) were indistinguishable from euploid embryos. The sex ratio was seen to be equal throughout preimplantation development. Interestingly, however, females were overrepresented amongst the fastest growing cleavage-stage embryos, whereas a sex-related skew in the opposite direction was noted for the most rapidly developing blastocysts. In summary, this study confirms that, at the cleavage stage, chromosome abnormalities have little if any effect on morphological scores assigned using traditional criteria. At the blastocyst stage some forms of aneuploidy begin to affect microscopic appearance, but in most instances the impact is subtle. In the case of the most clinically relevant aneuploidies (those capable of forming a pregnancy) there was no detectable effect on morphology at any preimplantation stage.

Health outcomes of children born after IVF/ICSI: a review of current expert opinion and literature.

The Sixth Evian Annual Reproduction (EVAR) Workshop Group Meeting was held to evaluate the impact of IVF/intracytoplasmic sperm injection on the health of assisted-conception children. Epidemiologists, reproductive endocrinologists, embryologists and geneticists presented data from published literature and ongoing research on the incidence of genetic and epigenetic abnormalities and congenital malformations in assisted-conception versus naturally conceived children to reach a consensus on the reasons for potential differences in outcomes between these two groups. IVF-conceived children have lower birthweights and higher peripheral fat, blood pressure and fasting glucose concentrations than controls. Growth, development and cognitive function in assisted-conception children are similar to controls. The absolute risk of imprinting disorders after assisted reproduction is less than 1%. A direct link between assisted reproduction and health-related outcomes in assisted-conception children could not be established. Women undergoing assisted reproduction are often older, increasing the chances of obtaining abnormal gametes that may cause deviations in outcomes between assisted-conception and naturally conceived children. However, after taking into account these factors, it is not clear to what extent poorer outcomes are due to the assisted reproduction procedures themselves. Large-scale, multicentre, prospective epidemiological studies are needed to investigate this further and to confirm long-term health consequences in assisted-conception children. Assisted reproduction treatment is a general term used to describe methods of achieving pregnancy by artificial means and includes IVF and sperm implantation. The effect of assisted reproduction treatment on the health of children born using these artificial methods is not fully understood. In April 2011, fertility research experts met to give presentations based on research in this area and to look carefully at the evidence for the effects of assisted reproduction treatment on children's health. The purpose of this review was to reach an agreement on whether there are differences in the health of assisted-conception children with naturally conceived children. The researchers discovered no increased risk in birth defects in assisted-conception children compared with naturally conceived children. They found that IVF-conceived children have lower birth weights and higher fat under the skin, higher blood pressure and higher fasting glucose concentrations than naturally conceived children; however, growth, development and cognitive function are similar between groups. A very low risk of disorders of genetic control was observed in assisted-conception children. Overall, there did not appear to be a direct link between assisted reproduction treatment and children's health. The researchers concluded that the cause of some differences in the health of children conceived using assisted reproduction treatment may be due to the age of the woman receiving treatment. Large-scale, research studies are needed to study the long-term health of children conceived using assisted reproduction treatment.

Increased numbers of DNA-damaged spermatozoa in samples presenting an elevated rate of numerical chromosome abnormalities.

STUDY QUESTION: Is there a relationship between DNA damage and numerical chromosome abnormalities in the sperm of infertile patients? SUMMARY ANSWER: A strong link between DNA fragmentation and the presence of numerical chromosome abnormalities was detected in human sperm. Chromosomally abnormal spermatozoa were more likely to be affected by DNA fragmentation than those that were chromosomally normal. WHAT IS KNOWN ALREADY: Several studies have described the presence of elevated levels of DNA damage or chromosome defects in the sperm of infertile or subfertile men. However, the nature of the relationship between sperm DNA damage and chromosome abnormalities is poorly understood. The fact that some assisted reproductive techniques have the potential to allow abnormal spermatozoa to achieve oocyte fertilization has led to concerns that pregnancies achieved using such methods may be at elevated risk of genetic anomalies. STUDY DESIGN, SIZE, DURATION: For this prospective study, semen samples were collected from 45 infertile men. PARTICIPANTS, SETTING, METHODS: Samples were assessed for DNA fragmentation using the Sperm Chromatin Dispersion Test (SCDt) and for chromosome abnormalities using multi-colour fluorescence in situ hybridization (FISH) with probes specific to chromosomes 13, 16, 18, 21, 22, X and Y. Additionally, both parameters were assessed simultaneously in 10 of the samples using a protocol combining SCDt and FISH. MAIN RESULTS AND THE ROLE OF CHANCE: A significant correlation between the proportion of sperm with a numerical chromosome abnormality and the level of DNA fragmentation was observed (P < 0.05). Data from individual spermatozoa subjected to combined chromosome and DNA fragmentation analysis indicated that chromosomally abnormal sperm cells were more likely to display DNA damage than those that were normal for the chromosomes tested (P < 0.05). Not only was this association detected in samples with elevated levels of numerical chromosome abnormalities, but it was also evident in samples with chromosome abnormality rates in the normal range. LIMITATIONS, REASONS FOR CAUTION: The inability to assess the entire chromosome complement is the main limitation of all studies aimed at assessing numerical chromosome abnormalities in sperm samples. As a result, some of the sperm classified as 'chromosomally normal' may be aneuploid for chromosomes that were not tested. WIDER IMPLICATIONS OF THE FINDINGS: During spermatogenesis, apoptosis (a process that involves active DNA degradation) acts to eliminate abnormal sperm. Failure to complete apoptosis may explain the coincident detection of aneuploidy and DNA fragmentation in some spermatozoa. In addition to shedding light on the biological mechanisms involved in the processing of defective sperm, this finding may also be of clinical relevance for the identification of patients at increased risk of miscarriage or chromosomally abnormal pregnancy. In some instances, detection of elevated sperm DNA fragmentation may indicate the presence of chromosomal abnormalities. It may be worth considering preimplantation genetic screening (PGS) of embryos produced using such samples in order to minimize the risk of aneuploidy.

Genomic DNA in human blastocoele fluid.

IVF often requires embryo cryopreservation through vitrification. During the vitrification process, the embryos can be collapsed by withdrawing the blastocoele fluid. The metabolomic profile of blastocoele fluid has been recently investigated by high-performance liquid chromatography-electrospray ionization-mass spectrometry to provide metabolite information that can help estimations of implantation efficiency. However, the presence of embryo DNA in blastocoele fluid has not been reported to date. This study shows using real-time PCR that genomic DNA was present in about 90% of blastocoele fluid samples harvested during the vitrification procedure. Moreover, the potential for determining embryo sex directly from blastocoele fluid is demonstrated by amplifying the multicopy genes TSPY1 (on the Y chromosome) and TBC1D3 (on chromosome 17). This opens up the possibility of screening embryos from couples carrying an X-linked disorder to identify male embryos at high risk of disease. The application of whole-genome amplification technologies to fluid samples is also shown to be feasible, potentially allowing more comprehensive genetic tests. As proof of principle, microarray comparative genomic hybridization was attempted to confirm the sex of embryos as well as detect several aneuploidies. However, further studies are needed to validate this approach and confirm that the accuracy is sufficient for diagnostic purposes.

Cloning of the putative tumour suppressor gene for hereditary multiple exostoses (EXT1).

Hereditary multiple exostoses is an autosomal dominant disorder that is characterized by short stature and multiple, benign bone tumours. In a majority of families, the genetic defect (EXT1) is linked to the Langer-Giedion syndrome chromosomal region in 8q24.1. From this region we have cloned and characterized a cDNA which spans chromosomal breakpoints previously identified in two multiple exostoses patients. Furthermore, the gene harbours frameshift mutations in affected members of two EXT1 families. The cDNA has a coding region of 2,238 bp with no apparent homology to other known gene sequences and thus its function remains elusive. However, recent studies in sporadic and exostosis-derived chondrosarcomas suggest that the 8q24.1-encoded EXT1 gene may have tumour suppressor function.

The social formation of fitness: lifetime consequences of prenatal nutrition and postnatal care in a wild mammal population.

Research in medicine and evolutionary biology suggests that the sequencing of parental investment has a crucial impact on offspring life history and health. Here, we take advantage of the synchronous birth system of wild banded mongooses to test experimentally the lifetime consequences to offspring of receiving extra investment prenatally versus postnatally. We provided extra food to half of the breeding females in each group during pregnancy, leaving the other half as matched controls. This manipulation resulted in two categories of experimental offspring in synchronously born litters: (i) 'prenatal boost' offspring whose mothers had been fed during pregnancy, and (ii) 'postnatal boost' offspring whose mothers were not fed during pregnancy but who received extra alloparental care in the postnatal period. Prenatal boost offspring lived substantially longer as adults, but postnatal boost offspring had higher lifetime reproductive success (LRS) and higher glucocorticoid levels across the lifespan. Both types of experimental offspring had higher LRS than offspring from unmanipulated litters. We found no difference between the two experimental categories of offspring in adult weight, age at first reproduction, oxidative stress or telomere lengths. These findings are rare experimental evidence that prenatal and postnatal investments have distinct effects in moulding individual life history and fitness in wild mammals. This article is part of the theme issue 'Evolutionary ecology of inequality'.

A biomechanical model of anther opening reveals the roles of dehydration and secondary thickening.

Understanding the processes that underlie pollen release is a prime target for controlling fertility to enable selective breeding and the efficient production of hybrid crops. Pollen release requires anther opening, which involves changes in the biomechanical properties of the anther wall. In this research, we develop and use a mathematical model to understand how these biomechanical processes lead to anther opening. Our mathematical model describing the biomechanics of anther opening incorporates the bilayer structure of the mature anther wall, which comprises the outer epidermal cell layer, whose turgor pressure is related to its hydration, and the endothecial layer, whose walls contain helical secondary thickening, which resists stretching and bending. The model describes how epidermal dehydration, in association with the thickened endothecial layer, creates forces within the anther wall causing it to bend outwards, resulting in anther opening and pollen release. The model demonstrates that epidermal dehydration can drive anther opening, and suggests why endothecial secondary thickening is essential for this process (explaining the phenotypes presented in the myb26 and nst1nst2 mutants). The research hypothesizes and demonstrates a biomechanical mechanism for anther opening, which appears to be conserved in many other biological situations where tissue movement occurs.

Development of a novel synthetic oligopeptide for the detection of DNA damage in human spermatozoa.

BACKGROUND: The integrity of DNA in spermatozoa is considered an additional parameter of semen quality and a potential fertility predictor. Significant progress has been made in recent years towards the development of reliable tests for sperm chromatin integrity and DNA damage assessment. However, most of the techniques available are labor intensive, require expensive instrumentation or utilize enzymes whose activity could be compromised by the highly condensed nature of sperm chromatin. In addition, all the methods currently available involve the destruction of the sperm tested; none is able to select intact spermatozoa that could then be used for fertilization. The aim of the present study was to create a peptide ligand-based stain, capable of binding specific DNA structures, thereby revealing the presence of DNA damage, preferably in living cells. METHODS: The peptide was bioinformatically modelled on the critical region of the p53 protein associated with DNA binding and fluorescently labeled with a terminal rhodamine B dye. The ability of this 21 amino acid synthetic peptide (DW1) to detect DNA damage in intact and fixed human spermatozoa was assessed in detail. Human sperm samples (n=20) were treated with reagents that induce single- and/or double-stranded DNA breaks. The effect of these treatments on peptide-labelling was measured and compared with results obtained using established tests for the evaluation of DNA damage, such as comet assay, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and sperm chromatin dispersion test. RESULTS: The peptide had a high affinity for single-stranded DNA, and DNA lesions such as double- and single-stranded breaks. The proportion of spermatozoa with intense staining was found to be closely associated with the percentage of cells possessing DNA damage. The analysis of 10 sperm samples using DW1 staining and TUNEL technique showed a significant correlation between the extent of DNA fragmentation for the two methods (r=0.892, Pearson's correlation, P<0.05). CONCLUSIONS: We have produced a novel peptide-based stain capable of detecting DNA damage in individual sperm cells. Evaluation of sperm DNA fragmentation using this peptide may be an inexpensive and easier to use alternative to the tests in current use. Additionally, although DW1 currently requires removal of the membrane using a detergent, further research may allow this approach to be applied to the selection of viable spermatozoa with intact DNA for use in ICSI and/or intra-cytoplasmic morphologically selected sperm injection.

A method for semi-automatic grading of human blastocyst microscope images.

BACKGROUND: The precise assessment of embryo viability is an extremely important factor for the optimization of IVF treatments. In order to assess embryo viability, several embryo scoring systems have been developed. However, they rely mostly on a subjective visual analysis of embryo morphological features and thus are subject to inter- and intra-observer variation. In this paper, we propose a method for image segmentation (the dividing of an image into its meaningful constituent regions) and classification of human blastocyst images with the aim of automating embryo grading. METHODS: The delineation of the boundaries (segmentation) of the zona pellucida, trophectoderm (TE) and inner cell mass (ICM) were performed using advanced image analysis techniques (level set, phase congruency and fitting of ellipse methods). The fractal dimension and mean thickness of TE and ICM image texture descriptors (texture spectrum and grey-level run lengths) were calculated to characterize the main morphological features of the blastocyst with the aim of automatic grading using Support Vector Machine classifiers. RESULTS: The fractal dimension calculated from the delineated TE boundary provided a good indication of cell number (presented a 0.81 Pearson correlation coefficient with the number of cells), a feature closely associated with blastocyst quality. The classifiers showed different accuracy levels for each grade. They presented accuracy ranges from 0.67 to 0.92 for the embryo development classification, 0.67-0.82 for the ICM classification and 0.53-0.92 for the TE classification. The value 0.92 was the highest accuracy achieved in the tests with 73 blastocysts. CONCLUSIONS: Semi-automatic grading of human blastocysts by a computer is feasible and may offer a more precise comparison of embryos, reducing subjectivity and allowing embryos with apparently identical morphological scores to be distinguished.

Prenatal and early sucking influences on dietary preference in newborn, weaning, and young adult cats.

Early experiences are of potential importance in shaping long-term behavior. This study examined the relative influence of prenatal and/or early postnatal experience of chemosensory stimuli on subsequent olfactory and dietary preferences of cats as newborns, at 9-10 weeks, and at 6 months. Cats were exposed to vanillin or 4-ethylguaiacol via their mother's diet either prenatally, postnatally, perinatally (prenatal and postnatal), or experienced no exposure to the stimuli (control). Newborns were given a two-choice olfactory test between the familiar "odor" and no odor; 9-10 week olds were tested for their preference between two food treats, one flavored with the familiar stimulus and the other unflavored; at 6 months, cats were given a choice of two bowls of food, one flavored with the familiar stimulus and the other unflavored. At all ages, cats preferred the familiar, and avoided the unfamiliar, stimulus. Perinatal exposure exerted the strongest influence on preference. Prenatal exposure influenced preference at all ages and postnatal exposure exerted a stronger effect as the cat aged. We conclude that long-term chemosensory and dietary preferences of cats are influenced by prenatal and early (nursing) postnatal experience, supporting a natural and biologically relevant mechanism for the safe transmission of diet from mother to young.

Aneuploidy in the human blastocyst.

Studies of human cleavage stage embryos, 3 days after fertilization of the oocyte, have revealed remarkably high levels of chromosome abnormality. In addition to meiotic errors derived from the gametes, principally the oocyte, mitotic errors occurring after fertilization are also common, leading to widespread chromosomal mosaicism. The prevalence of chromosome anomalies in embryos may explain the relatively poor fertility and fecundity in humans and the low success rates of assisted reproductive treatments (e.g., IVF). While much is known concerning the incidence of aneuploidy during the first 3 days following fertilization, it is only in the last couple of years that large numbers of embryos at the final stage of preimplantation development, the blastocyst stage, 5 days after fertilization, have been subjected to detailed analysis. Here we discuss the latest data from the comprehensive cytogenetic analysis of blastocysts. These findings indicate that the majority of selection against chromosome abnormalities does not occur until the time of implantation or shortly after, with aneuploidy typically affecting more than 50% of blastocysts. Additionally, clinical results presented suggest that screening of blastocyst stage embryos for chromosome abnormality, with preferential transfer to the uterus of those found to be euploid, may help to improve the success rates of assisted reproductive treatments.

Chromosome abnormalities in the human oocyte.

Aneuploidy is the most commonly occurring type of chromosome abnormality and the most significant clinically. It arises mostly due to segregation errors taking place during female meiosis and is also closely associated with advancing maternal age. Two main aneuploidy-causing mechanisms have been described: the first involves the non-disjunction of entire chromosomes and can take place during both meiotic divisions, whereas the second involves the premature division of a chromosome into its 2 sister chromatids, followed by their random segregation, upon completion of meiosis I. To elucidate the causal mechanisms of maternally derived aneuploidy and the manner with which they affect the 2 meiotic divisions, a large number of oocytes and their corresponding polar bodies have been examined. Various classical and molecular cytogenetic methods have been employed for this purpose, and valuable data have been obtained. Moreover, research into the gene expression patterns of oocytes according to maturity, maternal age, and chromosome status has provided a unique insight into the complex nature of the biological processes and genetic pathways regulating female meiosis. Findings obtained from the cytogenetic and molecular analysis of oocytes will be reviewed in this article.