Impact of male age on paternal aneuploidy: single-nucleotide polymorphism microarray outcomes following blastocyst biopsy.

RESEARCH QUESTION: Does advanced paternal age (APA; ≥40 years) contribute to a higher incidence of paternal origin aneuploidy in preimplantation embryos? DESIGN: This was a multicentre retrospective study of single-nucleotide polymorphism (SNP) microarray (Natera and Karyomapping) preimplantation genetic testing (PGT) outcomes of blastocyst-stage embryos. Whole-chromosome aneuploidy analysis was performed on 2409 embryos from 389 male patients undertaking 681 assisted reproductive technology (ART) cycles between 2012-2021. Segmental aneuploidy analysis was performed on 867 embryos from 140 men undertaking 242 ART cycles between 2016-2021. Embryos were grouped based on paternal age at sperm collection: <35, 35-39 and ≥40 years. Paternal and maternal origin aneuploidy rates were compared between groups using chi-squared and/or Fisher's exact tests. RESULTS: There was no significant difference across groups in paternal origin whole-chromosome aneuploidy rate, overall (P=0.7561) or when segregated by type (trisomy and monosomy: P=0.2235 and 0.8156) or complexity (single versus 2, 3 or ≥4 aneuploidies: P=0.9733, 0.7517, 0.669 and 0.1481). Conversely, maternal origin whole-chromosome aneuploidy rate differed across groups (P<0.0001) in alignment with differing mean maternal age (P<0.001). Paternal origin deletions were 2.9-fold higher than maternal origin deletions (P=0.0084), independent of age stratification. No significant difference in paternal origin deletions was observed with APA ≥40 compared with the younger age groups (4.8% versus 2.5% and 2.8%, P=0.5292). Individual chromosome aneuploidy rates were too low to perform statistical comparisons. CONCLUSIONS: No significant association was found between APA and the incidence of paternal origin aneuploidy in preimplantation embryos, irrespective of type or complexity. Thus, APA may not be an indication for PGT.

Mammalian embryo culture media: now and into the future.

For over 70years, since the culture of the first mammalian embryo in vitro , scientists have undertaken studies to devise and optimise media to support the manipulation and culture of gametes and embryos. This area of research became especially active in the late 1970s onwards following the successful birth of the first human in vitro fertilised embryo. This review summarises some of the key advances in mammalian embryo culture media over time based on a greater understanding of the biochemical milieu of the reproductive tract. It highlights how learnings from studies in mice and agricultural species have informed human culture media compositions, in particular the inclusion of albumin, growth factors, cytokines, and antioxidants into contemporary culture media formulations, and how these advances may then in turn help to inform and guide development of in vitro culture systems used in other arenas, in particular agriculture. Additionally, it will highlight how the introduction of new technologies, such as timelapse, can influence current trends in media composition and usage that may see a return to a single step medium.

Field-Based Distribution and Bioaccumulation Factors for Cyclic and Aliphatic Per- and Polyfluoroalkyl Substances (PFASs) in an Urban Sedentary Waterbird Population.

The field-based distribution and bioaccumulation factor (BAF) for per- and polyfluoroalkyl substances (PFASs) were determined in residential Black Swans (Cygnus atratus) from an urban lake (Melbourne, Australia). The concentrations of 46 aliphatic and cyclic PFASs were determined by HPLC-MS/MS in serum and excrement from swans, and water, sediment, aquatic macrophytes, soil, and grass samples in and around the lake. Elevated concentrations of ∑46PFASs were detected in serum (120 ng mL-1) and excrement (110 ng g-1 dw) were strongly related indicating a potential noninvasive sampling methodology. Environmental concentrations of PFASs were consistent with a highly impacted ecosystem and notably high concentrations of perfluoro-4-ethylcyclohexanesulfonate (PFECHS, 67584-42-3; C8HF15SO3) were detected in water (27 ng L-1) and swan serum (16 ng mL-1). In the absence of credible putative alternative sources of PFECHS input to the lake, we propose that the use of high-performance motorsport vehicles is a likely source of contamination to this ecosystem. The BAF of perfluorocarboxylic acids increased with each additional CF2 moiety from PFOA (15.7 L kg-1 ww) to PFDoDA (3615 L kg-1 ww). The BAF of PFECHS was estimated as 593 L kg-1 ww, which is lower compared with that of PFOS (1097 L kg-1 ww).

Evaluation and validation of methodologies for the extraction of per- and polyfluoroalkyl substances (PFASs) in serum of birds and mammals.

Advances in analytical techniques have allowed greater detection of environmental contaminants from small volumes of sample. Four methodologies were evaluated for the extraction of 53 per- and polyfluoroalkyl substances (PFASs) from eight classes in 200 µL of avian and mammal serum. Spiked serums at four concentrations (0, 0.5, 5.0 and 25 ng mL-1) were prepared by protein precipitation (PPT), enhanced matrix removal (EMR), weak anion exchange (WAX), and hydrophilic-lipophilic balance (HLB) solid-phase extraction cartridges. The extract from each methodology was analysed by high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS), and concentrations were compared with known concentrations in the spiked media. EMR performed the best overall, with 40 of 53 compounds effectively recovered at 5 ng mL-1. Furthermore, EMR was effective overall at concentrations ranging from 0.5 to 25 ng mL-1 for 39 out of 53. Similarly, PPT was effective for 35 of 53 compounds at all spiked serum concentrations. There was a negative correlation between internal standard recovery for compounds with increasing octanol-water coefficients (Kow) for WAX (R = - 0.65, p = 0.0043) and HLB (R = - 0.62, p = 0.0077) extractions, indicating methanol may not be a suitable solvent for long-chain PFAS extraction from protein-rich tissues. EMR and PPT represent fast and effective methodologies for the extraction of PFASs from low volumes of serum which allows greater accuracy and precision that can be applied to future human and wildlife biomonitoring programmes.

Effects of dietary supplementation of different oils and conjugated linoleic acid on the reproductive and metabolic aspects of male mice.

The present study was carried out to examine first, if diets enriched with 320 g of the base diet with common dietary oils including fish oil, olive oil, hydrogenated sunflower seed (H-SFS) oil, flaxseed oil and sunflower seed oil (SFS) could induce weight gain and alter reproductive and metabolic characteristics of male mice. Second, whether the addition of conjugated linoleic acid (CLA, 10% of the diet) could ameliorate any negative effects. In this cross-sectional study, 90 four-week-old male NMRI mice were used in two consecutive experiments. A high level of dietary oils negatively affected some reproductive and metabolic characteristics of male mice (p < 0.05), specifically, sunflower seed oil enrichment resulted in higher HDL levels and apoptosis of germinal epithelial cells. An olive oil-enriched diet caused an increase in plasma triglyceride concentrations and germinal cell apoptosis, as well as a decrease in sperm concentration and perturbed spermatogenesis. When CLA was fed in conjunction with dietary oils it successfully mitigated some of the negative reproductive and metabolic characteristics. We conclude that male reproductive processes are affected by high dietary oils, even before signs of obesity are evident. Inclusion of dietary CLA may provide some benefit to offset negative effects, although further studies are required.

Calcium chloride dihydrate supplementation at ICSI improves fertilization and pregnancy rates in patients with previous low fertilization: a retrospective paired treatment cycle study.

PURPOSE: To determine if 5mM calcium chloride dihydrate supplementation of the Polyvinylpyrrolidone (PVP) media at the time of ICSI (ICSI-Ca) improves fertilization, utilization, and clinical pregnancy rates compared to ICSI alone, particularly in patients with a history of low fertilization (< 50%). METHODS: Retrospective study between 2016 and 2021 at Monash IVF Victoria on a paired cohort of patients (n = 178 patients) where an ICSI cycle was analyzed coupled with the subsequent ICSI-Ca cycle. The paired cohort was further subdivided into a low-fertilization cohort (< 50% fertilization on previous cycles: n = 66 patients) compared to the remaining patients with fertilization ≥ 50% (n = 122). Exclusion criteria included donor cycles, PGT patients, surgical sperm retrieval, women ≥ 45 years old, patients with > 6 cycles, and patients with ≤ 5 inseminated oocytes. RESULTS: Calcium supplementation significantly increased both fertilization (28.8% ICSI vs 49.7% ICSI-Ca, P < 0.0001) and clinical pregnancy rate (4.9% ICSI vs 25.0% ICSI-Ca: P < 0.05) in the low-fertilization cohort but not in the normal-fertilization cohort. Interestingly, utilization rate significantly increased in the normal-fertilization cohort (32.6% ICSI vs ICSI-Ca: 44.9%, P < 0.01) but not in the low-fertilization cohort, although the number of embryos utilized per patient after ICSI-Ca increased in both groups. CONCLUSION: Calcium supplementation does not appear to be a detrimental addition to ICSI and may improve IVF outcomes, particularly for patients with a history of low fertilization. Further investigations including prospective case-matched studies or a RCT are required to confirm these findings.

A variant of human growth differentiation factor-9 that improves oocyte developmental competence.

Growth differentiation factor-9 (GDF9) and bone morphogenetic protein-15 (BMP15) are co-expressed exclusively in oocytes throughout most of folliculogenesis and play central roles in controlling ovarian physiology. Although both growth factors exist as homodimers, recent evidence indicates that GDF9 and BMP15 can also heterodimerize to form the potent growth factor cumulin. Within the cumulin complex, BMP15 "activates" latent GDF9, enabling potent signaling in granulosa cells via type I receptors (i.e. activin receptor-like kinase-4/5 (ALK4/5)) and SMAD2/3 transcription factors. In the cumulin heterodimer, two distinct type I receptor interfaces are formed compared with homodimeric GDF9 and BMP15. Previous studies have highlighted the potential of cumulin to improve treatment of female infertility, but, as a noncovalent heterodimer, cumulin is difficult to produce and purify without contaminating GDF9 and BMP15 homodimers. In this study we addressed this challenge by focusing on the cumulin interface formed by the helix of the GDF9 chain and the fingers of the BMP15 chain. We demonstrate that unique BMP15 finger residues at this site (Arg301, Gly304, His307, and Met369) enable potent activation of the SMAD2/3 pathway. Incorporating these BMP15 residues into latent GDF9 generated a highly potent growth factor, called hereafter Super-GDF9. Super-GDF9 was >1000-fold more potent than WT human GDF9 and 4-fold more potent than cumulin in SMAD2/3-responsive transcriptional assays in granulosa cells. Our demonstration that Super-GDF9 can effectively promote mouse cumulus cell expansion and improve oocyte quality in vitro represents a potential solution to the current challenges of producing and purifying intact cumulin.

Endocrine disrupting chemicals: Impacts on human fertility and fecundity during the peri-conception period.

It is becoming increasingly difficult to avoid exposure to man-made endocrine disrupting chemicals (EDCs) and environmental toxicants. This escalating yet constant exposure is postulated to partially explain the concurrent decline in human fertility that has occurred over the last 50 years. Controversy however remains as to whether associations exist, with conflicting findings commonly reported for all major EDC classes. The primary aim of this extensive work was to identify and review strong peer-reviewed evidence regarding the effects of environmentally-relevant EDC concentrations on adult male and female fertility during the critical periconception period on reproductive hormone concentrations, gamete and embryo characteristics, as well as the time to pregnancy in the general population. Secondly, to ascertain whether individuals or couples diagnosed as sub-fertile exhibit higher EDC or toxicant concentrations. Lastly, to highlight where little or no data exists that prevents strong associations being identified. From the greater than 1480 known EDCs, substantial evidence supports a negative association between exposure to phthalates, PCBs, PBDEs, pyrethroids, organochloride pesticides and male fertility and fecundity. Only moderate evidence exists for a negative association between BPA, PCBs, organochloride pesticides and female fertility and fecundity. Overall fewer studies were reported in women than men, with knowledge gaps generally evident for both sexes for all the major EDC classes, as well as a paucity of female fertility studies following exposure to parabens, triclosans, dioxins, PFAS, organophosphates and pyrethroids. Generally, sub-fertile individuals or couples exhibit higher EDC concentrations, endorsing a positive association between EDC exposure and sub-fertility. This review also discusses confounding and limiting factors that hamper our understanding of EDC exposures on fertility and fecundity. Finally, it highlights future research areas, as well as government, industry and social awareness strategies required to mitigate the negative effects of EDC and environmental toxicant exposure on human fertility and fecundity.

Maternal Heat Stress Alters Expression of Genes Associated with Nutrient Transport Activity and Metabolism in Female Placentae from Mid-Gestating Pigs.

Placental insufficiency is a known consequence of maternal heat stress during gestation in farm animals. The molecular regulation of placentae during the stress response is little known in pigs. This study aims to identify differential gene expression in pig placentae caused by maternal heat exposure during early to mid-gestation. RNA sequencing (RNA-seq) was performed on female placental samples from pregnant pigs exposed to thermoneutral control (CON; constant 20 °C; n = 5) or cyclic heat stress (HS; cyclic 28 to 33 °C; n = 5) conditions between d40 and d60 of gestation. On d60 of gestation, placental efficiency (fetal/placental weight) was decreased (p = 0.023) by maternal HS. A total of 169 genes were differentially expressed (FDR ≤ 0.1) between CON and HS placentae of female fetuses, of which 35 genes were upregulated and 134 genes were downregulated by maternal HS. The current data revealed transport activity (FDR = 0.027), glycoprotein biosynthetic process (FDR = 0.044), and carbohydrate metabolic process (FDR = 0.049) among the terms enriched by the downregulated genes (HS vs. CON). In addition, solute carrier (SLC)-mediated transmembrane transport (FDR = 0.008) and glycosaminoglycan biosynthesis (FDR = 0.027), which modulates placental stroma synthesis, were identified among the pathways enriched by the downregulated genes. These findings provide evidence that heat-stress induced placental inefficiency may be underpinned by altered expression of genes associated with placental nutrient transport capacity and metabolism. A further understanding of the molecular mechanism contributes to the identification of placental gene signatures of summer infertility in pigs.

Exposure to atrazine during puberty reduces sperm viability, increases weight gain and alters the expression of key metabolic genes in the liver of male mice.

Atrazine (ATZ) is one of the most widely used herbicides worldwide and is a common contaminant in human drinking water. It disrupts metabolic pathways in plants, and has metabolic and reproductive effects in vertebrates, including humans. Few studies have investigated the effects of exposure to low doses of ATZ, especially during sexual development in males. In this study, we exposed C57BL/6J male mice from weaning for 8 weeks to drinking water containing 0.5mgkg-1 bodyweight (BW) day-1 ATZ, the 'no observed effect' level used by the Australian government, or a 10-fold higher dose (5mgkg-1 BW day-1). Mice treated with the low dose of ATZ showed increased total and cumulative weight gain. At 12 weeks of age, there was a significant increase in the percentage of dead spermatozoa in both ATZ-exposed groups, as well as decreased epididymal sperm motility in the low-dose ATZ group. Significant changes in testis and liver gene expression were also observed following ATZ exposure. These data demonstrate that a low dose of ATZ can perturb metabolic and reproductive characteristics in male mice. A chronic reduction in sperm quality and increased weight gain could have negative consequences on the reproductive capacity of males, and further studies should consider the effects of long-term ATZ exposure on male reproductive health.

Artificial light at night prolongs juvenile development time in the black field cricket, Teleogryllus commodus.

A growing body of evidence exists to support a detrimental effect of the presence of artificial light at night (ALAN) on life-history and fitness traits. However, few studies simultaneously investigate multiple traits and the life stages at which changes manifest. We experimentally manipulated ALAN intensities, within those found in the natural environment, to explore the consequences for growth, survival, and reproductive success of the field cricket, Teleogryllus commodus. We reared crickets from egg to adult under a daily light-cycle consisting of 12 hr bright daylight (2,600 lx) followed by either 12 hr darkness (0 lx) or dim-light environments (1, 10, or 100 lx). We found egg hatch, adult survival, and reproductive measures were largely comparable for all treatments. However, juvenile development time (number of days from egg to adult) was on average 10 days (14%) longer and adults were also larger when crickets were exposed to any light at night (1, 10, or 100 lx). Our data demonstrate that chronic lifetime exposure to ALAN can modulate the timing of life-history events and may disrupt phenology to a similar extent as other abiotic factors.

Can physiological engineering/programming increase multi-generational thermal tolerance to extreme temperature events?

Organisms increasingly encounter higher frequencies of extreme weather events as a consequence of global climate change. Currently, few strategies are available to mitigate climate change effects on animals arising from acute extreme high-temperature events. We tested the capacity of physiological engineering to influence the intra- and multi-generational upper thermal tolerance capacity of a model organism, Artemia, subjected to extreme high temperatures. Enhancement of specific physiological regulators during development could affect thermal tolerance or life-history attributes affecting subsequent fitness. Using experimental Artemia populations, we exposed F0 individuals to one of four treatments: heat hardening (28°C to 36°C, 1°C per 10 min), heat hardening plus serotonin (0.056 µg ml-1), heat hardening plus methionine (0.79 mg ml-1) and a control treatment. Regulator concentrations were based on previous literature. Serotonin may promote thermal tolerance, acting upon metabolism and life history. Methionine acts as a methylation agent across generations. For all groups, measurements were collected for three performance traits of individual thermal tolerance (upper sublethal thermal limit, lethal limit and dysregulation range) over two generations. The results showed that no treatment increased the upper thermal limit during acute thermal stress, although serotonin-treated and methionine-treated individuals outperformed controls across multiple thermal performance traits. Additionally, some effects were evident across generations. Together, these results suggest that phenotypic engineering provides complex outcomes, and if implemented with heat hardening can further influence performance in multiple thermal tolerance traits, within and across generations. Potentially, such techniques could be up-scaled to provide resilience and stability in populations susceptible to extreme temperature events.

Oocyte mitochondrial deletions and heteroplasmy in a bovine model of ageing and ovarian stimulation.

STUDY HYPOTHESIS: Maternal ageing and ovarian stimulation result in the accumulation of mitochondrial DNA (mtDNA) deletions and heteroplasmy in individual oocytes from a novel bovine model for human assisted reproductive technology (ART). STUDY FINDING: The levels of mtDNA deletions detected in oocytes increased with ovarian ageing. Low levels of mtDNA heteroplasmy were apparent across oocytes and no relationship was identified with respect to ovarian ageing or ovarian stimulation. WHAT IS KNOWN ALREADY: Oocyte quality decreases with ovarian ageing and it is postulated that the mtDNA may have a role in this decline. The impact of ovarian stimulation on oocyte quality is poorly understood. Human studies investigating these effects are often limited by the use of low quality oocytes and embryos, variation in age and ovarian stimulation regimens within the patients studied, as well as genetic and environmental variability. Further, no study has investigated mtDNA heteroplasmy in individual oocytes using next-generation sequencing (NGS), and little is known about whether the oocyte accumulates heteroplasmic mtDNA mutations following ageing or ovarian stimulation. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: A novel bovine model for the effect of stimulation and age in human ART was undertaken using cows generated by somatic cell nuclear transfer (SCNT) from one founder, to produce a homogeneous population with reduced genetic and environmental variability. Oocytes and somatic tissues were collected from young (3 years of age; n = 4 females) and old (10 years of age; n = 5 females) cow clones following multiple natural ovarian cycles, as well as oocytes following multiple mild (FSH only) and standard (based on human a long GnRH agonist protocol) ovarian stimulation cycles. In addition, oocytes were recovered in a natural cycle from naturally conceived cows aged 4-13.5 years (n = 10) to provide a heterogeneous cohort for mtDNA deletion studies. The presence or absence of mtDNA deletions were investigated using long-range PCR in individual oocytes (n = 62). To determine the detection threshold for mtDNA heteroplasmy levels in individual oocytes, a novel NGS methodology was validated; artificial mixtures of the Bos taurus and Bos indicus mitochondrial genome were generated at 1, 2, 5, 15 and 50% ratios to experimentally mimic different levels of heteroplasmy. This NGS methodology was then employed to determine mtDNA heteroplasmy levels in single oocytes (n = 24). Oocyte mtDNA deletion and heteroplasmy data were analysed by binary logistic regression with respect to the effects of ovarian ageing and ovarian stimulation regimens. MAIN RESULTS AND THE ROLE OF CHANCE: Ovarian ageing, but not ovarian stimulation, increased the number of oocytes exhibiting mtDNA deletions (P = 0.04). A minimum mtDNA heteroplasmy level of 2% was validated as a sensitive (97-100%) threshold for variant detection in individual oocytes using NGS. Few mtDNA heteroplasmies were detected across the individual oocytes, with only 15 oocyte-specific variants confined to two of the 24 oocytes studied. There was no relationship (P > 0.05) evident between ovarian ageing or ovarian stimulation and the presence of mtDNA heteroplasmies. LIMITATIONS, REASON FOR CAUTION: The low number of oocytes collected from the natural ovarian cycles limited the analysis. Fertilization and developmental potential of the oocytes was not assessed as the oocytes were destroyed for mtDNA deletion and heteroplasmy analysis. WIDER IMPLICATIONS OF THE FINDINGS: If the findings of this model apply to the human, this study suggests that the incidence of mtDNA deletions increases with age, but not with degree of ovarian stimulation, while the frequency of mtDNA heteroplasmies may be low regardless of ovarian ageing or level of ovarian stimulation. STUDY FUNDING AND COMPETING INTERESTS: Funding was provided by Fertility Associates, the Nurture Foundation for Reproductive Research, the Fertility Society of Australia, and the Auckland Medical Research Foundation. J.C.P. is a shareholder of Fertility Associates and M.P.G. received a fellowship from Fertility Associates. The other authors of this manuscript declare no conflict of interest that could be perceived as prejudicing the impartiality of the reported research.

The effects of 2,4-dinitrophenol and d-glucose concentration on the development, sex ratio, and interferon-tau (IFNT) production of bovine blastocysts.

The preimplantation bovine embryo displays sexual dimorphism in glucose sensitivity and interferon-tau (IFNT) secretion that are negated by inhibition of the pentose phosphate pathway, suggesting that the association between glucose metabolism and IFNT likely underpins the selective loss of female embryos. The aim of this study was to determine if altered glucose metabolism, through glucose supplementation and/or uncoupling oxidative phosphorylation with 2,4-dinitrophenol (DNP), affected embryo development. Bovine blastocyst development, sex, and IFNT production were examined in embryos cultured in the presence or absence of glucose (0, 1.5, 4 mM) with or without exposure to DNP (0, 10, 100 µM) between Days 5 and 8 post-fertilization. The absence or presence of high (4 mM) glucose reduced blastocyst development and favored the development of male embryos (P < 0.001). DNP at 10 µM had no effect, whereas 100 µM had a negative impact on blastocyst development. Notably, in the presence or even absence of glucose, supplementation with 10 µM DNP further skewed the sex ratio toward males (P < 0.05). Sexually dimorphic IFNT production was maintained in these conditions, although total production was reduced in the presence of high glucose and DNP, irrespective of embryo sex. These data suggest that the pentose phosphate pathway can modulate embryonic sex ratio and development. Therefore, bovine embryo culture should be undertaken in a low glucose (<2.5 mM) medium to minimize potential embryonic stress, as higher concentrations have sexually dimorphic effects on development and an embryo's ability to signal to the maternal reproductive tract.

Combined parental obesity negatively impacts preimplantation mouse embryo development, kinetics, morphology and metabolism.

STUDY QUESTION: Does combined parental obesity, both an obese mother and father, have a greater effect on mouse preimplantation embryo development and quality than single-parent obesity? SUMMARY ANSWER: Combined parental obesity causes a greater reduction in the blastocyst rate and a greater delay to the timing of key embryonic developmental events than single-parental obesity, as well as altering embryonic characteristics, such as zona pellucida width. WHAT IS KNOWN ALREADY: Maternal or paternal obesity alone are known to have significant and detrimental impacts on preimplantation embryo development. Furthermore, these early embryonic perturbations can have long-term impacts on both offspring health and further generations. This is one of the first studies to examine the effects of having both an obese mother and an obese father. STUDY DESIGN, SIZE, DURATION: A cross-sectional control versus treatment mouse study of diet-induced obesity was employed, in which 300 embryos per group were generated and studied from reciprocal matings: (i) control female and control male (Lean Parented Embryos); (ii) control female and obese male (Paternal Obese Parented Embryos); (iii) obese female and control male (Maternal Obese Parented Embryos) and (iv) obese female and obese male (Combined Obese Parented embryos). Assessments of the embryonic development rate, timing of development, morphological characteristics, metabolic gene expression, metabolism and cell lineage allocation were made at selected time points and analysed in relation to parental obesity status. PARTICIPANTS/MATERIALS, SETTING, METHODS: Three-week-old C57BL6 male and female mice were fed control (7% total fat) or high fat (21% total fat) diets for a minimum of 8 weeks. Females were superovulated, mated, fertilized zygotes recovered and standard mouse in vitro embryo culture performed. Time-lapse monitoring was undertaken to compare developmental timings and morphological characteristics (embryonic area and zona pellucida width) for embryos from all four reciprocal matings. Differential staining identified cell lineage allocation. Real-time quantitative RT-PCR (qRT-PCR) and microfluorescence were used to measure gene expression and metabolism (glucose consumption and lactate production), respectively, in embryos from Lean Parented and Combined Obese Parented matings. This research was completed in a University research laboratory. MAIN RESULTS AND THE ROLE OF CHANCE: Blastocyst rate was reduced in Combined Obese Parented embryos when compared with both Single Obese (11% decrease for Maternal Obese Parented, P < 0.05; 15% for Paternal Obese Parented, P < 0.05) and Lean Parented embryos (25% decrease, P < 0.01). Time-lapse analysis of developmental kinetics highlighted a delay of 1 h at the 2-3 cell division, extending to 6 h delay by the blastocyst stage for Combined Obese Parented embryos (P < 0.05). A reduction in the total cell number of Combined Obese Parented blastocysts was a further manifestation of this developmental delay (P < 0.05). Zona pellucida width was reduced in Combined Obese Parented embryos (P < 0.05). Glucose consumption was increased in Combined Obese Parented embryos (P < 0.05), which was associated with the up-regulation of Glucose transporter 1 expression (P < 0.05). LIMITATIONS AND REASON FOR CAUTION: This study was completed in fertile C57BL/6 mice using a well-defined model of diet-induced obesity in which embryos were fertilized in vivo. Human obesity is complex, with many causes and co-morbidities, and therefore, the impact of combined obesity would require further investigation in human settings. WIDER IMPLICATIONS OF THE FINDINGS: This study demonstrates that combined parental obesity has a detrimental impact on mouse embryo development, a finding consistent with previous studies on individual parent obesity. Of note, the effect of combined parental obesity upon embryo development markers was greater than that of individual parental obesity. Plausibly, human embryos will be similarly impacted. The reduction in the blastocyst rate and delayed time to developmental events confirms that embryos of obese parents differ from those of lean parents. Allowance for this should therefore be incorporated into clinical practice when selecting the best embryo for the transfer of an obese couple. STUDY FUNDING/COMPETING INTERESTS: Funding was provided by University of Melbourne research monies. M.P.G. currently holds the position of Merck Serono Lecturer of Reproductive Biology. D.K.G. received research funds from Vitrolife AB Sweden. The other authors of this manuscript have nothing to declare and no conflicts of interest.

Maternal age and ovarian stimulation independently affect oocyte mtDNA copy number and cumulus cell gene expression in bovine clones.

STUDY QUESTION: Does maternal ageing and ovarian stimulation alter mitochondrial DNA (mtDNA) copy number and gene expression of oocytes and cumulus cells from a novel bovine model for human IVF? SUMMARY ANSWER: Oocytes collected from females with identical nuclear genetics show decreased mtDNA copy number and increased expression of an endoplasmic reticulum (ER) stress gene with repect to ovarian stimulation, whilst differences in the expression of genes involved in mitochondrial function, antioxidant protection and apoptosis were evident in relation to maternal ageing and the degree of ovarian stimulation in cumulus cells. WHAT IS KNOWN ALREADY: Oocyte quality declines with advancing maternal age; however, the underlying mechanism, as well as the effects of ovarian stimulation are poorly understood. Human studies investigating these effects are often limited by differences in age and ovarian stimulation regimens within a patient cohort, as well as genetic and environmental variability. STUDY DESIGN, SIZE, DURATION: A novel bovine cross-sectional maternal age model for human IVF was undertaken. Follicles were aspirated from young (3 years of age; n = 7 females) and old (10 years of age; n = 5 females) Holstein Freisian clones following multiple unstimulated, mild and standard ovarian stimulation cycles. These bovine cloned females were generated by the process of somatic cell nuclear transfer (SCNT) from the same founder and represent a homogeneous population with reduced genetic and environmental variability. Maternal age and ovarian stimulation effects were investigated in relation to mtDNA copy number, and the expression of 19 genes involved in mitochondrial function, antioxidant protection, oocyte-cumulus cell signalling and follicle development in both oocytes and cumulus cells. MATERIALS, SETTING, METHODS: Young (3 years of age; n = 7 females) and old (10 years of age; n = 5 females) Holstein Freisian bovine clones were maintained as one herd. Stimulation cycles were based on the long GnRH agonist down-regulation regimen used in human fertility clinics. Follicle growth rates, numbers and diameters were monitored by ultrasonography and aspirated when the lead follicles were >14 mm in diameter. Follicle characteristics were analysed using a mixed model procedure. Quantitative PCR (qPCR) was used to determine mtDNA copy number and reverse transcriptase-qPCR (RT-qPCR) was used to measure gene expression in oocytes and cumulus cells. MAIN RESULTS AND THE ROLE OF CHANCE: Method of ovarian stimulation (P = 0.04), but not maternal age (P > 0.1), was associated with a lower mtDNA copy number in oocytes. Neither factor affected mtDNA copy number in cumulus cells. In oocytes, maternal age had no effect on gene expression; however, ovarian stimulation in older females increased the expression of GRP78 (P = 0.02), a gene involved in ER stress. In cumulus cells, increasing maternal age was associated with the higher expression of genes involved in mitochondrial maintenance (TXN2 P = 0.008 and TFAM P = 0.03), whereas ovarian stimulation decreased the expression of genes involved in mitochondrial oxidative stress and apoptosis (TXN2 P = 0.002, PRDX3 P = 0.03 and BAX P = 0.03). LIMITATIONS, REASON FOR CAUTION: The low number of oocyte and cumulus cell samples collected from the unstimulated cycles limited the analysis. Fertilization and developmental potential of the oocytes was not assessed because these were used for mtDNA and gene expression quantification. WIDER IMPLICATIONS OF THE FINDINGS: Delineation of the independent effects of maternal age and ovarian stimulation regimen on mtDNA copy number gene expression in oocytes and cumulus cells was enabled by the removal of genetic and environmental variability in this bovine model for human IVF. Therefore, these extend upon previous knowledge and findings provide relevant insights that are applicable for improving human ovarian stimulation regimens. STUDY FUNDING/COMPETING INTERESTS: Funding was provided by Fertility Associates and the University of Auckland. J.C.P. is a shareholder of Fertility Associates and M.P.G. received a fellowship from Fertility Associates. The other authors of this manuscript declare no conflict of interest that could be perceived as prejudicing the impartiality of the reported research.

Exposure to endocrine disrupting chemicals from beverage packaging materials and risk assessment for consumers.

This study investigated the influence of beverage packaging materials on the presence of endocrine disrupting chemicals (EDCs) in plastic, glass, carton, aluminium, and tin canned non-alcoholic beverages. Results showed that 63 EDCs including perfluoroalkyl and polyfluoroalkyl substances (PFAS), bisphenols, parabens, benzophenone-type UV-filters, biocides, nitrophenols, and alkylphenols, were detected in 144/162 screened products. Detected ∑63EDC concentrations ranged from 1.3 to 19,600 ng/L. EDC concentrations were higher in beverages packaged in metal cans while lower or no levels were detected in glass, plastic, and carton packaged drinks. Bisphenol levels were higher on average in canned beverages compared to glass (p < 0.01) and plastic products (p < 0.05) produced by the same brand and manufacturer. Two structural isomers of bisphenol A (BPA) were identified in 19 beverages, constituting the first detection in foodstuffs. The calculated daily intake of detected EDCs showed that exposure to BPA from per capita beverage consumption of 364 mL/day are up to 2000-fold higher than the newly revised safety guideline for BPA recommended by the EFSA (European Food Safety Authority). Overall, these findings suggest that BPA exposure poses a potential health hazard for individuals who regularly consume non-alcoholic beverages packaged in aluminium or tin cans, particularly young children.

The perils of poly- and perfluorinated chemicals on the reproductive health of humans, livestock, and wildlife.

Poly- and perfluoroalkyl substances (PFAS) are a prominent class of persistent synthetic compound. The widespread use of these substances in various industrial applications has resulted in their pervasive contamination on a global scale. It is therefore concerning that PFAS have a propensity to accumulate in bodily tissues whereupon they have been linked with a range of adverse health outcomes. Despite this, the true extent of the risk posed by PFAS to humans, domestic animals, and wildlife remains unclear. Addressing these questions requires a multidisciplinary approach, combining the fields of chemistry, biology, and policy to enable meaningful investigation and develop innovative remediation strategies. This article combines the perspectives of chemists, soil scientists, reproductive biologists, and health policy researchers, to contextualise the issue of PFAS contamination and its specific impact on reproductive health. The purpose of this article is to describe the challenges associated with remediating PFAS-contaminated soils and waters and explore the consequences of PFAS contamination on health and reproduction. Furthermore, current actions to promote planetary health and protect ecosystems are presented to instigate positive social change among the scientific community.

Assessment of the impact of direct in vitro PFAS treatment on mouse spermatozoa.

Abstract: Poly- and per-fluoroalkyl substances (PFAS) are synthetic environmentally persistent chemicals. Despite the phaseout of specific PFAS, their inherent stability has resulted in ubiquitous and enduring environmental contamination. PFAS bioaccumulation has been reported globally with omnipresence in most populations wherein they have been associated with a range of negative health effects, including strong associations with increased instances of testicular cancer and reductions in overall semen quality. To elucidate the biological basis of such effects, we employed an acute in vitro exposure model in which the spermatozoa of adult male mice were exposed to a cocktail of PFAS chemicals at environmentally relevant concentrations. We hypothesized that direct PFAS treatment of spermatozoa would induce reactive oxygen species generation and compromise the functional profile and DNA integrity of exposed cells. Despite this, post-exposure functional testing revealed that short-term PFAS exposure (3 h) did not elicit a cytotoxic effect, nor did it overtly influence the functional profile, capacitation rate, or the in vitro fertilization ability of spermatozoa. PFAS treatment of spermatozoa did, however, result in a significant delay in the developmental progression of the day 4 pre-implantation embryos produced in vitro. This developmental delay could not be attributed to a loss of sperm DNA integrity, DNA damage, or elevated levels of intracellular reactive oxygen species. When considered together, the results presented here raise the intriguing prospect that spermatozoa exposed to a short-term PFAS exposure period potentially harbor an alternate stress signal that is delivered to the embryo upon fertilization. Lay summary: PFAS are synthetic chemicals widely used in non-stick cookware, food packaging, and firefighting foam. Such extensive use has led to concerning levels of environmental contamination and reports of associations with a spectrum of negative health outcomes, including testicular cancer and reduced semen quality. To investigate the effects of PFAS on male reproduction, we incubated mouse sperm in a cocktail of nine PFAS at environmentally relevant concentrations before checking for a range of functional outcomes. This treatment strategy was not toxic to the sperm; it did not kill them or reduce their motility, nor did it affect their fertilization capacity. However, we did observe developmental delays among pre-implantation embryos created using PFAS-treated sperm. Such findings raise the intriguing prospect that PFAS-exposed sperm harbor a form of stress signal that they deliver to the embryo upon fertilization.

High-resolution temporal wastewater treatment plant investigation to understand influent mass flux of per- and polyfluoroalkyl substances (PFAS).

This study aims to identify sources of per- and polyfluoroalkyl substances (PFAS) to wastewater treatment plants (WWTPs) and reveals previously undescribed variability in daily PFAS concentrations by measuring their occurrence in WWTP influent each hour over the course of a week. ∑50PFAS concentrations ranged between 89 ± 38 on Monday and 173 ± 110 ng L-1 on Friday, where perfluoroalkyl carboxylic acids (PFCAs), disubstituted phosphate esters (diPAPs), and perfluoroalkyl sulfonic acids (PFSAs) contributed the largest proportion to overall weekly concentrations 37%, 30%, and 17% respectively. Simultaneous pulse events of perfluorooctanesulfonic acid (PFOS; 400 ng L-1) and perfluoroheptanesulfonic acid (PFHpS; 18 ng L-1) indicate significant industrial or commercial waste discharge that persists for up to 3 h. The minimum number of hourly grab samples required to detect variation of PFOS and PFHpS concentrations are 7 and 9 samples respectively, indicating a high degree of variability in PFAS concentrations between days. Overall, the risk of sampling bias from grab samples is high given the variability in PFAS concentrations and more frequent sampling campaigns must be balanced against the cost of analysis carefully to avoid the mischaracterisation of mass flux to receiving surface waters.