Review of human oocyte cryopreservation in ART programs: Current challenges and opportunities.

Oocyte cryopreservation has notably increased in recent times, to become an essential part of clinical infertility treatment. Since the 1980s, many improvements in oocyte cryopreservation (OC) have been adopted, including the great advance with the application of vitrification. The commonly used vitrification protocol applies different cryoprotectants (Ethylene glycol and/or DMSO and/or PROH and sucrose and/or Trehalose) and two different steps: firstly, exposure in equilibration solution for 5-15 min, followed by a vitrification solution for 60-90 s at room temperature. The warming method includes a first step for 1 min at 37 °C and 3 subsequent steps at room temperature to remove the cryoprotectant for a total of 9-12 min. In addition, biosafety is a critical aspect to mention, and it is related to devices used during the vitrification, mainly in terms of whether the biological vitrified material comes in direct contact with liquid nitrogen (open vitrification) or not (closed vitrification), where LN2 may contain potentially contaminating viruses or pathogens. Furthermore, during early development major waves of epigenetic reprogramming take place. Recent literature suggests that epigenetic and transcriptomic profiles are sensitive to the stress induced by vitrification, including osmotic shock, temperature, rapid changes of pH and toxicity of cryoprotectants. It is, therefore, important to better understand the potential perturbations of epigenetic modifications that may be associated with the globally used vitrification methods. Therefore, we here discuss the benefits and efficiency of human oocyte vitrification; we also review the evidence surrounding oocyte cryopreservation-related epigenetic modifications and potential epigenetic dysregulations, together with long-term consequences for offspring health.

Bet-hedgers commit to the hedge: Zooplankton in ephemeral semiarid wetlands of tropical Brazil that widely spread risk.

Bet-hedging is an ecological risk-aversion strategy in which a population does not commit all its effort toward a single reproductive event or specific environmental condition, and instead spreads the risk to include multiple reproductive events or conditions. For aquatic invertebrates in dry wetlands, this often takes the form of some propagules hatching in the first available flood, while remaining propagules hatch in subsequent floods (the "hedge"); this better ensures that a subset of propagules will hatch in a flood of sufficient duration to successfully complete development. Harsh environmental conditions are believed to promote an increased reliance on bet-hedging. Bet-hedging studies have typically been restricted to single sites or single populations. Community-level assessments may provide more robust support for the range of hatching strategies that exist in nature. Here, we tested whether freshwater zooplankton assemblages inhabiting ephemeral and unpredictable wetlands of a semiarid zone of tropical Brazil employ hatching strategies suggestive of bet-hedging; few efforts have addressed bet-hedging in the tropics where the unique conditions may influence the strategy. We collected dry sediments from six ephemeral wetlands, and flooded them across a sequence of three hydrations under similar laboratory conditions to assess whether hatching patterns conform to some of the predictions of the bet-hedging theory. We found that taxa showing hatching patterns akin to bet-hedging associated with delayed hatching numerically dominated the assemblages that emerged from dry sediments, although there was large heterogeneity in the hatching rate among sites and across taxa. While some populations distributed their hatching across all three floods and committed most of their hatching fraction to the first hydration, others committed as much or more effort to the second hydration (the "hedge") or the third hydration (another substantial "hedge"). Thus, in the harsh study wetlands, hatching patterns akin to bet-hedging associated with delayed hatching were common and occurred at multiple temporal scales. Our community assessment found that a commitment to the "hedge" was greater than the current theory would predict. Our findings have broader implications; bet-hedger taxa seem especially well equipped to tolerate stress if conditions become harsher as environments change.

Cost-saving population genomic investigation of Daphnia longispina complex resting eggs using whole-genome amplification and pre-sequencing screening.

Resting stages of aquatic organisms that accumulate in the sediment over time are an exceptional resource that allows direct insights into past populations and addressing evolutionary questions. This is of particular interest in taxa that face relatively new environmental challenges, e.g., climate change and eutrophication, such as the Daphnia longispina species complex, a keystone zooplankton group in European freshwater ecosystems. However, genomic analysis might be challenging as DNA yield from many of these resting stages can be low and the material degraded. To reliably allow the resequencing of single Daphnia resting eggs from different sediment layers and characterize genomic changes through time, we performed whole-genome amplification to obtain DNA amounts suitable for genome resequencing and tested multiple protocols involving egg isolation, whole-genome amplification kits, and library preparation. A pre-sequencing contamination screening was developed, consisting of amplifying mitochondrial Daphnia and bacterial markers, to quickly assess and exclude possibly contaminated samples. In total, we successfully amplified and sequenced nine genomes from Daphnia resting eggs that could be identified as Daphnia longispina species. We analyzed the genome coverage and heterozygosity of these samples to optimize this method for future projects involving population genomic investigation of the resting egg bank.

A catastrophic change in a european protected wetland: From harmful phytoplankton blooms to fish and bird kill.

Understanding the processes that underlay an ecological disaster represents a major scientific challenge. Here, we investigated phytoplankton and zooplankton community changes before and during a fauna mass kill in a European protected wetland. Evidence on gradual development and collapse of harmful phytoplankton blooms, allowed us to delineate the biotic and abiotic interactions that led to this ecological disaster. Before the mass fauna kill, mixed blooms of known harmful cyanobacteria and the killer alga Prymnesium parvum altered biomass flow and minimized zooplankton resource use efficiency. These blooms collapsed under high nutrient concentrations and inhibitory ammonia levels, with low phytoplankton biomass leading to a dramatic drop in photosynthetic oxygenation and a shift to a heterotrophic ecosystem phase. Along with the phytoplankton collapse, extremely high numbers of red planktonic crustaceans-Daphnia magna, visible through satellite images, indicated low oxygen conditions as well as a decrease or absence of fish predation pressure. Our findings provide clear evidence that the mass episode of fish and birds kill resulted through severe changes in phytoplankton and zooplankton dynamics, and the alternation on key abiotic conditions. Our study highlights that plankton-related ecosystem functions mirror the accumulated heavy anthropogenic impacts on freshwaters and could reflect a failure in conservation and restoration measures.

Racial and ethnic disparities among donor oocyte banks in the United States.

OBJECTIVE: To determine whether racial and ethnic distributions of oocyte donors contributing to US oocyte banks differ from the demographics of US women and donor oocyte recipients. DESIGN: Cross-sectional study. SETTING: United States donor oocyte banks, US census, and fertility clinics reporting to the Society for Assisted Reproductive Technology Clinic Outcome Reporting System. PATIENTS: Oocyte donors from 12 banks, women aged 18-44 years based on the 2019 census, and US recipients of cryopreserved donor oocytes from 2012 to 2015. INTERVENTION: None. MAIN OUTCOME MEASURE: Proportions of donors identifying as each racial and ethnic group. RESULTS: Of the 1,574 oocyte donors, 678 (43.1%) identified as white compared with 54.8% of US women and 69.1% of donor oocyte recipients. Proportions of donors identifying as Hispanic or two or more races were larger than those of US women and donor oocyte recipients (Hispanic: 24.1% vs. 20.8%, and 24.1% vs. 8.8%, respectively; two or more races: 16.1% vs. 2.3%, and 16.1% vs. 0.5%, respectively). African American donors were underrepresented compared with US women (8.9% vs. 14.0%) and oocyte recipients (8.9% vs. 10.8%). Although the proportion of Asian donors was similar to that of US women (7.7% vs. 7.1%), Asian donors were underrepresented compared with donor oocyte recipients (7.7% vs. 10.6%). CONCLUSION: Racial and ethnic distribution of oocyte donors differs significantly from the demographics of US women and cryopreserved donor oocyte recipients. These data suggest a need for targeted recruitment of African American and Asian oocyte donors.

Embryo ecology: Developmental synchrony and asynchrony in the embryonic development of wild annual fish populations.

Embryo-environment interactions are of paramount importance during the development of all organisms, and impacts during this period can echo far into later stages of ontogeny. African annual fish of the genus Nothobranchius live in temporary pools and their eggs survive the dry season in the dry bottom substrate of the pools by entering a facultative developmental arrest termed diapause. Uniquely among animals, the embryos (encased in eggs) may enter diapause at three different developmental stages. Such a system allows for the potential to employ different regulation mechanisms for each diapause. We sampled multiple Nothobranchius embryo banks across the progressing season, species, and populations. We present important baseline field data and examine the role of environmental regulation in the embryonic development of this unique system. We describe the course of embryo development in the wild and find it to be very different from the typical development under laboratory conditions. Development across the embryo banks was synchronized within and across the sampled populations with all embryos entering diapause I during the rainy season and diapause II during the dry season. Asynchrony occurred at transient phases of the habitat, during the process of habitat desiccation, and at the end of the dry season. Our findings reveal the significance of environmental conditions in the serial character of the annual fish diapauses.

Next-generation sequencing of DNA from resting eggs: signatures of eutrophication in a lake's sediment.

Hatching resting stages of ecologically important organisms such as Daphnia from lake sediments, referred to as resurrection ecology, is a powerful approach to assess changes in alleles and traits over time. However, the utility of the approach is constrained by a few obstacles, including low and/or biased hatching among genotypes. Here, we eliminated such bottlenecks by investigating DNA sequences isolated directly (i.e. without hatching) from resting eggs found in the sediments of Lake Constance spanning pre-, peri-, and post-eutrophication. While we expected genome-wide changes, we specifically expected changes in alleles related to pathways involved in mitigating effects of cyanobacterial toxins. We used pairwise FST-analyses to identify transcripts that showed strongest divergence among the four different populations and a clustering analysis to identify correlations between allele frequency shifts and changes in abiotic and biotic lake parameters. In a cluster that correlated with the increased abundance of cyanobacteria in Lake Constance we find genes that have been reported earlier to be differentially expressed in response to the cyanobacterial toxin microcystin and to microcystin-free cyanobacteria. We further reveal the enrichment of gene ontology terms that have been shown to be involved in microcystin-related responses in other organisms but not yet in Daphnia and as such are candidate loci for adaptation of natural Daphnia populations to increased cyanobacterial abundances. In conclusion this approach of investigating DNA extracted from Daphnia resting stages allowed to determine frequency changes of loci in a natural population over time.

Centennial clonal stability of asexual Daphnia in Greenland lakes despite climate variability.

Climate and environmental condition drive biodiversity at many levels of biological organization, from populations to ecosystems. Combined with paleoecological reconstructions, palaeogenetic information on resident populations provides novel insights into evolutionary trajectories and genetic diversity driven by environmental variability. While temporal observations of changing genetic structure are often made of sexual populations, little is known about how environmental change affects the long-term fate of asexual lineages. Here, we provide information on obligately asexual, triploid Daphnia populations from three Arctic lakes in West Greenland through the past 200-300 years to test the impact of environmental change on the temporal and spatial population genetic structure. The contrasting ecological state of the lakes, specifically regarding salinity and habitat structure may explain the observed lake-specific clonal composition over time. Palaeolimnological reconstructions show considerable regional environmental fluctuations since 1,700 (the end of the Little Ice Age), but the population genetic structure in two lakes was almost unchanged with at most two clones per time period. Their local populations were strongly dominated by a single clone that has persisted for 250-300 years. We discuss possible explanations for the apparent population genetic stability: (a) persistent clones are general-purpose genotypes that thrive under broad environmental conditions, (b) clonal lineages evolved subtle genotypic differences unresolved by microsatellite markers, or (c) epigenetic modifications allow for clonal adaptation to changing environmental conditions. Our results motivate research into the mechanisms of adaptation in these populations, as well as their evolutionary fate in the light of accelerating climate change in the polar regions.

Bet hedging in stochastic habitats: an approach through large branchiopods in a temporary wetland.

Organisms evolve to maintain fitness across generations, while short-term fitness in stochastic habitats such as temporary wetlands may be highly varied. As typical temporary wetland inhabitants, large branchiopods rely on bet hedging hatching that helps them survive throughout generations. An optimal hatching rate is predicted to be approximate to the successful reproduction probability (SRP). We tested the difference between hatching rate and SRP of large branchiopods Branchinella kugenumaensis and Eulimnadia braueriana in a temporary wetland in Taiwan, through field surveys and climatic records to evaluate their SRP. Comparisons were performed under two proposed scenarios, where a population's hatching was bet hedged for a hydroperiod or for a wet season (with several hydroperiods), respectively. Population size fluctuations were simulated for these two scenarios under assumed egg mortalities and reproductive replenishments. Results showed that the hatching rates only fitted to SRP for E. braueriana under the scenario of bet hedging on a wet season, not for B. kugenumaensis, nor for both species under the scenario of bet hedging on a hydroperiod. Bet hedging on a wet season would have a smaller range of population size fluctuation and a lower rate of population size decrease. This implies that large branchiopods adopt a conservative hatching strategy, lowering the hatching fraction in each hydroperiod to reduce long-term egg bank size fluctuation. Bet hedging strategies could occur during other life cycle stages, coexist with other life history strategies, and lead to the diversified hatching fraction distribution rather than a single, optimal fraction throughout hydroperiods.

Acute and chronic effects of exposure to the juvenile hormone analog fenoxycarb during sexual reproduction in Daphnia magna.

Recent studies have demonstrated that insect growth regulating insecticides are able to affect reproductive endpoints in zooplankton species at very low levels. For the cyclic parthenogenetic water flea Daphnia, most of this research has focused on the asexual part of the life cycle and induction of male offspring. Even though Daphnia and many other aquatic invertebrates rely on sexual reproduction and subsequent production of dormant eggs to recover from environmentally harsh conditions, much less is known about the effects of toxicants on the sexual reproductive phase. Using fenoxycarb as a model pesticide, we exposed male and female neonate Daphnia magna, under conditions inducing a switch to sexual reproduction, and tested for effects on dormant egg (ephippia) production and sex ratio of parthenogenetic offspring. Subsequently, we assessed whether fenoxycarb exposure affected the quality of the produced dormant eggs and viability of the hatchlings. Our results showed that exposure to sub-lethal concentrations of fenoxycarb caused a sharp decrease in parthenogenetic reproduction, while inducing male offspring. Dormant egg production was marginally negatively affected, but survival and fitness of the hatched individuals were not significantly affected. This indicates that under pesticide stress, surviving adult females invested in sexual reproduction at the expense of parthenogenetic reproduction. Exposure to toxicants during the sexual reproductive phase, could affect the active aquatic phase as well as the dormant phase in natural zooplankton populations. This indicates the need for further ecotoxicological research and development of test protocols taking into account the full life cycle of zooplankton species.

Whole genome amplification and sequencing of a Daphnia resting egg.

Resting eggs banks are unique windows that allow us to directly observe shifts in population genetics, and phenotypes over time as natural populations evolve. Though a variety of planktonic organisms also produce resting stages, the keystone freshwater consumer, Daphnia, is a well-known model for paleogenetics and resurrection ecology. Nevertheless, paleogenomic investigations are limited largely because resting eggs do not contain enough DNA for genomic sequencing. In fact, genomic studies even on extant populations include a laborious preparatory phase of batch culturing dozens of individuals to generate sufficient genomic DNA. Here, we furnish a protocol to generate whole genomes of single ephippial (resting) eggs and single daphniids. Whole genomes of single ephippial eggs and single adults were amplified using Qiagen REPLI-g Single Cell kit reaction, followed by NEBNext Ultra DNA Library Prep Kit for library construction and Illumina sequencing. We compared the quality of the single-egg and single-individual amplified genomes to the standard batch genomic DNA extraction in the absence of genome amplification. At mean 20× depth, coverage was essentially identical for the amplified single individual relative to the unamplified batch extracted genome (>90% of the genome was covered and callable). Finally, while amplification resulted in the slight loss of heterozygosity for the amplified genomes, estimates were largely comparable and illustrate the utility and limitations of this approach in estimating population genetic parameters over long periods of time in natural populations of Daphnia and also other small species known to produce resting stages.

Barcoding rotifer biodiversity in Mediterranean ponds using diapausing egg banks.

The biodiversity of Mediterranean freshwater bodies is among the most threatened worldwide; therefore, its accurate estimation is an urgent issue. However, traditional methods are likely to underestimate freshwater zooplankton biodiversity due to its high species seasonality and cryptic diversity. We test the value of applying DNA barcoding to diapausing egg banks, in combination with the creation of a reference collection of DNA barcodes using adult individual samples, to characterize rotifer communities. We use monogonont rotifers from two lakes in Doñana National Park and one from Ruidera Natural Park in Spain as models to create a reference collection of DNA barcodes for taxonomically diagnosed adult individuals sampled from the water column, to compare with the sequences obtained from individual eggs from the diapausing egg banks. We apply two different approaches to carry out DNA taxonomy analyses, the generalized mixed Yule coalescent method (GMYC) and the Automatic Barcode Gap Discovery (ABGD), to the obtained sequences and to publicly available rotifer sequences. We obtained a total of 210 new rotifer COI sequences from all three locations (151 diapausing eggs and 59 adults). Both GMYC and ABGD generated the same 35 operational taxonomic units (OTUs), revealing four potential cryptic species. Most sequences obtained from diapausing eggs (85%) clustered with sequences obtained from morphologically diagnosed adults. Our approach, based on a single sediment sample, retrieved estimates of rotifer biodiversity higher than or similar to those of previous studies based on a number of seasonal samples. This study shows that DNA barcoding of diapausing egg banks is an effective aid to characterize rotifer diversity in Mediterranean freshwater bodies.

Temporal genetic stability in natural populations of the waterflea Daphnia magna in response to strong selection pressure.

Studies monitoring changes in genetic diversity and composition through time allow a unique understanding of evolutionary dynamics and persistence of natural populations. However, such studies are often limited to species with short generation times that can be propagated in the laboratory or few exceptional cases in the wild. Species that produce dormant stages provide powerful models for the reconstruction of evolutionary dynamics in the natural environment. A remaining open question is to what extent dormant egg banks are an unbiased representation of populations and hence of the species' evolutionary potential, especially in the presence of strong environmental selection. We address this key question using the water flea Daphnia magna, which produces dormant stages that accumulate in biological archives over time. We assess temporal genetic stability in three biological archives, previously used in resurrection ecology studies showing adaptive evolutionary responses to rapid environmental change. We show that neutral genetic diversity does not decline with the age of the population and it is maintained in the presence of strong selection. In addition, by comparing temporal genetic stability in hatched and unhatched populations from the same biological archive, we show that dormant egg banks can be consulted to obtain a reliable measure of genetic diversity over time, at least in the multidecadal time frame studied here. The stability of neutral genetic diversity through time is likely mediated by the buffering effect of the resting egg bank.

Local human pressures influence gene flow in a hybridizing Daphnia species complex.

Anthropogenic environmental changes are considered critical drivers of the genetic structure of populations and communities through, for example, the facilitation of introgressive hybridization between syntopic species. However, the mechanisms by which environmental perturbations trigger changes in the genetic structure of populations and communities, such as the processes that determine the directionality of hybridization and patterns of mitochondrial introgression over many generations, remain largely unexplored. In this study, the changes in genetic structure of hybridizing members of the Daphnia longispina species complex were reconstructed over the last 100 years for three large temperate lakes under strong anthropogenic pressures via palaeogenetic analyses of resting egg banks. Drastic changes in the genetic structure of the Daphnia community, associated with hybridization events between D. longispina and D. galeata and subsequent introgression, were detected in Lakes Geneva and Bourget. In Lake Bourget, these changes were induced by the successful establishment of D. galeata with rising phosphorus levels and reinforced by the sensitivity of D. longispina to fish predation pressure. In Lake Geneva, the pattern of hybridization during eutrophication is more likely a function of the original taxonomic composition of the species complex in this lake. Lakes seem to require at least a meso-oligotrophic status to allow D. galeata populations to establish and accordingly no D. galeata genotypes were found in the egg bank of oligotrophic Lake Annecy. In contrast to the generally assumed pattern of unidirectional hybridization in this species complex, bidirectional hybridization was recorded in Lakes Geneva and Bourget. Our results also demonstrate complex genetic trajectories within this species complex and highlight the irreversibility of changes in the genotypic architecture of populations driven by local human pressures. Finally, we show that extensive hybridization and introgression do not necessarily result in a large and homogenous hybrid swarm.

Population genetic dynamics of an invasion reconstructed from the sediment egg bank.

Biological invasions are a global issue with far-reaching consequences for single species, communities and whole ecosystems. Our understanding of modes and mechanisms of biological invasions requires knowledge of the genetic processes associated with successful invasions. In many instances, this information is particularly difficult to obtain as the initial phases of the invasion process often pass unnoticed and we rely on inferences from contemporary population genetic data. Here, we combined historic information with the genetic analysis of resting eggs to reconstruct the invasion of Daphnia pulicaria into Lower Lake Constance (LLC) in the 1970s from the resting egg bank in the sediments. We identified the invader as 'European D. pulicaria' originating from meso- and eutrophic lowland lakes and ponds in Central Europe. The founding population was characterized by extremely low genetic variation in the resting egg bank that increased considerably over time. Furthermore, strong evidence for selfing and/or biparental inbreeding was found during the initial phase of the invasion, followed by a drop of selfing rate to low levels in subsequent decades. Moreover, the increase in genetic variation was most pronounced during early stages of the invasion, suggesting additional introductions during this period. Our study highlights that genetic data covering the entire invasion process from its beginning can be crucial to accurately reconstruct the invasion history of a species. We show that propagule banks can preserve such information enabling the study of population genetic dynamics and sources of genetic variation in successful invasive populations.

A mixture of environmental organic contaminants in lake sediments affects hatching from Daphnia resting eggs.

Despite the relevance of resting eggs for ecology and evolution of many aquatic organisms and their exposure to contaminants accumulating in sediments, ecotoxicological studies using resting eggs are vastly underrepresented. The authors established a method to perform exposure assays with resting eggs produced by the Daphnia longispina species complex, key species in large lake ecosystems. A mixture of organic contaminants previously detected in sediments of Lake Greifensee was selected to test the potential effect of organic contaminants present in sediments on the hatching process. Resting eggs were exposed to a mix of 10 chemicals, which included corrosion inhibitors, biocides, pesticides, and personal care products, for a period of 15 d. Using an automated counting software, the authors found a significant increase in hatching success in the exposed resting eggs compared with controls. Such an effect has not yet been reported from ecotoxicological assays with resting eggs. Possible mechanistic explanations as well as the potential implications on the ecology and evolution of aquatic species that rely on a resting egg banks are discussed. Observed increased mortality and developmental abnormalities for hatchlings in the exposure treatments can be explained by toxic contaminant concentrations. The results of the present study highlight the need for additional studies assessing the effects of organic contaminants on resting egg banks and aquatic ecosystems.

Timing matters: sensitivity of Daphnia magna dormant eggs to fenoxycarb exposure depends on embryonic developmental stage.

Although Daphnia magna is a key species in many lentic freshwater ecosystems and is commonly used as model organism in ecology and ecotoxicology, very little is known about the effects of chemicals on their dormant life stages. Dormant eggs (ephippia) are produced when environmental conditions deteriorate, and Daphnia switch from clonal to sexual reproduction. Ephippia produced over different growing seasons can accumulate in the sediment of ponds and lakes, where they can be exposed to pesticides and other (anthropogenic) stressors. In the present study, we have investigated the effects of pesticide exposure on dormant eggs at different embryonic developmental stages and evaluated the degree of protection against pollution provided by the ephippial case. We therefore conducted a hatching experiment in which decapsulated and encapsulated dormant eggs were exposed to an insect growth regulator (fenoxycarb) at different stages during their development, both before and after activation of the eggs. In addition, we developed an analytical method to measure fenoxycarb concentrations in the dormant eggs. Fenoxycarb negatively affected development and hatching success and changed the timing of hatching in activated and in dormant eggs. Hatching characteristics as well as fenoxycarb concentrations inside the eggs differed significantly between exposure treatments. Final stages of embryonic development were most sensitive to pesticide exposure and had the highest tissue concentrations of fenoxycarb. Tissue concentrations did not differ significantly between decapsulated and encapsulated eggs, suggesting that the ephippial case offers limited or no direct protection against pesticide exposure. With this study we provide new evidence showing that pesticides can bioconcentrate in and affect D. magna dormant eggs. The severity of the effects on developing embryos depends on the timing of pesticide exposure. Our results stress the importance of considering the full life-cycle of model organisms used in ecotoxicological studies, since these are ultimately aimed at assessing risks of chemical exposure on natural aquatic ecosystems.