First observation of a skate egg case nursery in the Ross Sea.

Areas of importance to Southern Ocean skates are poorly defined. Here, we identify a deepwater skate egg case nursery in a discrete location at ~460 m depth off Cape Adare in the Southern Ocean. This is the first confirmed observation of a skate nursery area in the Ross Sea and only the second observation for the Southern Ocean. The morphology and size of the egg cases were consistent with the genus Bathyraja and most likely belong to the Bathyraja sp. (cf. eatonii). The nursery occurs within the "no take" General Protection Zone of the Ross Sea region marine protected area, where commercial fishing is prohibited.

Sharks checking in to the sponge hotel: First internal use of sponges of the genus Agelas and family Irciniidae by banded sand catsharks Atelomycterus fasciatus.

Trawl surveys within and surrounding two northwestern Australian marine parks revealed banded sand catsharks Atelomycterus fasciatus (family Atelomycteridae) taking refuge within large sponges of the family Irciniidae (Demospongiae: Dictyoceratida) and the genus Agelas (Demospongiae: Agelasida: Agelasidae). Five sponges contained a total of 57 A. fasciatus, comprising both sexes and both immature and mature individuals ranging from 102 to 390 mm total length (TL). In the same surveys, only five A. fasciatus were captured unassociated with sponges, suggesting that sponges are an important microhabitat for A. fasciatus and may provide a daytime refuge from predators. A southerly range extension is also reported for this species.

A Placebo-Controlled Randomized Trial of Vigabatrin in the Management of Acute Alcohol Withdrawal.

OBJECTIVE: To undertake a double blinded randomised placebo-controlled trial to assess the efficacy of vigabatrin, a GABA-transaminase inhibitor, as a benzodiazepine sparing agent in the management of acute alcohol withdrawal syndrome in a residential setting. METHODS: We enrolled 120 patients with alcohol use disorder who were randomly assigned to either treatment with vigabatrin (2g/day for 4 days) or placebo. The primary outcome was defined as the number of participants in each treatment arm needing diazepam for withdrawal management. A secondary outcome prespecified was the total dose of diazepam received by participants in each treatment arm. Participants were recruited on admission to a residential withdrawal unit at St Vincent's Hospital Melbourne from December 2014 to April 2019. RESULTS: No significant difference was observed in the number of participants requiring benzodiazepines during their residential withdrawal stay with 44 participants (78.6%) in placebo arm requiring at least one dose of diazepam compared to 38 (66.7%) in vigabatrin arm (p = .156). An 18.1% difference was observed between the proportion of participants who received a total dose of >100mg of diazepam during their residential withdrawal stay in placebo arm (32.1%), compared to vigabatrin arm (14.0%, p = .022). There were higher rates of reported adverse events in placebo arm with nine (15.0%) participants reporting adverse events compared with two (3.3%) participants in vigabatrin arm (p = .027). CONCLUSION: Vigabatrin significantly reduced the number of participants requiring >100mg diazepam over the course of their alcohol withdrawal and was associated with a reduction in adverse effects when compared to placebo.

Seamount coral reefs are egg case nurseries for deep-sea skates.

Egg case nurseries of the boreal skate (Amblyraja hyperborea) and Richardson's skate (Bathyraja richardsoni) were defined and mapped on a bathyal seascape (c. 500-1900 m depths) south of Tasmania, Australia, using 99 towed-camera transects (157 linear km; N = 50,858 images). In total, 738 skate egg cases were observed (present in 240 images, absent in 50,618); among 113 egg cases examined to identify parent species, 70% were A. hyperborea, 10% B. richardsoni and 20% unidentified Bathyraja species. "Recently laid" egg cases were differentiated from "aged" ones by classifying their colour and condition. The great majority (98%) of egg cases were observed in c. 1100-1400 m depths on seamounts (15 of 36 surveyed), not seamount bases or adjacent continental slope. Egg cases were associated with reefs formed by accumulated skeletal matrix of the stony coral Solenosmilia variabilis, with >90% egg cases (including most of those recently laid) observed on living S. variabilis that characterises a "coral zone" in c. 950-1350 m depths. Water in the coral zone is warmer (+0.66 to 2.37°C) than at the deep distributional limits of adult A. hyperborea and B. richardsoni (2000 and 3000 m, respectively), potentially providing for accelerated embryonic development. Co-occurrence with living coral infers an energetically favourable local-scale hydrodynamic environment for egg cases, particularly on seamount peaks, where increased water flow over egg cases would avert smothering by suspended sediment, and compensate for lower oxygen concentration compared to deeper depths occupied by adult skates. Criteria identifying egg case nurseries are strongly met for A. hyperborea at Seamount Z110 (468 egg cases of varied ages, maximum density of 5.47 m-2 ) and to a lesser extent on five others (Seamounts K1, Z16, Hill U, Z5 and Hill V). An abundance (density) criterion for defining nurseries needs to be flexible because it is a spatially scale-dependent measure that differs between surveys according to the tools and design employed. Off Australia, coral reef egg case nursery habitat is restricted to a narrow depth range in temperate latitudes where it is scarce and impacted by historical bottom trawl fishing in many locations. There has been effective conservation of nursery habitat, however, because four of the six nursery sites identified here and extensive coral reef areas are protected within marine parks.

What came first, the shark or the egg? Discovery of a new species of deepwater shark by investigation of egg case morphology.

Apristurus ovicorrugatus, a new species of deepwater catshark, is described from northwestern Australia. Unique egg cases belonging to an unknown species of Apristurus prompted a more detailed investigation of Apristurus specimens off northwestern Australia. One specimen previously identified as A. sinensis collected off Dampier Archipelago was found gravid with a single egg case. Removal of this egg case confirmed that this species was responsible for producing the unique egg cases previously recorded. The egg cases of this species have strong T-shaped longitudinal ridges on the dorsal and ventral surfaces which are unique in the genus Apristurus. The ridges most closely resemble those present in Bythaelurus canescens from South America, but are larger and always T-shaped. The holotype is closest morphologically to A. sinensis but differs in having a medium brown buccal cavity (vs. jet black), ridged egg cases (vs. smooth egg cases), fewer intestinal spiral valve turns and larger pectoral fins. The holotype is also similar, and closest on a molecular level, to A. nakayai with which it shares a unique synapomorphic character, the white shiny iris (apomorphic within the genus). A late-term embryo removed from an egg case superficially resembled the holotype except in having two parallel rows of enlarged dermal denticles on the dorsolateral predorsal surface. Recent nomenclatural changes to the genera Apristurus and Pentanchus are discussed and challenged. This study highlights the important contribution that egg case morphology has on oviparous elasmobranch taxonomy.

Age-related fertility decline: is there a role for elective ovarian tissue cryopreservation?

Age-related fertility decline (ARFD) is a prevalent concern amongst western cultures due to the increasing age of first-time motherhood. Elective oocyte and embryo cryopreservation remain the most established methods of fertility preservation, providing women the opportunity of reproductive autonomy to preserve their fertility and extend their childbearing years to prevent involuntary childlessness. Whilst ovarian cortex cryopreservation has been used to preserve reproductive potential in women for medical reasons, such as in pre- or peripubertal girls undergoing gonadotoxic chemotherapy, it has not yet been considered in the context of ARFD. As artificial reproductive technology (ART) and surgical methods of fertility preservation continue to evolve, it is a judicious time to review current evidence and consider alternative options for women wishing to delay their fertility. This article critically appraises elective oocyte cryopreservation as an option for women who use it to mitigate the risk of ARFD and introduces the prospect of elective ovarian cortex cryopreservation as an alternative.

SARS-CoV-2 infection in the first trimester and the risk of early miscarriage: a UK population-based prospective cohort study of 3041 pregnancies conceived during the pandemic.

STUDY QUESTION: Does maternal infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the first trimester affect the risk of miscarriage before 13 week's gestation? SUMMARY ANSWER: Pregnant women with self-reported diagnosis of SARS-CoV-2 in the first trimester had a higher risk of early miscarriage. WHAT IS KNOWN ALREADY: Viral infections during pregnancy have a broad spectrum of placental and neonatal pathology. Data on the effects of the SARS-CoV-2 infection in pregnancy are still emerging. Two systematic reviews and meta-analyses reported an increased risk of preterm birth, caesarean delivery, maternal morbidity and stillbirth. Data on the impact of first trimester infection on early pregnancy outcomes are scarce. This is the first study, to our knowledge, to investigate the rates of early pregnancy loss during the SARS-CoV-2 outbreak among women with self-reported infection. STUDY DESIGN, SIZE, DURATION: This was a nationwide prospective cohort study of pregnant women in the community recruited using social media between 21 May and 31 December 2020. We recruited 3545 women who conceived during the SARS-CoV-2 pandemic who were <13 week's gestation at the time of recruitment. PARTICIPANTS/MATERIALS, SETTING, METHODS: The COVID-19 Contraception and Pregnancy Study (CAP-COVID) was an on-line survey study collecting longitudinal data from pregnant women in the UK aged 18 years or older. Women who were pregnant during the pandemic were asked to complete on-line surveys at the end of each trimester. We collected data on current and past pregnancy complications, their medical history and whether they or anyone in their household had symptoms or been diagnosed with SARS-CoV-2 infection during each trimester of their pregnancy. RT-PCR-based SARS-CoV-2 RNA detection from respiratory samples (e.g. nasopharynx) is the standard practice for diagnosis of SARS-CoV-2 in the UK. We compared rate of self-reported miscarriage in three groups: 'presumed infected', i.e. those who reported a diagnosis with SARS-CoV-2 infection in the first trimester; 'uncertain', i.e. those who did not report a diagnosis but had symptoms/household contacts with symptoms/diagnosis; and 'presumed uninfected', i.e. those who did not report any symptoms/diagnosis and had no household contacts with symptoms/diagnosis of SARS-CoV-2. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 3545 women registered for the CAP-COVID study at <13 weeks gestation and were eligible for this analysis. Data for the primary outcome were available from 3041 women (86%). In the overall sample, the rate of self-reported miscarriage was 7.8% (238/3041 [95% CI, 7-9]). The median gestational age (GA) at miscarriage was 9 weeks (interquartile range 8-11). Seventy-seven women were in the 'presumed infected' group (77/3041, 2.5% [95% CI 2-3]), 295/3041 were in the uncertain group (9.7% [95% CI 9-11]) and the rest in the 'presumed uninfected' (87.8%, 2669/3041 [95% CI 87-89]). The rate of early miscarriage was 14% in the 'presumed infected' group, 5% in the 'uncertain' and 8% in the 'presumed uninfected' (11/77 [95% CI 6-22] versus 15/295 [95% CI 3-8] versus 212/2669 [95% CI 7-9], P = 0.02). After adjusting for age, BMI, ethnicity, smoking status, GA at registration and the number of previous miscarriages, the risk of early miscarriage appears to be higher in the 'presumed infected' group (relative rate 1.7, 95% CI 1.0-3.0, P = 0.06). LIMITATIONS, REASONS FOR CAUTION: We relied on self-reported data on early pregnancy loss and SARS-CoV-2 infection without any means of checking validity. Some women in the 'presumed uninfected' and 'uncertain' groups may have had asymptomatic infections. The number of 'presumed infected' in our study was low and therefore the study was relatively underpowered. WIDER IMPLICATIONS OF THE FINDINGS: This was a national study from the UK, where infection rates were one of the highest in the world. Based on the evidence presented here, women who are infected with SARS-CoV-2 in their first trimester may be at an increased risk of a miscarriage. However, the overall rate of miscarriage in our study population was 8%. This is reassuring and suggests that if there is an effect of SARS-CoV-2 on the risk of miscarriage, this may be limited to those with symptoms substantial enough to lead to a diagnostic test. Further studies are warranted to evaluate a causal association between SARS-CoV-2 infection in early pregnancy and miscarriage risk. Although we did not see an overall increase in the risk of miscarriage, the observed comparative increase in the presumed infected group reinforces the message that pregnant women should continue to exercise social distancing measures and good hygiene throughout their pregnancy to limit their risk of infection. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by a grant from the Elizabeth Garrett Anderson Hospital Charity (G13-559194). The funders of the study had no role in study design, data collection, data analysis, data interpretation or writing of the report. J.A.H. is supported by an NIHR Advanced Fellowship. A.L.D. is supported by the National Institute for Health Research University College London Hospitals Biomedical Research Centre. All authors have completed the ICMJE uniform disclosure form at www.icmje.org/coi_disclosure.pdf and declare: support to J.A.H. and A.L.D. as above; no financial relationships with any organizations that might have an interest in the submitted work in the previous 3 years; no other relationships or activities that could appear to have influenced the submitted work. TRIAL REGISTRATION NUMBER: N/A.

Further description of the Kerguelen sandpaper skate Bathyraja irrasa (Rajiformes: Arhynchobatidae) based on additional specimens, including egg cases and embryos.

Adult specimens, additional juvenile specimens, egg cases and embryos were used to provide a more detailed anatomical description of the Kerguelen sandpaper skate Bathyraja irrasa, a species of skate endemic to the Kerguelen Plateau and listed as Vulnerable by the IUCN Red List of Threatened Species. The morphological and meristic data reveal a relatively high level of intraspecific variation, mostly related to size. Egg cases are described for the first time and were shown to vary in colour and fouling depending on the length of time spent in the marine environment. Embryos removed from egg cases represent all stages of embryonic development in this species and include the largest embryo recorded for this species, i.e., 230 mm total length (LT ), which increases the range of size at hatching of B. irrasa to 178-230 mm LT . A number of morphometric and meristic characters varied ontogenetically in B. irrasa, in particular relative tail length, number of tail thorns in the median row and the size of orbits. This study highlights the importance of describing intraspecific variation in species and the importance of egg cases to taxonomic and biological research on oviparous species.

Targeting the Spleen as an Alternative Site for Hematopoiesis.

Bone marrow is the main site for hematopoiesis in adults. It acts as a niche for hematopoietic stem cells (HSCs) and contains non-hematopoietic cells that contribute to stem cell dormancy, quiescence, self-renewal, and differentiation. HSC also exist in resting spleen of several species, although their contribution to hematopoiesis under steady-state conditions is unknown. The spleen can however undergo extramedullary hematopoiesis (EMH) triggered by physiological stress or disease. With the loss of bone marrow niches in aging and disease, the spleen as an alternative tissue site for hematopoiesis is an important consideration for future therapy, particularly during HSC transplantation. In terms of harnessing the spleen as a site for hematopoiesis, here the remarkable regenerative capacity of the spleen is considered with a view to forming additional or ectopic spleen tissue through cell engraftment. Studies in mice indicate the potential for such grafts to support the influx of hematopoietic cells leading to the development of normal spleen architecture. An important goal will be the formation of functional ectopic spleen tissue as an aid to hematopoietic recovery following clinical treatments that impact bone marrow. For example, expansion or replacement of niches could be considered where myeloablation ahead of HSC transplantation compromises treatment outcomes.

Within- and between-group dynamics in an obligate cooperative breeder.

Cooperative behaviour can have profound effects on demography. In many cooperative species, components of fitness (e.g. survival, reproductive success) are diminished in smaller social groups. These effects (termed group-level component Allee effects) may lead smaller groups to grow relatively slowly or fail to persist (termed group-level demographic Allee effects). If these group-level effects were to propagate to the population level, small populations would grow slowly or decline to extinction (termed population-level demographic Allee effects). However, empirical studies have revealed little evidence of such population-level effects. Theoretical studies suggest that dispersal behaviour could either cause or prevent the propagation of group-level Allee effects to the population level. We therefore characterized within- and between-pack dynamics in a population of African wild dogs (Lycaon pictus) to test these contrasting model predictions. Larger wild dog packs produced more pups, and their members experienced higher survival than those in smaller packs. Nevertheless, larger packs grew more slowly than smaller packs, because natal adults dispersed away from them. Most packs either died out in whole-pack death events or broke up when their founders died, irrespective of pack size. Overall, packs showed negative density dependence rather than group-level demographic Allee effects. Larger packs produced more, but not larger, dispersal groups and hence generated more, but not larger, new packs. Larger packs thus contributed more than smaller packs to the number of packs in the population, but their large size did not propagate to their daughter packs. This pattern helps to explain the absence of population-level Allee effects in this species. Dispersal behaviour, itself driven by natural selection on individual reproductive strategies, played a pivotal role in population dynamics, leading to the formation of new packs and limiting the size of established packs. Understanding dispersal processes is likely to be important to understanding the population dynamics of other cooperatively breeding species.

Clinical Germline Genome Editing: When Will Good be Good Enough?

Ensuring experimental outcomes are of the highest clinical caliber is crucial prior to the introduction of germline genome editing. However, if we are to police scientific progress using probability or the potential to go wrong, then we must account for the specious standards of human reproduction. With 15% of clinically recognized pregnancies estimated to end in spontaneous miscarriage within the first trimester, and 25% of all pregnancies ending in miscarriage, human reproduction has a high failure rate. These figures, coupled with the percentage of all births with congenital defects and the number of these who will die in the first year of life, paint two scenarios: one, that evolutionary checkpoints are cruel but critical, and two, that for the seemingly inevitable 3%, or 8 million babies born annually with congenital disorders, perhaps more must be done for prevention, when methods exist for prediction. Unifying progress in three coevolving technologies-assisted reproduction, genome editing, and genome sequencing-could produce revolutionary clinical changes in the harsh global statistics of hereditary disease. A historical perspective on the rocky foundations upon which IVF was built suggests that lessons should be learned from the misalignment of research and clinical practice due to funding and research restrictions. At present, it seems likely that history will repeat itself, and that progress in research will be hampered by hypocritical hesitation.

Normal Levels of Sox9 Expression in the Developing Mouse Testis Depend on the TES/TESCO Enhancer, but This Does Not Act Alone.

During mouse sex determination, transient expression of the Y-linked gene Sry up-regulates its direct target gene Sox9, via a 3.2 kb testis specific enhancer of Sox9 (TES), which includes a core 1.4 kb element, TESCO. SOX9 activity leads to differentiation of Sertoli cells, rather than granulosa cells from the bipotential supporting cell precursor lineage. Here, we present functional analysis of TES/TESCO, using CRISPR/Cas9 genome editing in mice. Deletion of TESCO or TES reduced Sox9 expression levels in XY fetal gonads to 60 or 45% respectively relative to wild type gonads, and reduced expression of the SOX9 target Amh. Although human patients heterozygous for null mutations in SOX9, which are assumed to have 50% of normal expression, often show XY female sex reversal, mice deleted for one copy of Sox9 do not. Consistent with this, we did not observe sex reversal in either TESCO-/- or TES-/- XY embryos or adult mice. However, embryos carrying both a conditional Sox9 null allele and the TES deletion developed ovotestes. Quantitative analysis of these revealed levels of 23% expression of Sox9 compared to wild type, and a significant increase in the expression of the granulosa cell marker Foxl2. This indicates that the threshold in mice where sex reversal begins to be seen is about half that of the ~50% levels predicted in humans. Our results demonstrate that TES/TESCO is a crucial enhancer regulating Sox9 expression in the gonad, but point to the existence of additional enhancers that act redundantly.

Correction: Normal Levels of Sox9 Expression in the Developing Mouse Testis Depend on the TES/TESCO Enhancer, but This Does Not Act Alone.

[This corrects the article DOI: 10.1371/journal.pgen.1006520.].

The global decline of cheetah Acinonyx jubatus and what it means for conservation.

Establishing and maintaining protected areas (PAs) are key tools for biodiversity conservation. However, this approach is insufficient for many species, particularly those that are wide-ranging and sparse. The cheetah Acinonyx jubatus exemplifies such a species and faces extreme challenges to its survival. Here, we show that the global population is estimated at ∼7,100 individuals and confined to 9% of its historical distributional range. However, the majority of current range (77%) occurs outside of PAs, where the species faces multiple threats. Scenario modeling shows that, where growth rates are suppressed outside PAs, extinction rates increase rapidly as the proportion of population protected declines. Sensitivity analysis shows that growth rates within PAs have to be high if they are to compensate for declines outside. Susceptibility of cheetah to rapid decline is evidenced by recent rapid contraction in range, supporting an uplisting of the International Union for the Conservation of Nature (IUCN) Red List threat assessment to endangered. Our results are applicable to other protection-reliant species, which may be subject to systematic underestimation of threat when there is insufficient information outside PAs. Ultimately, conserving many of these species necessitates a paradigm shift in conservation toward a holistic approach that incentivizes protection and promotes sustainable human-wildlife coexistence across large multiple-use landscapes.

Description of the egg cases and juvenile colouration in two catsharks of the genus Atelomycterus (Carcharhiniformes: Scyliorhinidae).

Descriptions of egg cases and hatchlings of the Bali catshark Atelomycterus baliensis and the Australian marbled catshark Atelomycterus macleayi are provided. Comparisons are made with two of their congeners, banded sand catshark Atelomycterus fasciatus and coral catshark Atelomycterus marmoratus. The Atelomycterus egg cases have the same general morphology, i.e., elongate with anterior and posterior waists, a depressed and truncate anterior end sometimes with attachment fibres, a medially tapered posterior end with two horns sometimes terminating in short, tightly curled tendrils and four respiratory fissures (one at each left corner on opposing sides). Morphometric measurements taken of the egg cases of the four species differed significantly when subjected to multivariate analysis, with unique characters enabling distinction between them. The morphological characters that best distinguished the Atelomycterus species from each other were anterior border width, posterior waist width and posterior case width. Thus, egg case morphology could be used to distinguish the egg cases of the four Atelomycterus species examined, consistent with other studies on scyliorhinid and pentanchid egg cases. Colouration of A. baliensis and A. macleayi juveniles was similar to that of other Atelomycterus juveniles in being pale with a series of dark-brown distinct dorsal saddles along the body, blotches and sometimes with a low number of small dark spots.

A novel myeloid cell in murine spleen defined through gene profiling.

A novel myeloid antigen presenting cell can be generated through in vitro haematopoiesis in long-term splenic stromal cocultures. The in vivo equivalent subset was recently identified as phenotypically and functionally distinct from the spleen subsets of macrophages, conventional (c) dendritic cells (DC), resident monocytes, inflammatory monocytes and eosinophils. This novel subset which is myeloid on the basis of cell surface phenotype, but dendritic-like on the basis of cell surface marker expression and antigen presenting function, has been tentatively labelled "L-DC." Transcriptome analysis has now been employed to determine the lineage relationship of this cell type with known splenic cDC and monocyte subsets. Principal components analysis showed separation of "L-DC" and monocytes from cDC subsets in the second principal component. Hierarchical clustering then indicated a close lineage relationship between this novel subset, resident monocytes and inflammatory monocytes. Resident monocytes were the most closely aligned, with no genes specifically expressed by the novel subset. This subset, however, showed upregulation of genes reflecting both dendritic and myeloid lineages, with strong upregulation of several genes, particularly CD300e. While resident monocytes were found to be dependent on Toll-like receptor signalling for development and were reduced in number in Myd88-/- and Trif-/- mutant mice, both the novel subset and inflammatory monocytes were unaffected. Here, we describe a novel myeloid cell type closely aligned with resident monocytes in terms of lineage but distinct in terms of development and functional capacity.

Improved cryopreservation of spermatozoa using vitrification: comparison of cryoprotectants and a novel device for long-term storage.

STUDY QUESTION: Does cryoprotection of spermatozoa using a vitrification protocol with improved cryoprotective agents and a novel device for large storage lead to better outcomes than conventional slow freezing? Vitrification of human sperm using sucrose and dextran-based cryoprotectant (CPA4) with a new vitrification device resulted in significantly better sperm motility and progressive motility and improved DNA integrity with lower DNA fragmentation compared with conventional slow freezing. WHAT IS KNOWN ALREADY: A major limitation to clinical implementation of vitrification is the right balance between the volume of spermatozoa suspension cryopreserved and a standardised use of CPAs for survival of spermatozoa. STUDY DESIGN, SIZE, DURATION: This was a control versus current clinical practice study using 30 fresh human semen samples to carry out the different cryoprotectant analyses followed by a further 23 semen samples to test the novel vitrification protocol. PARTICIPANTS/MATERIALS, SETTING, METHODS: All human specimens fulfilled the following criteria: > 5 million spermatozoa/mL, > 20% total motility, ≥ 1.8 mL in volume, with all participants falling within the age range of 25-45 inclusively. The concentration, progressive motility, non-progressive motility, immotility, and various morphokinetic variables including DAP, DCL, DSL, LIN, and STR were then determined using the IVOS II™ Clinical CASA system (Hamilton Thorne, Beverly, MA, USA) on the basis of the 5th Edition of WHO Laboratory Manual for the Examination and Processing of Human Semen. MAIN RESULTS AND THE ROLE OF CHANCE: Among the 6 cryopreservation methods in this study, vitrification with the funnel-shaped device using CPA4 best preserves the 13 sperm parameters evaluated by CASA system. Conventional slow freezing and vitrification with the device using seminal plasma also protects sperm quality, but the overall motilities are statistically lower in comparison with the novel vitrification approach with cryoprotective media using the device. DNA fragmentation significantly increased after cryopreservation through the method of conventional slow freezing (p = 0.07). There was no significant difference in DNA fragmentation between fresh control and vitrification (p = 1.000). LIMITATIONS, REASONS FOR CAUTION: Extensive training is required to minimise the human error in using the vitrification device to perform cryopreservation. Each operator can only handle one sample at a time with device vitrification, whereas several samples can be processed without the need for special training with conventional slow freezing. WIDER IMPLICATIONS OF THE FINDINGS: The presented study shows that a new vitrification method could improve survival sperm rate. Human sperm vitrification using our novel protocol gives higher motility and progression and lower percentage of DNA fragmentation than conventional slow freezing. Our findings indicate that this method could supersede the current clinical practice in particular for patients with oligospermia as it reduces osmotic damage, time, and cost.

In assisted reproduction by IVF or ICSI, the rate at which embryos develop to the blastocyst stage is influenced by the fertilization method used: a split IVF/ICSI study.

PURPOSE: To compare in vitro fertilization (IVF) with intracytoplasmic sperm injection (ICSI) in regard to post-fertilization development and outcome with the purpose of ascertaining the most effective fertilization method for assisted reproduction. METHODS: A retrospective cohort study of 136 split IVF/ICSI cycles (where sibling oocytes are fertilized by two different methods using the same sperm sample). RESULTS: IVF-derived embryos developed to the blastocyst stage at a significantly faster rate than ICSI-derived embryos. There was no significant difference in fertilization or livebirth rates between the two fertilization methods. CONCLUSIONS: For patients with sperm progressive motility ≥ 1.0 × 106/ml (who usually constitute the majority of patients), no significant difference between the two fertilization methods was found in regard to fertilization rate or livebirth rate. Remaining factors influencing choice between the two methods appear to be restricted to convenience, financial considerations and concern with regard to possible perpetuation of genetically linked infertility to future generations.

MCSF drives regulatory DC development in stromal co-cultures supporting hematopoiesis.

BACKGROUND: Splenic stroma overlaid with hematopoietic progenitors supports in vitro hematopoiesis with production of dendritic-like cells. Co-cultures of murine lineage-depleted bone marrow over the 5G3 stromal line produce two populations of cells, characterised as CD11b+CD11c+MHC-II- dendritic-like 'L-DC', and CD11b+CD11c+MHC-II+ cells, resembling conventional dendritic cells (cDC). To date, the functional capacity of these two subsets has not been clearly distinguished. RESULTS: Here we show both the L-DC and cDC-like subsets can be activated and induce proliferation of OT-I CD8+ T cells, being strong inducers of IL-2 and IFN-γ production. Both subsets lack ability to induce proliferation of OT-II CD4+ T cells. The cDC-like population is shown here to resemble regulatory DC in that they induce FoxP3 expression and IL-10 production in OT-II CD4+ T cells, in line with their function as regulatory DC. L-DC did not activate or induce the proliferation of CD4+ T cells and did not induce FoxP3 expression in CD4+ T cells. L-DC can be distinguished from cDC-like cells through their superior endocytic capacity and expression of 4-1BBL, F4/80 and Sirp-α. A comparison of gene expression by the two subsets was consistent with L-DC having an activated or immunostimulatory DC phenotype, while cDC-like cells reflect myeloid dendritic cells with inflammatory and suppressive properties, also consistent with functional characteristics as regulatory DC. When a Transwell membrane was used to prevent hematopoietic cell contact with stroma, only cDC-like cells and not L-DC were produced, and cell production was dependent on M-CSF production by stroma. CONCLUSION: Co-cultures of hematopoietic progenitors over splenic stroma produce two distinct subsets of dendritic-like cells. These are here distinguished phenotypically and through gene expression differences. While both resemble DC, there are functionally distinct. L-DC activate CD8+ but not CD4+ T cells, while the cDC-like population induce regulatory T cells, so reflecting regulatory DC. The latter can be enriched through Transwell co-cultures with cell production dependent on M-CSF.