ART in Europe, 2019 : results generated from European registries by ESHRE

Study question: What are the data and trends on ART and IUI cycle numbers and their outcomes, and on fertility preservation (FP) interventions, reported in 2019 as compared to previous years? Summary answer: The 23rd ESHRE report highlights the rising ART treatment cycles and children born, alongside a decline in twin deliveries owing to decreasing multiple embryo transfers; fresh IVF or ICSI cycles exhibited higher delivery rates, whereas frozen embryo transfers (FET) showed higher pregnancy rates (PRs), and reported IUI cycles decreased while maintaining stable outcomes. What is known already: ART aggregated data generated by national registries, clinics, or professional societies have been gathered and analyzed by the European IVF-Monitoring (EIM) Consortium since 1997 and reported in a total of 22 manuscripts published in Human Reproduction and Human Reproduction Open. Study design, size, duration: Data on medically assisted reproduction (MAR) from European countries are collected by EIM for ESHRE each year. The data on treatment cycles performed between 1 January and 31 December 2019 were provided by either national registries or registries based on initiatives of medical associations and scientific organizations or committed persons in one of the 44 countries that are members of the EIM Consortium. Participants/materials, setting, methods: Overall, 1487 clinics offering ART services in 40 countries reported, for the second time, a total of more than 1 million (1 077 813) treatment cycles, including 160 782 with IVF, 427 980 with ICSI, 335 744 with FET, 64 089 with preimplantation genetic testing (PGT), 82 373 with egg donation (ED), 546 with IVM of oocytes, and 6299 cycles with frozen oocyte replacement (FOR). A total of 1169 institutions reported data on IUI cycles using either husband/partner's semen (IUI-H; n = 147 711) or donor semen (IUI-D; n = 51 651) in 33 and 24 countries, respectively. Eighteen countries reported 24 139 interventions in pre- and post-pubertal patients for FP, including oocyte, ovarian tissue, semen, and testicular tissue banking. Main results and the role of chance: In 21 countries (21 in 2018) in which all ART clinics reported to the registry 476 760 treatment cycles were registered for a total population of approximately 300 million inhabitants, allowing the best estimate of a mean of 1581 cycles performed per million inhabitants (range: 437-3621). Among the reporting countries, for IVF the clinical PRs per aspiration slightly decreased while they remained similar per transfer compared to 2018 (21.8% and 34.6% versus 25.5% and 34.1%, respectively). In ICSI, the corresponding PRs showed similar trends compared to 2018 (20.2% and 33.5%, versus 22.5% and 32.1%) When freeze-all cycles were not considered for the calculations, the clinical PRs per aspiration were 28.5% (28.8% in 2018) and 26.2% (27.3% in 2018) for IVF and ICSI, respectively. After FET with embryos originating from own eggs, the PR per thawing was at 35.1% (versus 33.4% in 2018), and with embryos originating from donated eggs at 43.0% (41.8% in 2018). After ED, the PR per fresh embryo transfer was 50.5% (49.6% in 2018) and per FOR 44.8% (44.9% in 2018). In IVF and ICSI together, the trend toward the transfer of fewer embryos continues with the transfer of 1, 2, 3, and ≥4 embryos in 55.4%, 39.9%, 2.6%, and 0.2% of all treatments, respectively (corresponding to 50.7%, 45.1%, 3.9%, and 0.3% in 2018). This resulted in a reduced proportion of twin delivery rates (DRs) of 11.9% (12.4% in 2018) and a similar triplet DR of 0.3%. Treatments with FET in 2019 resulted in twin and triplet DR of 8.9% and 0.1%, respectively (versus 9.4% and 0.1% in 2018). After IUI, the DRs remained similar at 8.7% after IUI-H (8.8% in 2018) and at 12.1% after IUI-D (12.6% in 2018). Twin and triplet DRs after IUI-H were 8.7% and 0.4% (in 2018: 8.4% and 0.3%) and 6.2% and 0.2% after IUI-D (in 2018: 6.4% and 0.2%), respectively. Eighteen countries (16 in 2018) provided data on FP in a total number of 24 139 interventions (20 994 in 2018). Cryopreservation of ejaculated sperm (n = 11 592 versus n = 10 503 in 2018) and cryopreservation of oocytes (n = 10 784 versus n = 9123 in 2018) were most frequently reported. Limitations, reasons for caution: Caution with the interpretation of results should remain as data collection systems and completeness of reporting vary among European countries. Some countries were unable to deliver data about the number of initiated cycles and/or deliveries. Wider implications of the findings: The 23rd ESHRE data collection on ART, IUI, and FP interventions shows a continuous increase of reported treatment numbers and MAR-derived livebirths in Europe. Although it is the largest data collection on MAR in Europe, further efforts toward optimization of both the collection and the reporting, from the perspective of improving surveillance and vigilance in the field of reproductive medicine, are awaited. Study funding/competing interest(s): The study has received no external funding and all costs are covered by ESHRE. There are no competing interests. Keywords: ICSI; IUI; IVF; data collection; egg donation; fertility preservation; frozen embryo transfer; registry; surveillance; vigilance.peer-reviewe

Plasmodium falciparum histidine-rich protein II causes vascular leakage and exacerbates experimental cerebral malaria in mice.

A devastating complication of Plasmodium falciparum infection is cerebral malaria, in which vascular leakage and cerebral swelling lead to coma and often death. P. falciparum produces a protein called histidine-rich protein II (HRPII) that accumulates to high levels in the bloodstream of patients and serves as a diagnostic and prognostic marker for falciparum malaria. Using a human cerebral microvascular endothelial barrier model, we previously found that HRPII activates the endothelial cell inflammasome, resulting in decreased integrity of tight junctions and increased endothelial barrier permeability. Here, we report that intravenous administration of HRPII induced blood-brain barrier leakage in uninfected mice. Furthermore, HRPII infusion in P. berghei-infected mice increased early mortality from experimental cerebral malaria. These data support the hypothesis that HRPII is a virulence factor that contributes to cerebral malaria by compromising the integrity of the blood-brain barrier

Palmitoyl transferases have critical roles in the development of mosquito and liver stages of Plasmodium.

As the Plasmodium parasite transitions between mammalian and mosquito host, it has to adjust quickly to new environments. Palmitoylation, a reversible and dynamic lipid post-translational modification, plays a central role in regulating this process and has been implicated with functions for parasite morphology, motility and host cell invasion. While proteins associated with the gliding motility machinery have been described to be palmitoylated, no palmitoyl transferase responsible for regulating gliding motility has previously been identified. Here, we characterize two palmityol transferases with gene tagging and gene deletion approaches. We identify DHHC3, a palmitoyl transferase, as a mediator of ookinete development, with a crucial role for gliding motility in ookinetes and sporozoites, and we co-localize the protein with a marker for the inner membrane complex in the ookinete stage. Ookinetes and sporozoites lacking DHHC3 are impaired in gliding motility and exhibit a strong phenotype in vivo; with ookinetes being significantly less infectious to their mosquito host and sporozoites being non-infectious to mice. Importantly, genetic complementation of the DHHC3-ko parasite completely restored virulence. We generated parasites lacking both DHHC3, as well as the palmitoyl transferase DHHC9, and found an enhanced phenotype for these double knockout parasites, allowing insights into the functional overlap and compensational nature of the large family of PbDHHCs. These findings contribute to our understanding of the organization and mechanism of the gliding motility machinery, which as is becoming increasingly clear, is mediated by palmitoylation

Antibody-Mediated Protection against Plasmodium Sporozoites Begins at the Dermal Inoculation Site

Studies in experimental animal models and humans have shown that antibodies against Plasmodium sporozoites abolish parasite infectivity and provide sterile immunity. While it is well documented that these antibodies can be induced after immunization with attenuated parasites or subunit vaccines, the mechanisms by and location in which they neutralize parasites have not been fully elucidated. Here, we report studies indicating that these antibodies display a significant portion of their protective effect in the skin after injection of sporozoites and that one mechanism by which they work is by impairing sporozoite motility, thus diminishing their ability to reach blood vessels. These results suggest that immune protection against malaria begins at the earliest stages of parasite infection and emphasize the need of performing parasite challenge in the skin for the evaluation of protective immunity.Plasmodium sporozoites are injected into the skin as mosquitoes probe for blood. From here, they migrate through the dermis to find blood vessels which they enter in order to be rapidly carried to the liver, where they invade hepatocytes and develop into the next life cycle stage, the exoerythrocytic stage. Once sporozoites enter the blood circulation, they are found in hepatocytes within minutes. In contrast, sporozoite exit from the inoculation site resembles a slow trickle and occurs over several hours. Thus, sporozoites spend the majority of their extracellular time at the inoculation site, raising the hypothesis that this is when the malarial parasite is most vulnerable to antibody-mediated destruction. Here, we investigate this hypothesis and demonstrate that the neutralizing capacity of circulating antibodies is greater at the inoculation site than in the blood circulation. Furthermore, these antibodies are working, at least in part, by impacting sporozoite motility at the inoculation site. Using actively and passively immunized mice, we found that most parasites are either immobilized at the site of injection or display reduced motility, particularly in their net displacement. We also found that antibodies severely impair the entry of sporozoites into the bloodstream. Overall, our data suggest that antibodies targeting the migratory sporozoite exert a large proportion of their protective effect at the inoculation site

HRPII causes vascular leakage <i>in vivo</i>.

<p><b>(A)</b> Scheme of experimental design. Two doses of HRPII or BSA (200 μg) were injected into 4-week old female C57Bl/6 mice at 0 and 24 hours. At 48 hours, fluorescein levels in the cortex (<b>B</b>) and cerebellum (<b>C</b>) of the mice was measured. HRPII treatment was significantly different from control by two-tailed t-test, p = 0.01 (cortex) and p = 0.02 (cerebellum). Data are mean values +/-SEM for 8–16 mice per group accumulated over 3 independent experiments.</p

HRPII-mediated vascular leakag<i>e</i> is blocked by antibody to IL-1β.

<p>Mice were infused as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177142#pone.0177142.g001" target="_blank">Fig 1</a>, with HRPII plus an isotype antibody (Iso, positive control), HRPII plus anti-IL-1β antibody (experimental condition) or anti-IL-1β antibody alone (negative control). Untreated mice (no HRPII, no antibody) served as a further control. Vascular leakage in mice infused with HRPII/isotype is statistically significantly different from mice infused with HRPII/ anti-IL-1β, p = 0.01 (cerebellum) and p = 0.01 (cortex), by two-tailed t-test; p = 0.003 (cerebellum) and p = 0.06 (cortex) by ANOVA one-way variance with significance between HRPII/isotype and HRPII/ anti-IL-1β. Data are mean values +/-SEM for 6–12 mice per group accumulated over 3 independent experiments.</p

HRPII reduces survival time in an experimental cerebral malaria model.

<p>(<b>A</b>) Survival curves of 4-week old female mice infused with 50 μg of BSA or HRPII prior to infection with <i>P</i>. <i>berghei</i> ANKA (10⁵ parasites). Shown are the means for n = 24 to 27 mice pooled from four independent experiments. Curves are significantly different, p = 0.03, by the log-rank (Mantel-Cox) test. Mean time to death for HRPII = 11.5 days and for BSA = 16 days, p = 0.018, by two tailed t-test. (<b>B</b>) Mice displaying cerebral malaria-like symptoms died at low parasitemia by day 10, yet parasitemias between HRPII-infused mice and controls were closely matched on each day. Representative data from one of three experiments shown in panel A, 10 mice per group.</p

HRPII causes vascular leakage <i>in vivo</i>.

<p><b>(A)</b> Scheme of experimental design. Two doses of HRPII or BSA (200 μg) were injected into 4-week old female C57Bl/6 mice at 0 and 24 hours. At 48 hours, fluorescein levels in the cortex (<b>B</b>) and cerebellum (<b>C</b>) of the mice was measured. HRPII treatment was significantly different from control by two-tailed t-test, p = 0.01 (cortex) and p = 0.02 (cerebellum). Data are mean values +/-SEM for 8–16 mice per group accumulated over 3 independent experiments.</p

Pantagruel

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Electron micrographs illustrating the process of cell death observed in oocysts of the ΔRep and ΔNΔRep mutants.

<p><b>A.</b> Low power of a ΔNΔRep oocyst with a degenerating, undifferentiated central cytoplasmic mass. Bar is 10 µm. <b>B.</b> Detail from the degeneration of a ΔNΔRep oocyst similar to that in <b>A</b> showing a dilated nuclear membrane containing a number of nuclei (N) exhibiting peripheral chromatin condensation. Bar is 100 nm. <b>C.</b> Low power of a ΔRep oocyst in which sporozoite formation had occurred but now exhibited features of cell degeneration. Bar is 10 µm. <b>D.</b> Detail of the nuclei observed in an intact ΔNΔRep oocyst showing the absence of any peripheral nuclear condensation. Bar is 100 nm. <b>E.</b> Longitudinal section through a sporozoite showing the nucleus with peripheral chromatin condensation and dilatation of the nuclear membranes. N – nucleus. Bar is 500 nm. <b>F.</b> Low power of a ΔRep oocyst in which there is a cross section of a central mass of degenerating sporozoites (S). Bar is 10 µm. <b>G.</b> Histogram comparing the relative number of immature mature and degenerate oocysts at two time points (12–14 days and 18–21 days) for WT, ΔRep and ΔNΔRep oocysts. (Based on EM examination of multiple midguts from multiple experiments – number of oocysts evaluated: 405 wild type; 236 ΔRep mutant; 165 ΔNΔRep mutant).</p