Engineered phage with antibacterial CRISPR-Cas selectively reduce E. coli burden in mice.

Antibiotic treatments have detrimental effects on the microbiome and lead to antibiotic resistance. To develop a phage therapy against a diverse range of clinically relevant Escherichia coli, we screened a library of 162 wild-type (WT) phages, identifying eight phages with broad coverage of E. coli, complementary binding to bacterial surface receptors, and the capability to stably carry inserted cargo. Selected phages were engineered with tail fibers and CRISPR-Cas machinery to specifically target E. coli. We show that engineered phages target bacteria in biofilms, reduce the emergence of phage-tolerant E. coli and out-compete their ancestral WT phages in coculture experiments. A combination of the four most complementary bacteriophages, called SNIPR001, is well tolerated in both mouse models and minipigs and reduces E. coli load in the mouse gut better than its constituent components separately. SNIPR001 is in clinical development to selectively kill E. coli, which may cause fatal infections in hematological cancer patients.

Oocyte maturation. Basic and clinical aspects of in vitro maturation (IVM) with special emphasis of the role of FF-MAS.

In Vitro maturation (IVM) of human oocytes is an emerging infertility treatment with great promise. To be successful this future assisted reproductive technology must entail both nuclear and cytoplasmic maturation of the oocytes and give rise to human embryos that have the same developmental potential as embryos resulting from the golden standard of human IVF. The aspiration of immature oocytes from small to medium size antral follicles followed by their maturation In Vitro present an attractive alternative to the hormonal stimulation of patients in In Vitro fertilization (IVF) treatment, since administration of exogenous hormones is a costly treatment and may cause severe health problems. Of the long list of side effect and health concern ovarian hyper-stimulation syndrome (OHSS) is by far the most severe although long term effect on cancer prevalence is another concern. Another potential group of patients that could benefit from future IVM treatments are the young women undergoing anticancer therapy (radiation- or chemotherapy). Thus, ovarian and oocyte cryopreservation techniques are emerging, however such treatments can only be fully realized when IVM becomes an efficient means of obtaining healthy birth. At present, the In Vitro maturation techniques are highly successful in mice, variable successful in domestic species and still regarded experimental in the human clinic due to suboptimal fertilization rates and embryo quality. This review discusses comparative studies of the processes of oocyte maturation In Vivo and In Vitro, in various mammalian species including human. Among the substances that have been reported to influence oocyte maturation there is an interesting endogenous signaling molecule: FF-MAS (4,4-dimethyl-5 alpha-cholest-8,14,24-trien-3 beta -ol), an intermediate in the cholesterol biosynthetic pathway present in all cells. This review gives special focus to FF-MAS, the effect seen in animal and human studies so far and its potential use in treatment of human infertility is being discussed, including both the safety and efficacy issues that need to be addressed. It is being reviewed how FF-MAS and related MAS analogues by our group and other scientific groups have been observed to mediate a dose-dependant response on both the nuclear maturation and especially the cytoplasmic maturation during oocyte maturation In Vitro thus giving rise to pre-embryos of higher developmental potential. Studies are reviewed regarding the family of meiosis activating sterols, its In Vivo regulation by gonadotropins (especially LH) and suggestions to the signaling pathways as the putative MAS receptor eliciting the important cytoplasmic maturation signaling cascade that involves mos/MAP kinase. The pharmacological effect of synthetic FF-MAS has been observed in various models and species, including murine, porcine and humane oocytes. Finally, the chromosome status of IVM human oocytes has been the focus of a large prospective clinical trial, documenting that FF-MAS acting on human oocytes during In Vitro maturation presents a safe procedure evaluated on numerical chromosome aberration rates in metaphase-II oocytes. In conclusion the In Vitro maturation of human oocytes is already now a valuable clinical treatment alternative for a subset of infertile patients, especially the Polycystic Ovarian Syndrome (PCOS) patients. IVM has the promise of being tomorrow's gold standard in treatment of human infertility if most of the important components of oocyte maturation are understood and can be adequately addressed In Vitro. Considering the present low frequency of successful fertilization and pre-implantation development following In Vitro maturation of human oocytes, the addition of FF-MAS or MAS analogues to the maturation medium to improve the cytoplasmic maturation and to yield higher quality pre-embryos may prove highly beneficial.

Effect of prematuration, meiosis activating sterol and enriched maturation medium on the nuclear maturation and competence to development of calf oocytes.

New strategies were proposed to improve the developmental competence of calf oocytes through in vitro technologies. Cumulus-oocyte complexes were first prematured for 24 h in the presence of meiosis inhibitors. Both Roscovitine alone (50 microM) or in combination with Butyrolactone-I (12.5 microM Rosco+6.25 microM BL-I) prevented the progression of meiosis. Their effect on nuclear maturation was reversible after a further 17 or 24 h maturation step. However, a dramatic decrease in embryo development was observed after fertilization (abattoir oocytes: 4-9% blastocyst rate versus 14-17% for control embryos). Similar results were obtained with oocytes collected by Ovum Pick Up from living donors. No pregnancy was obtained after single transfer of two blastocysts obtained from prematured oocytes (0/2 versus 4/12 for control embryos). Adding low concentrations (1, 3 or 10 microM) of follicular fluid-meiosis activating sterol (FF-MAS) during the maturation step had a beneficial effect on nuclear maturation (73-86% metaphase II versus 58% for control oocytes). However, subsequent embryo development was not improved. Enriching the maturation medium, namely with hormones, growth factors and precursors of glutathione, induced a sixfold increase in glutathione in the oocyte and had a beneficial effect on embryo development (38% increase in blastocyst rate). In conclusion, in opposition to the results reported with adult oocytes, prematuring calf oocytes had a negative impact on their developmental potential. Although FF-MAS improved nuclear maturation, its addition in the maturation medium did not increase embryo development. However, enriching the maturation medium had a positive effect on embryo development, indicating that cytoplasmic maturation was improved.

Effect of rescuing donated immature human oocytes derived after FSH/hCG stimulation following in vitro culture with or without Follicular Fluid Meiosis Activating Sterol (FF-MAS)--an embryo chromosomal and morphological analysis.

PURPOSE: Studies in mice and humans have shown that Follicular Fluid - Meiosis Activating Sterol (FF-MAS) induces meiotic maturation of immature oocytes in vitro. A multicenter, prospectively randomised study evaluated chromosomal status of embryos from FSH/hCG primed human immature oocytes, cultured with or without FF-MAS. METHODS: Denuded immature oocytes (n=365) were randomly allocated into inert control, FF-MAS 5 microM or 20 microM. Seventy +/-2 hours after ICSI on matured oocytes, all cleaved embryos were fixed for fluorescence in situ hybridisation analysis. RESULTS: Only 15% of oocytes resulted in cleaved embryos. GV oocytes matured at significantly lower rates (14% and 7%) in the two FF-MAS groups compared to the inert control group (47%). High rates of chromosomal abnormalities were found in all groups. CONCLUSION: Immature oocytes showed poor development with high rates of embryo chromosomal abnormalities. Exposure to FF-MAS in the concentrations, duration and/or formulation used in this study did not improve the results.

Principal findings from a multicenter trial investigating the safety of follicular-fluid meiosis-activating sterol for in vitro maturation of human cumulus-enclosed oocytes.

OBJECTIVE: To evaluate the safety of applying follicular-fluid meiosis-activating sterol (FF-MAS) in vitro to immature human oocytes. DESIGN: Phase I bicenter, randomized, parallel-group, controlled, partially blinded trial. SETTING: Third-level referral academic centers, including reproductive biology and genetics laboratories. PATIENTS: Endocrinologically normal women with a medical indication for IVF or intracytoplasmic sperm injection, or healthy volunteers. INTERVENTION(S): Subjects were randomized at a ratio 1 to 6 into either conventional GnRH-agonist and recombinant FSH stimulation (IVO) for oocyte retrieval, or minimally stimulated in vitro maturation (IVM) with the use of recombinant FSH. Retrieved immature oocyte cumulus complexes were cultured for 30 or 36 hours in one of six IVM culture conditions containing FF-MAS (range, 0.1-20 microM). Polar body-extruded oocytes from the IVO and IVM groups were processed for chromosomal analysis. MAIN OUTCOME MEASURE(S): The primary endpoint was the incidence of metaphase II stage oocytes with numeric chromosomal abnormalities, using full (spectral karyotyping) or partial (fluorescent in situ hybridization with seven probes) karyotyping or Giemsa count. A secondary objective was to document the frequency of metaphase II oocytes after IVM with FF-MAS supplements. RESULT(S): Oocyte cumulus complexes obtained from the IVO (mean, 8.9) and IVM (mean, 6.2) groups had equal maturation rates. Compared to IVO, exposure of germinal-vesicle oocytes for a maturation period of 30 hours did not increase aneuploidy. An exposure period of 36 hours doubled the aneuploidy rate, but this was significant only for the 20-muM dose of FF-MAS. CONCLUSION: Inclusion of 1-10 microM FF-MAS in a 30-hour IVM protocol is safe.

The effect of FF-MAS on porcine cumulus-oocyte complex maturation, fertilization and pronucleus formation in vitro.

Follicular fluid meiosis-activating sterol (FF-MAS) has been isolated from the follicular fluid (FF) of several species including man. FF-MAS increases the quality of in vitro oocyte maturation, and thus the developmental potential of oocytes exposed to FF-MAS during in vitro maturation is improved. The aim of the present study was to investigate the effects of FF-MAS on porcine oocyte maturation and pronucleus formation in vitro. Porcine cumulus-oocyte complexes (COCs) were isolated from abattoir ovaries and in vitro matured for 48 h in NCSU 37 medium supplemented with 1 mg/l cysteine, 10 ng/ml epidermal growth factor and 50 microM 2-mercaptoethanol with or without 10% porcine follicular fluid (pFF). For the first 22 h, 1 mM db-cAMP and 10 I.E PMSG/hCG was added. The medium was supplemented with 1 microM, 3 microM, 10 microM, 30 microM or 100 microM FF-MAS dissolved in ethanol. After maturation the COCs were denuded mechanically using a fine glass pipette under constant pH and in vitro fertilized with fresh semen (5 x 10(5) spermatozoa/ml). The presumptive zygotes were evaluated 18 h after fertilization. The addition of pFF increased the monospermic as well as the polyspermic penetration of oocytes. In the absence of pFF, the addition of FF-MAS decreased the polyspermic penetration rate, whereas FF-MAS in combination with pFF decreased monospermic and increased polyspermic penetration. The degeneration rate of ova decreased in the presence of FF-MAS irrespective of the presence or absence of pFF. In the absence of pFF, FF-MAS at 3-10 microM increased the number of zygotes with advanced maternal pronuclear stages. In supraphysiological doses, i.e. 30-100 microM, FF-MAS dose-dependently and reversibly inhibited nuclear maturation in the absence of pFF.

Impact of follicular-fluid meiosis-activating sterol in an albumin-based formulation on the incidence of human pre-embryos with chromosome abnormalities.

OBJECTIVE: To evaluate the effect of adding follicular-fluid meiosis-activating sterol (FF-MAS) in a novel 0.2% recombinant human albumin-based formulation to cumulus-enclosed oocytes on chromosomal status and development of pre-embryos. DESIGN: Multicenter, prospective, randomized, open (double-blind for vehicle and FF-MAS groups), four parallel groups, controlled trial. SETTING: Four public IVF clinics in Denmark. PATIENT(S): Two hundred eighteen women undergoing IVF donated 483 oocytes. INTERVENTION(S): Follicle-stimulating hormone/hCG-primed cumulus-enclosed oocytes randomized to 4 hours of exposure to medium with 1 or 10 micromol/L of FF-MAS dissolved in 0.2% recombinant human albumin, medium with 0.2% recombinant human albumin (vehicle control), or medium alone (control) before insemination. MAIN OUTCOME MEASURE(S): Primary endpoint: incidence of human pre-embryos with chromosomal abnormalities. Secondary endpoint: fertilization rate, cleavage rate, and pre-embryo quality assessed after 68 hours of culture. RESULT(S): At pre-embryo level, the overall abnormality rates in the control, vehicle control, and 1- and 10-micromol/L FF-MAS groups were 53%, 39%, 42%, 53%, respectively, and at blastomere level 49%, 44%, 44%, and 48%, respectively. After 20 and 26 hours, the fertilization rates were between 67% and 71% in all groups. No differences in the cleavage rates were observed. CONCLUSION(S): The concentrations of FF-MAS in a novel 0.2% recombinant human albumin-based formulation of FF-MAS did not increase the risk of chromosomal abnormalities in pre-embryos or blastomeres. No statistically significant differences in fertilization rate, cleavage rate, or number of good quality pre-embryos were found among the four groups.

Meiosis activating sterols derived from diosgenin.

Continuing research based on the meiosis activation properties of the endogenous sterol FF-MAS is reported. The synthesis and SAR of 16- and 26-substituted sterols are described, utilising diosgenin as starting material. Selected sterols were tested for their ability to induce oocyte maturation in hypoxanthine arrested mouse oocytes in vitro.

Meiosis-activating sterol promotes the metaphase I to metaphase II transition and preimplantation developmental competence of mouse oocytes maturing in vitro.

The objective of this study was to determine the effects of a sterol found in ovarian follicular fluid, known as meiosis-activating sterol (FF-MAS), on the maturation of mouse oocytes in vitro. Possible effects of FF-MAS in promoting the metaphase I (MI) to metaphase II (MII) transition (nuclear maturation) and the competence of oocytes to complete preimplantation embryo development to the blastocyst stage (cytoplasmic maturation) were assessed. Cumulus cell-enclosed oocytes that were compromised in their ability to undergo nuclear maturation and subsequent development because of the age or genotype of the female were isolated at the germinal vesicle stage and matured in vitro using media supplemented with 0 to 20 microM FF-MAS. Oocytes that progressed to MII were inseminated in vitro, and the percentages developing to the 2-cell and blastocyst stages were determined. The sterol was omitted from the media used for oocyte insemination or preimplantation development. FF-MAS promoted a significantly higher percentage of oocytes in all groups to progress to MII in vitro. Moreover, FF-MAS treatment of oocytes maturing in vitro dramatically increased the competence of all but one of the groups of oocytes to complete preimplantation development. Therefore, FF-MAS improved mouse oocyte quality by promoting both nuclear and cytoplasmic maturation in vitro.

Chromosomal abnormality rate in human pre-embryos derived from in vitro fertilization cycles cultured in the presence of Follicular-Fluid Meiosis Activating Sterol (FF-MAS).

BACKGROUND: The objective of the study was to investigate the effect of Follicular-Fluid Meiosis Activating Sterol (FF-MAS) when added to the culture media on the incidence of chromosomal abnormalities and pre-embryo development in human pre-embryos. METHODS: 243 women undergoing IVF/ICSI treatment donated 353 oocytes in a multicentre, prospective, randomized, double blind, four-arm, controlled trial performed at Danish and Swedish public and private IVF centers. Metaphase II oocytes were randomly assigned to: FF-MAS 5 microM, FF-MAS 20 microM, ethanol 0.2% (vehicle control) or water for injection (inert control). The exposure regimen of FF-MAS to the human oocytes was 4 h prior to fertilization by ICSI and 20 h exposure post ICSI. The primary endpoint was the incidence of numerical chromosomal abnormalities. Secondary endpoints were cleavage rate and pre-embryo quality. RESULT: On the pre-embryo level, no significant differences in chromosomal abnormality rate were observed among the four groups. However, the percentage of uniformly normal pre-embryos was significantly lower in the pooled FF-MAS group (5 microM: 12% and 20 microM: 17%) than in the pooled control group (inert control 32% and vehicle control 42%). A high level of mosaicism (41-60%) was found in all groups. At the blastomere level, the percentage of blastomeres categorized as normal was significantly lower in the FF-MAS 5 microM group (41%) and the FF-MAS 20 microM (29%) group versus the inert (52%) and the vehicle (61%) groups. Significantly reduced cleavage and good quality pre-embryo rates were found in both FF-MAS groups. CONCLUSION: FF-MAS increased the rate of aneuploidy and had detrimental effects on cleavage and pre-embryo development, when exposed both before and after fertilization.

Physiology of meiosis-activating sterol: endogenous formation and mode of action.

BACKGROUND: In the context of mammalian oocyte maturation, it has been suggested that intermediates of cholesterol biosynthesis may represent the physiological signal that instructs the oocyte to reinitiate meiosis. METHODS: Endogenous levels of follicular fluid meiosis-activating sterol (FF-MAS) were monitored in rabbit ovarian tissue, and the influence of exogenous gonadotrophins on sterol formation was assessed. The involvement of cAMP in FF-MAS-induced versus spontaneous oocyte maturation in vitro in mice was also investigated, as was the direct microinjection of FF-MAS into mouse oocytes. RESULTS: Levels of FF-MAS in rabbit ovaries were significantly elevated 1 h after hCG/LH induction and remained so for 4 and 12 h after induction. In naked oocytes undergoing spontaneous maturation, a significant decrease in cAMP was detected after 30 min of culture. However, FF-MAS-mediated induction of oocyte maturation in hypoxanthine-arrested naked oocytes was not associated with any detectable decrease in intracellular cAMP levels. Microinjected FF-MAS failed to induce any noticeable meiosis. CONCLUSIONS: A rapid increase in FF-MAS level occurred in vivo in the rabbit ovary in response to LH, and clear differences were seen in the cAMP pattern during spontaneous and induced oocyte maturation in mice.