Transcriptomic integrity of human oocytes used in ARTs: technical and intrinsic factor effects.

BACKGROUND: Millions of children have been born throughout the world thanks to ARTs, the harmlessness of which has not yet been fully demonstrated. For years, efforts to evaluate the specific effects of ART have focused on the embryo; however, it is the oocyte quality that mainly dictates first and foremost the developmental potential of the future embryo. Ovarian stimulation, cryopreservation, and IVM are sometimes necessary steps to obtain a mature oocyte, but they could alter the appropriate expression of the oocyte genome. Additionally, it is likely that female infertility, environmental factors, and lifestyle have a significant influence on oocyte transcriptomic quality, which may interfere with the outcome of an ART attempt. OBJECTIVE AND RATIONALE: The objective of this review is to identify transcriptomic changes in the human oocyte caused by interventions specific to ART but also intrinsic factors such as age, reproductive health issues, and lifestyle. We also provide recommendations for future good practices to be conducted when attempting ART. SEARCH METHODS: An in-depth literature search was performed on PubMed to identify studies assessing the human oocyte transcriptome following ART interventions, or in the context of maternal aging, suboptimal lifestyle, or reproductive health issues. OUTCOMES: ART success is susceptible to external factors, maternal aging, lifestyle factors (smoking, BMI), and infertility due to endometriosis or polycystic ovary syndrome. Indeed, all of these are likely to increase oxidative stress and alter mitochondrial processes in the foreground. Concerning ART techniques themselves, there is evidence that different ovarian stimulation regimens shape the oocyte transcriptome. The perturbation of processes related to the mitochondrion, oxidative phosphorylation, and metabolism is observed with IVM. Cryopreservation might dysregulate genes belonging to transcriptional regulation, ubiquitination, cell cycle, and oocyte growth pathways. For other ART laboratory factors such as temperature, oxygen tension, air pollution, and light, the evidence remains scarce. Focusing on genes involved in chromatin-based processes such as DNA methylation, heterochromatin modulation, histone modification, and chromatin remodeling complexes, but also genomic imprinting, we observed systematic dysregulation of such genes either after ART intervention or lifestyle exposure, as well as due to internal factors such as maternal aging and reproductive diseases. Alteration in the expression of such epigenetic regulators may be a common mechanism linked to adverse oocyte environments, explaining global transcriptomic modifications. WIDER IMPLICATIONS: Many IVF factors and additional external factors have the potential to impair oocyte transcriptomic integrity, which might not be innocuous for the developing embryo. Fortunately, it is likely that such dysregulations can be minimized by adapting ART protocols or reducing adverse exposure.

In Vivo Anticoagulant and Antithrombic Activity of Depolymerized Glycosaminoglycan from Apostichopus japonicus and Dynamic Effect-Exposure Relationship in Rat Plasma.

Glycosaminoglycan from Apostichopus japonicus (AHG) and its depolymerized fragments (DAHGs) are anticoagulant fucosylated chondroitin sulfate. The aim of this study was to further evaluate the anticoagulant and antithrombic activity of AHG and DAHGs, as well as reveal the dynamic relationship between exposure and effect in vivo. The results demonstrated that AHG100 (Mw~100 kDa), DAHG50 (Mw~50 kDa), and DAHG10 (Mw~10 kDa) exhibited potent anticoagulant activity by inhibiting intrinsic factor Xase complex (FXase) as well as antithrombin-dependent factor IIa (FIIa) and factor Xa (FXa). These glycosaminoglycans markedly prevented thrombosis formation and thrombin-induced platelet aggregation in a dose- and molecular weight-dependent manner in vitro and in vivo. The further bleeding time measurement indicated that DAHG10 exhibited obviously lower hemorrhage risks than native AHG100. Following oral administration, DAHG10 could be absorbed into blood, further dose-dependently prolonging activated partial thromboplastin time (APTT) and thrombin time (TT) as well as inhibiting FXa and FIIa partially through FXase. Anticoagulant activity was positively associated with plasma concentration following oral administration of DAHG10. Our study proposed a new point of view to understand the correlation between effects and exposure of fucosylated chondroitin sulfate as an effective and safe oral antithrombotic agent.

Cubilin- and megalin-mediated uptake of albumin in cultured proximal tubule cells of opossum kidney.

BACKGROUND: Reabsorption of albumin from the glomerular filtrate occurs via receptor-mediated endocytosis in the proximal tubule. This process is initiated by binding of albumin in apical clathrin-coated pits, followed by endocytosis and degradation in lysosomes. Although binding sites have been characterized by kinetic studies, the receptors responsible for the binding of albumin have not been fully identified. Two giant glycoproteins, cubilin and megalin, constitute important endocytic receptors localized to the kidney proximal tubule. METHODS: In the present study, we examined the colocalization of cubilin and megalin in the endocytic pathway and the relationship between the uptake of albumin and the expression of cubilin and megalin in opossum kidney (OK) proximal tubule cells by immunocytochemistry and immunoblotting. RESULTS: OK cells expressed both cubilin and megalin. The light microscope labeling patterns for cubilin and megalin were almost identical and were mainly located at the surface area of the cells. Cubilin and megalin were also shown to colocalize on cell surface microvilli, in coated pits, and in endocytic compartments at the electron microscope level. Endocytosed bovine serum albumin (BSA) was identified exclusively in cells expressing megalin and cubilin. Uptake of BSA-FITC was saturable and inhibited by receptor-associated protein (RAP) and by intrinsic factor-vitamin B12 complex (IF-B12) at high concentrations. Significant inhibition was also observed by specific antibodies to cubilin, and megalin and cubilin antisense oligonucleotides likewise significantly reduced albumin uptake. Egg albumin did not affect the uptake of BSA. CONCLUSION: The present observations suggest that the two receptors cubilin and megalin are both involved in the endocytic uptake of albumin in renal proximal tubule cells.

Vitamin B12 absorption in some selected pathological states of the gastrointestinal tract.

The absorption of vitamin B12 in selected pathological states of the gastrointestinal tract was studied. Schilling test was performed with 37 kBq (1 microCi) of 57Co-labelled vitamin B12 as an analysis of urinary radioactivity. No increase in cobalamin absorption was present after exogenous IF had been administrated to patients after resection of the upper part the stomach and total gastrectomy. This suggests that there is another factor likely to affect vitamin B12 absorption.

Mechanism of uptake of cobalamin into enterocyte mitochondria.

To study the mechanism which cobalamin is taken up by the mitochondria, 57Co-cyanocobalamin-binder complex and freshly prepared mitochondria were prepared from the enterocytes of rats. Subsequently, the binder complex was incubated together with mitochondria in a calcium-containing medium in vitro. Uptake of cobalamin was determined by measuring the radioactivities bound to the mitochondria. Consequently, lysosomal and microsomal binders enhanced cobalamin uptake into the mitochondria, but intrinsic factor did not. It was found that the uptake into the mitochondria was inhibited by previous treatment with calcium-chelating agents. The uptake was completely restored after addition of calcium ions to the mitochondria.

Intrinsic factor-mediated intestinal absorption of cobalamin in the dog.

The purpose of these studies was to determine whether gastric intrinsic factor and the ileal intrinsic factor receptor participate in the process of cobalamin absorption in the dog. Physicochemical analysis of gastrointestinal fluids and mucosal extracts obtained 3-5 h after cyano[57Co]-cobalamin was fed to dogs demonstrated that 1) all cyano-[57Co]cobalamin became bound to proteins during intraluminal transport; and 2) mucosal cyano[57Co]cobalamin in the extract of the ileal mucosa was bound to intrinsic factor, to intrinsic factor coupled to receptor protein, and to proteins with properties similar to R protein and transcobalamin II. A significant fraction of the cyano[57Co]cobalamin in the mucosal extract was membrane bound and, upon solubilization with Triton X-100, was found to contain immunoreactive intrinsic factor that, however, could no longer couple to the isolated receptor. The formation of the complex of cobalamin with intrinsic factor and the receptor protein and the selective accumulation of cobalamin in the ileum indicate that the intrinsic factor-mediated mechanism for absorption of this vitamin is active in the dog.

Clinical significance of serum vitamin B12 measured by radioassay using pure intrinsic factor.

Serum vitamin B12 (B12) levels of 53 patients (15 with pernicious anemia) and 42 healthy volunteers were determined using crude intrinsic factor (IF), pure IF, and a mixture of crude IF + R-protein blocking agent (block IF). The radioassay using pure IF showed less sample-to-sample variation in nonspecific binding than the radioassay using block IF. The mean B12 levels in 42 healthy subjects were significantly higher with crude IF (499 +/- 23 pg/ml, 1 s.e.m.) than with pure IF (408 +/- 29 pg/ml) or with block IF (407 +/- 22 pg/ml). B12 levels were abnormally low in all 15 patients with pernicious anemia by pure IF (less than 100 pg/ml), in 14 patients by block IF (less than 150 pg/ml), and in only seven patients by crude IF (less than 200 pg/ml). Our data confirm previous reports that B12 deficiency can be diagnosed more reliably by measuring serum B12 levels with etiher pure IF or block IF.

Clinically significant vitamin B12 deficiency secondary to malabsorption of protein-bound vitamin B12.

Protein- (chicken serum) bound [57Co]cyanocobalamin absorption was evaluated in five hypochlorhydric patients who had developed B12 deficiency despite having normal absorption of unbound crystalline vitamin B12. All five patients had decreased urinary excretion of protein-bound B12 (0.06--0.34%) as compared to twelve normal controls (0.61--5.6%), P less than .001. Improvement in protein-bound B12 absorption in four of the five patients occurred with the exogenous administration of hydrochloric acid, pepsin, gastric intrinsic factor, or a combination thereof. Vitamin B12 deficiency developing in the setting of hypochlorhydria may result from deficiency of acid-peptic digestion of B12 bound to protein and/or a relative deficiency of intrinsic factor. This digestive defect is not detected with tests which measure the absorption of unbound crystalline B12 but is detected by a simple test which employs B12 bound to chicken serum as the form of protein-bound B12.

Influence of duodenal intrinsic factor on the absorption of labelled vitamin B12.

The intrinsic factor activity of powdered hog duodenal mucosa was confirmed by measurements of the absorption of labelled vitamin B 12 (Schilling-test) in 7 cases of Addisonian pernicious anaemia. In view of the fact that, according to earlier studies of the authors based on a bacteriological test, human duodenal juice also exhibits an intrinsic factor activity, it is suggested that the duodenal intrinsic factor may be involved in the pathogenesis of pernicious anaemia. The results of the Schilling-test are critically reviewed in the light of recent published evidence.

[Blocking and binding antibodies to intrinsic factor and their behaviour during oral therapy with intrinsic factor-vitamin B 12 complex (author's transl)]. / Uber blockierende und bindende Antikörper gegen Intrinsic-Factor und deren Verhalten unter peroraler Intrinsic-Factor B 12-Therapie

13 patients with pernicious anaemia and 2 patients who had been subjected to total gastrectomy were treated orally with a hog instrinsic factor preparation (IF) for 1 to 4.5 years. During this therapy 11 of the patients with pernicious anaemia and both gastrectomized patients developed blocking and binding antibodies to IF. Antibodies already present before the commencement of therapy showed an increase in titre. All patients remained in complete haematological remission. The conclusion is drawn that circulating antibodies to IF do not play any significant role in the absorption of the hog IF-B 12 complex.