Assisted oocyte activation does not overcome recurrent embryo developmental problems.

STUDY QUESTION: Can recurrent embryo developmental problems after ICSI be overcome by assisted oocyte activation (AOA)? SUMMARY ANSWER: AOA did not improve blastocyst formation in our patient cohort with recurrent embryo developmental problems after ICSI. WHAT IS KNOWN ALREADY: The use of AOA to artificially induce calcium (Ca2+) rises by using Ca2+ ionophores (mainly calcimycin and ionomycin) has been reported as very effective in overcoming fertilization failure after ICSI, especially in patients whose Ca2+ dynamics during fertilization are deficient. However, there is only scarce and contradictory literature on the use of AOA to overcome embryo developmental problems after ICSI, and it is not clear whether abnormal Ca2+ patterns during fertilization disturb human preimplantation embryo development. Moreover, poor embryo development after ICSI has also been linked to genetic defects in the subcortical maternal complex (SCMC) genes. STUDY DESIGN, SIZE, DURATION: This prospective cohort single-center study compared ICSI-AOA cycles and previous ICSI cycles in couples with normal fertilization rates (≥60%) but impaired embryonic development (≤15% blastocyst formation) in at least two previous ICSI cycles. In total, 42 couples with embryo developmental problems were included in this study from January 2018 to January 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: Of the 42 couples included, 17 underwent an ICSI-AOA cycle consisting of CaCl2 injection and double ionomycin exposure. Fertilization, blastocyst development, pregnancy, and live birth rates after ICSI-AOA were compared to previous ICSI cycles. In addition, the calcium pattern induced by the male patient's sperm was investigated by mouse oocyte calcium analysis. Furthermore, all 42 couples underwent genetic screening. Female patients were screened for SCMC genes (TLE6, PADI6, NLRP2, NLRP5, NLRP7, and KHDC3L) and male patients were screened for the sperm-oocyte-activating factor PLCZ1. MAIN RESULTS AND THE ROLE OF CHANCE: We compared 17 AOA cycles to 44 previous ICSI cycles from the same patient cohort. After AOA, a total fertilization rate of 68.95% (131/190), a blastocyst development rate of 13.74% (18/131), a pregnancy rate of 29.41% (5/17), and a live birth rate of 23.53% (4/17) were achieved, which was not different from the previous ICSI cycles (76.25% (321/421, P-value = 0.06); 9.35% (30/321, P-value = 0.18), 25.00% (11/44, P-value = 0.75), and 15.91% (7/44, P-value = 0.48), respectively). Calcium analysis showed that patient's sperm induced calcium patterns similar to control sperm samples displaying normal embryo developmental potential. Genetic screening revealed 10 unique heterozygous variants (in NLRP2, NLRP5, NLRP7, TLE6, and PADI6) of uncertain significance (VUS) in 14 females. Variant NLRP5 c.623-12_623-11insTTC (p.?) was identified in two unrelated individuals and variant NLRP2 c.1572T>C (p.Asp524=) was identified in four females. Interestingly, we identified a previously reported homozygous mutation PLCZ1, c.1499C>T (p.Ser500Leu), in a male patient displaying impaired embryonic development, but not showing typical fertilization failure. LIMITATIONS, REASONS FOR CAUTION: Our strict inclusion criteria, requiring at least two ICSI cycles with impaired embryo development, reduced cycle-to-cycle variability, while the requirement of a lower blastocyst development not influenced by a poor fertilization excluded couples who otherwise would be selective cases for AOA; however, these criteria limited the sample size of this study. Targeted genetic screening might be too restricted to identify a genetic cause underlying the phenotype of poor embryo development for all patients. Moreover, causality of the identified VUS should be further determined. WIDER IMPLICATIONS OF THE FINDINGS: Strong evidence for AOA overcoming impaired embryonic development is still lacking in the literature. Thus far, only one article has reported a beneficial effect of AOA (using calcimycin) compared to previous ICSI cycles in this patient population, whilst two more recent sibling-oocyte control studies (one using calcimycin and the other ionomycin) and our research (using ionomycin) could not corroborate these findings. Although no major abnormalities have been found in children born after AOA, this technique should be reserved for couples with a clear Ca2+-release deficiency. Finally, genetic screening by whole-exome sequencing may reveal novel genes and variants linked to embryo developmental problems and allow the design of more personalized treatment options, such as wild-type complementary RNA or recombinant protein injection. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Flemish Fund for Scientific Research (grant FWO.OPR.2015.0032.01 to B.H. and grant no. 1298722N to A.B.). A.C.B., D.B., A.B., V.T., R.P., F.M., I.D.C., L.L., D.S., P.D.S., P.C., and F.V.M. have nothing to disclose. B.H. reports a research grant from the Flemish Fund for Scientific Research and reports being a board member of the Belgian Society for Reproductive Medicine and the Belgian Ethical Committee on embryo research. TRIAL REGISTRATION NUMBER: NCT03354013.

Phosphorus Balance in Adolescent Girls and the Effect of Supplemental Dietary Calcium.

There are limited data on phosphorus balance and the effect of dietary calcium supplements on phosphorus balance in adolescents. The purpose of this study was to determine phosphorus balance and the effect of increasing dietary calcium intake with a supplement on net phosphorus absorption and balance in healthy adolescent girls. This study utilized stored urine, fecal, and diet samples from a previously conducted study that focused on calcium balance. Eleven healthy girls ages 11 to 14 years participated in a randomized crossover study, which consisted of two 3-week periods of a controlled diet with low (817 ± 19.5 mg/d) or high (1418 ± 11.1 mg/d) calcium, separated by a 1-week washout period. Phosphorus intake was controlled at the same level during both placebo and calcium supplementation (1435 ± 23.5 and 1453 ± 28.0 mg/d, respectively, p = 0.611). Mean phosphorus balance was positive by about 200 mg/d and was unaffected by the calcium supplement (p = 0.826). Urinary phosphorus excretion was lower with the calcium supplement (535 ± 42 versus 649 ± 41 mg/d, p = 0.013), but fecal phosphorus and net phosphorus absorption were not significantly different between placebo and calcium supplement (553 ± 60 versus 678 ± 63 versus mg/d, p = 0.143; 876 ± 62 versus 774 ± 64 mg/d, p = 0.231, respectively). Dietary phosphorus underestimates using a nutrient database compared with the content measured chemically from meal composites by ~40%. These results show that phosphorus balance is positive in girls during adolescent growth and that a calcium dietary supplement to near the current recommended level does not affect phosphorus balance when phosphorus intake is at 1400 mg/d, a typical US intake level.

Measurement of intracellular ions by flow cytometry.

Using flow cytometry, single-cell measurements of calcium can be made on isolated populations identified by one or more phenotypic characteristics. Most earlier techniques for measuring cellular activation parameters determined the mean value for a population of cells, which did not permit optimal resolution of the responses. The flow cytometer is particularly useful for this purpose because it can measure ion concentrations in large numbers of single cells and thereby allows ion concentration to be correlated with other parameters such as immunophenotype and cell cycle stage. A limitation of flow cytometry, however, is that it does not permit resolution of certain complex kinetic responses such as cellular oscillatory responses. This unit describes the preparation of cells, including labeling with antibodies and with calcium probes, and discusses the principles of data analysis and interpretation.

An evidence-based guide for coronary calcium scoring in asymptomatic patients without coronary heart disease.

As public awareness and clinical use of CAC screening increases, physicians should, at a minimum, know the following information: 1) The presence of CAC indicates underlying CHD but does not predict luminal obstruction. 2) Non-contrast, prospectively ECG-gated cardiac EBCT and MDCT are sensitive, reproducible, rapid, and essentially equivalent imaging techniques commonly used to screen for CAC. 3) Currently, CAC screening is appropriate for all intermediate- risk patients and low-risk patients with a family history of premature CHD, and might be appropriate for all low-risk women. 4) The risks associated with CAC screening are a small but measurable excess risk of cancer and the risk of unnecessary downstream tests and procedures. 5) A CAC score of zero has a very high negative predictive value for CHD events. 6) Increasingly positive (non-zero) CAC scores are directly proportional to increased CHD event risk, and a CAC score >100 or greater than the 75th percentile indicates high risk. 7) Repeat screening to determine CAC progression or regression is not currently recommended.

Plaque distribution in clopidogrel responders and low responders as determined by multislice computed tomography.

Via multidetector computed tomography (MDCT) with retrospective electrographic gating, we sought to evaluate whether plaque distribution differs between responders and low responders to clopidogrel treatment. Low response was defined as a post-treatment aggregation of 35% to 70%. In this observational study, we enrolled 62 patients (mean age, 64.8 ± 8.9 yr; 51 men). In addition to determining coronary calcium scores, we performed noninvasive coronary angiography with MDCT before stent implantation. Plaques were visually classified as calcified, mixed, or completely noncalcified. Mean density was measured. Residual platelet aggregation (RPA) was evaluated by aggregometry 6 hr after administration of a 600-mg loading dose of clopidogrel. Patients with an RPA of less than 35% were defined as responders.The median calcium score was 736 Agatston score equivalent (ASE) (range, 0-5,772) and mean platelet inhibition was 35% ± 19% (range, 0-70%). A total of 494 coronary plaques were detected (responders: calcified, 197; mixed, 47, noncalcified, 5; and low responders: calcified, 177; mixed, 65; noncalcified, 3). Responders (n = 35) had significantly lower ASEs and fewer mixed but more calcified plaques than did low responders. In mean plaque density (measured within the noncalcified part of the plaques), no statistically significant difference existed between the 2 patient groups. By use of MDCT, we showed that ASE and plaque distribution were associated with RPA after clopidogrel treatment. Patients with a low coronary plaque burden and a small proportion of mixed plaques were more likely to have low RPA after administration of clopidogrel.

The protection of aminoguanidine,silymarin and anisodamine on growth and cytosolic free calcium changes of retinal capillary pericytes cultured in glycation products / 中华眼底病杂志

Objective To investigate the protective effect of aminoguanidine(AG),silymarin (Sil) and anisodamine (Ani) on retinal capillary pericytes cultured in glycosylation products. Methods MTT cololrimetric assay, thymidine incorporating and fluorescent indicator fura 2 acetoxy methyl ester (Fura 2AM) were used to study the influence of AG,Sil and Ani on the growth,DNA synthesis,and cytosolic free calcium([Ca 2+ ]i)changes of pericytes cultured in the medium contained early glycation products (EGs) or advanced glycation end products (AGEs). Results Cultured in the medium contained EGs,the A value by MTT assayed and amount of thymidine incorporating in AG group and Sil group were obviously elevated than those of control group(P

Breast-milk calcium concentrations during prolonged lactation in British and rural Gambian mothers.

Detailed investigation of breast-milk calcium concentrations during 2 years of lactation have been conducted in Cambridge, UK, and rural Gambia. Mature milk concentrations remained steady for 3 months but declined during months 4-12 by over 25% (p less than 0.001). The pattern was identical in both communities despite differences in breast-feeding practices. Calcium concentrations were not related to feed frequency or breast-milk sodium concentrations, suggesting that breast involution is not responsible for the decrease after 3 months. Breast-milk calcium concentrations were characteristic of the individual, varied twofold between mothers and were independent of maternal age, parity and milk output. Gambian breast-milk contained 19% less calcium than Cambridge milk, throughout lactation (p less than 0.001). The extent to which this reflected the lower calcium intakes of Gambian mothers is unknown. Further studies are required to determine factors regulating breast-milk calcium secretion.

Auger and pixe microanalysis of intrauterine devices (IUDs).

Auger Emission Spectroscopy (AES) and Particle Induced X-ray Emission (PIXE) analysis were employed in a study on the calcification and oxidation processes of the copper wires used in Intra-Uterine Device (IUDs) contraceptives. A strong copper release (approximately 50 micrograms/day) has been observed in the first year of IUDS application during the high device antifertility effect. The copper release is reduced with the IUD-use time, because of the calcification process on the surface by organic liquids. This effect produces a drastic reduction in the contraceptive efficiency, and a new IUDs replacement is needed after about 2 years of use. Auger, X-ray spectrometry and scanning electron microscopy (SEM) have been used to investigate the lifetime and efficiency of IUDS in order to understand the surface modification of copper wire during its permanence in the intrauterine environment.

PIP: Auger Emission Spectroscopy (AES) and Particle Induced X-ray Emission (PIXE) analysis were employed in a study on the calcification and oxidation processes of the copper wires used in IUDs. A strong copper release (approximately 50 mcg/day) was observed in the 1st year of IUD use, during the high device antifertility effect. Copper release is reduced over time due to the calcification process on the surface by organic liquids. This effect produces a drastic reduction in contraceptive efficiency and new IUDs are necessary after 2 years of use. Auger, x-ray spectrometry, and scanning electron microscopy have been used to investigate IUD lifetime and efficiency in order to understand the surface modifications of copper wire during its performance in the intrauterine environment. (author's modified).

Premenopausal bone mineral content relates to height, weight and calcium intake during early adulthood.

The influence of dietary and anthropometric factors on bone mineral content was studied in 183 healthy premenopausal French-Canadian women aged between 40 and 50 years and living in the same area. Dietary evaluation of their calcium (Ca), caffeine and alcohol intake since the age of 20 was performed in all cases. Age, height, weight, exercise level, cigarette smoking, parity and estrogen were also recorded. Bone mineral content of the L2 to L4 vertebrae (BMCL, n = 183) and forearm (BMCF, n = 137) were measured respectively by dual and single photon absorptiometry. While stepwise regression analysis showed a significant relationship between total BMCL and height, weight and Ca intake, this only occurred with weight and Ca intake when BMCL was corrected for height of L2-L4 (BMCL/cm) or for bone scan area of L2-L4 (BMCL/cm2). BMCF expressed per unit of length correlated to height only. When subjects were divided into three groups according to their Ca intake (less than 500 mg/day, between 500 and 1000 mg/day and greater than 1000 mg/day), the mean BMC adjusted for significant covariables (height and weight) was statistically different for the low and high intake groups at both sites (BMCF, F = 3.9, P = 0.02; BMCL, F = 4.2, P less than 0.02; BMCL/cm, F = 6.1, P less than 0.005; BMCL/cm2, F = 4.4, P less than 0.02). These findings indicate that, of the variables considered, Ca intake, height and weight were the only significant factors related to bone mass in our homogeneous population. It is therefore suggested that Ca intake in early adulthood influences the axial and appendicular bone mass in premenopausal women.

Effects of increased dietary calcium intake upon the calcium and bone mineral status of lactating adolescent and adult women.

We studied the effectiveness of an increased calcium (Ca) diet in preventing bone mineral loss in lactating adolescent mothers. Three groups of lactating women were studied: 15 control adolescents consuming their usual Ca diet (900 mg/d), 21 experimental adolescents consuming a high-Ca diet (greater than 1600 mg/d), and 12 adults. At 2 and 16 wk postpartum, serum calcium, phosphate, magnesium, albumin, alkaline phosphatase, vitamin D, parathyroid hormone (PTH), and calcitonin (CT) were determined. Bone mineral analyses were performed by photon absorptiometry. By 16 wk the control adolescent group had a 10% decrease in bone mineral content (BMC) and increased PTH and CT. The experimental adolescent and adult groups had no significant change in BMC during the study. There was a positive correlation (r = 0.45, p less than 0.01) between dietary Ca intake and BMC in all adolescents. Data suggest that bone loss during lactation in adolescents may be prevented with adequate dietary Ca intakes.

A population study on the relationship between blood pressure and the excretion of urinary cations.

The relationships between blood pressure and the 24-h excretion of three urinary cations (Ca2+, K+, and Na+) were investigated in a random sample of 528 adults (greater than or equal to 20 years) inhabitants of two Belgian towns. In these adults both systolic and diastolic blood pressures were strongly and independently correlated with age and body weight. Systolic pressure in women was significantly and positively related to pulse rate. After adjusting for age and body weight, systolic and diastolic pressures in men were significantly (p less than 0.001 and p less than 0.01, respectively) and negatively correlated with urinary potassium excretion. Diastolic pressure in men was weakly (p less than 0.05 after adjusting for body weight, age, and urinary potassium excretion) but positively correlated with calcium excretion. In a subsample of 162 premenopausal women, blood pressure was higher in current contraceptive pill users than in women not taking oral contraceptives. In 56 current pill users there was a positive association between systolic pressure and 24-h urinary sodium (p less than 0.01 after adjusting for age, body weight, and pulse rate). The present study indicates that urinary potassium is a consistent and negative predictor of both systolic and diastolic pressures in adult men, whose diastolic pressure is weakly and positively associated with urinary calcium. The positive association between systolic pressure and urinary sodium in current users of contraceptive pills may suggest that their blood pressure becomes responsive to their usual salt intake.

PIP: The relationships between blood pressure and the 24-hour excretion of 3 urinary cations (Ca2+, K+, and Na+) were investigated in a random sample of 528 adults (or= 20 years), inhabitants of 2 Belgian towns. In these adults, both systolic and diastolic blood pressures were strongly and independently correlated with age and body weight. Systolic pressure in women was significantly and positively related to pulse rate. After adjusting for age and body weight, systolic and diastolic pressures in men were significantly (P .001 and P .01, respectively) and negatively correlated with urinary potassium excretion. Diastolic pressure in men was weakly (P .05 after adjusting for boyd weight, age, and urinary potassium excretion) but positively correlated with calcium excretion. In a subsample of 162 premenopausal women, blood pressure was higher in current contraceptive pill users than in women not taking OCs. In 56 current OC users, there was a positive association between systolic pressure and 24-hour urinary sodium (P .01 after adjusting for age, body weight, and pulse rate). The present study indicates that urinary potassium is a consistent and negative predictor of both systolic and diastolic pressures in adult men, whose diastolic pressure is weakly and positively associated with urinary calcium. The positive association between systolic pressure and urinary sodium in curren OC users may suggest that their blood pressure becomes responsive to usual salt intake.

Dental caries in South African rural black women who had large families and long lactations.

A general recommendation for good bone and teeth formation is that a high intake of calcium is essential. Some consider that pregnancy and lactation, with associated calcium depletion, could aggravate caries development. South African black mothers in rural areas have habitually low calcium intakes, and usually several pregnancies and long periods of lactation. To learn whether high parity is detrimental to caries scores, investigations were made on a selected total of 521 black mothers, 250 aged 35-44 years, and 271 aged 45-54 years. Each group was sub-divided into those with small families (1-3 children) or large families (5 + children). The numbers involved were 122 and 133 mothers, and 128 and 138 mothers, respectively. Studies revealed: black mothers had very much lower mean caries scores than values reported for white mothers, who are accustomed, inter alia, to high calcium intakes, have few children, and often no or short lactation periods; black mothers of large compared with small families did not have significantly higher mean caries scores; and upper and lower thirds of black mothers respecting calcium intake, and sugar intake, did not have significantly different mean caries scores. Hence, in the context studied, it would seem that high parity has no obvious effect on caries scores.

PIP: A general recommendation for good bone and teeth formation is that a high intake of calcium is essential. Some consider that pregnancy and lactation, with associated calcium depletion, could aggravate caries development. South African black mothers in rural areas have habitually low calcium intakes, and usually several pregnancies and long periods of lactation. To learn whether high parity is detrimental to caries scores, investigations were made on a selected total of 521 black mothers, 250 between ages 35-44 and 271 ages 45-54. Each group was subdivided into those with small families (1-3 children) or large families (5+ children). The numbers involved were 122 and 133 mothers, and 128 and 138 mothers, respectively. Studies revealed that black mothers had much lower mean caries scores than values reported for white mothers, who are accustomed, inter alia, to high calcium intakes, have fewer children, and often no or short periods of lactation; black mothers of large compared to small families did not have significantly higher mean caries scores; and upper and lower thirds of black mothers respecting calcium intake and sugar intake did not have significantly different mean caries scores. Thus, in the context studied, it would seem that high parity has no obvious effect on caries scores.