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Reproduction technologies could be improved by the development of methods to evaluate oocyte or embryo quality in a non-invasive, quantitative manner. Since human embryos secrete a factor that inhibits granulosa cell progesterone production, an interspecies bioassay was established to investigate whether the presence of this progesterone-inhibitory factor (PIF) in human embryo-conditioned (HEC) media is related to the health and developmental capacity of the embryos. Oocytes were microsurgically removed from oocyte-cumulus complexes isolated from superovulated mouse ovaries, and the oocytectomized complexes were cultured in HEC media in the presence of follicle stimulating hormone and testosterone. Progesterone accumulation in the media was determined by radioimmunoassay. Despite the potential limitations of very small volumes of HEC media to evaluate, and the need to freeze these media at the source, the bioassay was able to detect PIF activity in HEC media. Most embryos produced PIF activity, but the degree of inhibition was not correlated with the ability of oocytes to be fertilized, nor with embryo morphology or ability to cleave and develop after transfer. These results demonstrate that secretion of PIF by human embryos can be measured by this bioassay and that human PIF can inhibit murine granulosa cell steroidogenesis; however, PIF activity is not correlated with human embryo quality or developmental competence. [ABSTRACT FROM AUTHOR]

We report here that intracellular pH (pH(i)) in cleavage-stage human embryos (2-8-cell) is regulated by at least two mechanisms: the HCO(3)(-)/Cl(-) exchanger (relieves alkalosis) and the Na(+)/H(+) antiporter (relieves acidosis). The mean pH(i) of cleavage-stage embryos was 7.12 +/- 0.008 (n = 199) with little variation between different stages. Embryos demonstrated robust recovery from alkalosis that was appropriately Cl(-)-dependent, indicating the presence of the HCO(3)(-)/Cl(-) exchanger. This was further confirmed by measuring the rate of intracellular alkalinization upon Cl(-) removal, which was markedly inhibited by the anion transport inhibitor, 4,4'-diisocyanatostilbene-2,2'-disulphonic acid, disodium salt. The set-point of the HCO(3)(-)/Cl(-) exchanger was between pH(i) 7.2 and 7.3. Embryos also exhibited Na(+)-dependent recovery from intracellular acidosis. Na(+)/H(+) antiporter activity appeared to regulate recovery up to about pH(i) 6.8; this recovery was HCO(3)(-)-independent and amiloride-sensitive, with a pH(i) set-point of approximately 6.8-6.9. A second system that was both Na(+)- and HCO(3)(-)-dependent appeared to mediate further recovery from acidosis up to about pH(i) 7.1. Thus, pH(i) of early human preimplantation embryos appears to be regulated by opposing mechanisms (HCO(3)(-)/Cl(-) exchanger, Na(+)/H(+) antiporter, and possibly a third acid-alleviating transporter that was both Na(+)- and HCO(3)(-)-dependent) resulting in the maintenance of pH(i) within a narrow range. [ABSTRACT FROM AUTHOR]

Mouse zygotes and early cleavage-stage embryos have previously been shown to utilize glycine as an organic osmolyte, accumulating it to oppose any decrease in cell volume. Such glycine uptake in early cleavage-stage mouse embryos is via the glycine-specific Gly transporter. Mouse embryos also possess swelling-activated channels which function to release osmotically active glycine and other osmolytes when cell volume becomes too large. In this study it was found that human cleavage-stage embryos also transported glycine via a similarly saturable, sarcosine-inhibitable transporter, implying that the Gly transporter also mediates glycine transport in human embryos. Mouse zygotes have previously been shown to accumulate more intracellular glycine when cultured at increased osmolarities for 24 h. It was found in the current study that this ability was lost as preimplantation mouse embryo development proceeded, and that early cleavage-stage human embryos may also be capable of such osmosensitive accumulation of glycine. Finally, using spare human eggs which had failed to fertilize or cleave, the presence of swelling-activated currents resembling those in mouse zygotes was demonstrated. These data indicate that osmoregulation in early human embryos occurs via similar mechanisms as in the mouse. [ABSTRACT FROM AUTHOR]

Mouse zygotes and early cleavage-stage embryos have previously been shown to utilize glycine as an organic osmolyte, accumulating it to oppose any decrease in cell volume. Such glycine uptake in early cleavage-stage mouse embryos is via the glycine-specific Gly transporter. Mouse embryos also possess swelling-activated channels which function to release osmotically active glycine and other osmolytes when cell volume becomes too large. In this study it was found that human cleavage-stage embryos also transported glycine via a similarly saturable, sarcosine-inhibitable transporter, implying that the Gly transporter also mediates glycine transport in human embryos. Mouse zygotes have previously been shown to accumulate more intracellular glycine when cultured at increased osmolarities for 24 h. It was found in the current study that this ability was lost as preimplantation mouse embryo development proceeded, and that early cleavage-stage human embryos may also be capable of such osmosensitive accumulation of glycine. Finally, using spare human eggs which had failed to fertilize or cleave, the presence of swelling-activated currents resembling those in mouse zygotes was demonstrated. These data indicate that osmoregulation in early human embryos occurs via similar mechanisms as in the mouse. [ABSTRACT FROM PUBLISHER]

Intrauterine insemination is a common procedure used for the treatment of different causes of infertility. Adverse reactions associated with this procedure are very rare and usually the procedure is well tolerated by the patient. We report a case of an allergic reaction after intrauterine insemination. The patient developed fever, difficulty breathing and wheezing in both lung fields. Although a low concentration of penicillin in the medium was used, it caused a significant allergic reaction. When intrauterine insemination was performed in subsequent cycles with an antibiotic-free medium, no allergic reaction occurred, and the procedure was well tolerated by the patient. A careful allergy history is essential in patients pursuing infertility treatment where antibiotics are utilized. Patients who are known to be allergic to penicillin should have semen prepared by an antibiotic-free medium. [ABSTRACT FROM AUTHOR]

Nonalcoholic fatty liver disease (NAFLD) is a growing epidemic linked to metabolic disease. The first stage of NAFLD is characterized by lipid accumulation in hepatocytes, but this can progress into nonalcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma (HCC). Western diets, high in fats, sugars, and cholesterol, are linked to NAFLD development. Murine models are often used to study NAFLD; however, there remains debate on which diet-induced model best mimics both human disease progression and pathogenesis. In this study, we performed a side-by-side comparison of two popular diet models of murine NAFLD/NASH and associated HCC, a high-fat diet supplemented with 30% fructose water (HFHF) and a Western diet high in cholesterol (WDHC), and these were compared with a common grain-based chow diet (GBD). Mice on both experimental diets developed liver steatosis, and WDHC-fed mice had greater levels of hepatic inflammation and fibrosis than HFHF-fed mice. In contrast, HFHF-fed mice were more obese and developed more severe metabolic syndrome, with less pronounced liver disease. Despite these differences, WDHC-fed and HFHF-fed mice had similar tumor burdens in a model of diet-potentiated liver cancer. Response to diet and resulting phenotypes were generally similar between sexes, albeit delayed in females. This study shows that modest differences in diet can significantly uncouple glucose homeostasis and liver damage. In conclusion, long-term feeding of either HFHF or WDHC is a reliable method to induce NASH and diet-potentiated liver cancer in mice of both sexes; however, the choice of diet involves a trade-off between severity of metabolic syndrome and liver damage.

Objective: The liver is regularly exposed to changing metabolic and inflammatory environments. It must sense and adapt to metabolic need while balancing resources required to protect itself from insult. Peroxisome proliferator activated receptor gamma coactivator-1 alpha (PGC-1α) is a transcriptional coactivator expressed as multiple, alternatively spliced variants transcribed from different promoters that coordinate metabolic adaptation and protect against inflammation. It is not known how PGC-1α integrates extracellular signals to balance metabolic and anti-inflammatory outcomes., Methods: Primary mouse hepatocytes were used to evaluate the role(s) of different PGC-1α proteins in regulating hepatic metabolism and inflammatory signaling downstream of tumor necrosis factor alpha (TNFα). Gene expression and signaling analysis were combined with biochemical measurement of apoptosis using gain- and loss-of-function in vitro and in vivo., Results: Hepatocytes expressed multiple isoforms of PGC-1α, including PGC-1α4, which microarray analysis showed had common and isoform-specific functions linked to metabolism and inflammation compared with canonical PGC-1α1. Whereas PGC-1α1 primarily impacted gene programs of nutrient metabolism and mitochondrial biology, TNFα signaling showed several pathways related to innate immunity and cell death downstream of PGC-1α4. Gain- and loss-of-function models illustrated that PGC-1α4 uniquely enhanced expression of anti-apoptotic gene programs and attenuated hepatocyte apoptosis in response to TNFα or lipopolysaccharide (LPS). This was in contrast to PGC-1α1, which decreased the expression of a wide inflammatory gene network but did not prevent hepatocyte death in response to cytokines., Conclusions: PGC-1α variants have distinct, yet complementary roles in hepatic responses to metabolism and inflammation, and we identify PGC-1α4 as an important mitigator of apoptosis., (Copyright © 2020 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

The liver constantly adapts to meet energy requirements of the whole body. Despite its remarkable adaptative capacity, prolonged exposure of liver cells to harmful environmental cues (such as diets rich in fat, sugar, and cholesterol) results in the development of chronic liver diseases (including non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH)) that can progress to hepatocellular carcinoma (HCC). The pathogenesis of these diseases is extremely complex, multifactorial, and poorly understood. Emerging evidence suggests that mitochondrial dysfunction or maladaptation contributes to detrimental effects on hepatocyte bioenergetics, reactive oxygen species (ROS) homeostasis, endoplasmic reticulum (ER) stress, inflammation, and cell death leading to NASH and HCC. The present review highlights the potential contribution of altered mitochondria function to NASH-related HCC and discusses how agents targeting this organelle could provide interesting treatment strategies for these diseases.

Background & Aims: Inefficient fatty acid oxidation in mitochondria and increased oxidative damage are features of non-alcoholic fatty liver disease (NAFLD). In rodent models and patients with NAFLD, hepatic expression of peroxisome proliferator-activated receptor-γ (PPARG) coactivator 1α (PPARGC1A or PGC1A) is inversely correlated with liver fat and disease severity. A common polymorphism in this gene (rs8192678, encoding Gly482Ser) has been associated with NAFLD. We investigated whether reduced expression of PGC1A contributes to development of NAFLD using mouse models, primary hepatocytes, and human cell lines., Methods: HepG2 cells were transfected with variants of PPARGC1A and protein and messenger RNA levels were measured. Mice with liver-specific hemizygous or homozygous disruption of Ppargc1a (Ppargc1a f/+ Alb-cre +/0 and Ppargc1a f/f Alb-cre +/0 mice, respectively) were fed regular chow (control) or a high-fat diet supplemented with 30% d-fructose in drinking water (obesogenic diet) for 25-33 weeks. Liver tissues were analyzed by histology and by immunoblotting. Primary hepatocytes were analyzed for insulin signaling, reactive oxygen species, and estrogen response. Luciferase reporter expression was measured in transfected H2.35 cells expressing an estrogen receptor reporter gene, estrogen receptor 1, and/or PGC1A/B., Results: The serine 482 variant of the human PGC1A protein had a shorter half-life than the glycine 482 variant when expressed in HepG2 cells. Liver tissues from mice with liver-specific hemizygous disruption of Ppargc1a placed on an obesogenic diet expressed increased markers of inflammation and fibrosis and decreased levels of antioxidant enzymes compared with the Ppargc1a +/+ on the same diet. Oxidative damage was observed in livers from Ppargc1a f/+ Alb-cre +/0 mice of each sex, in a cell-autonomous manner, but was greater in livers from the female mice. Expression of PGC1A in H2.35 cells coactivated estrogen receptor 1 and was required for estrogen-dependent expression of genes that encode antioxidant proteins. These findings could account for the increased liver damage observed in female Ppargc1a f/+ Alb-cre +/0 mice; while, compensatory increases in PPARG coactivator 1β could prevent oxidative damage associated with complete loss of PGC1A expression in Ppargc1a f/f Alb-cre +/0 female mice., Conclusions: In mice, loss of estrogen signaling contributes to oxidative damage caused by low levels of PGC1A in liver, exacerbating steatohepatitis associated with diets high in fructose and fat., (Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Purpose: This study examined the reliability and validity of the Test of Infant Motor Performance Screening Items (TIMPSI) in infants with type I spinal muscular atrophy (SMA)., Methods: After training, 12 evaluators scored 4 videos of infants with type I SMA to assess interrater reliability. Intrarater and test-retest reliability was further assessed for 9 evaluators during a SMA type I clinical trial, with 9 evaluators testing a total of 38 infants twice. Relatedness of the TIMPSI score to ability to reach and ventilatory support was also examined., Results: Excellent interrater video score reliability was noted (intraclass correlation coefficient, 0.97-0.98). Intrarater reliability was excellent (intraclass correlation coefficient, 0.91-0.98) and test-retest reliability ranged from r = 0.82 to r = 0.95. The TIMPSI score was related to the ability to reach (P ≤ .05)., Conclusion: The TIMPSI can reliably be used to assess motor function in infants with type I SMA. In addition, the TIMPSI scores are related to the ability to reach, an important functional skill in children with type I SMA.

Objective: The goal of our study was to compare Doppler sonography and renal scintigraphy as tools for predicting the therapeutic response in patients after undergoing renal angioplasty. SUBJECTS AND METHODS. Seventy-four hypertensive patients underwent clinical examination, Doppler sonography, and renal scintigraphy before and after receiving captopril in preparation for renal revascularization. The patients were evaluated for the status of hypertension 3 months after the procedure. The predictive values of the findings of clinical examination, Doppler sonography, renal scintigraphy, and angiography were assessed., Results: For prediction of a favorable therapeutic outcome, abnormal results from renal scintigraphy before and after captopril administration had a sensitivity of 58% and specificity of 57%. Findings of Doppler sonography had a sensitivity of 68% and specificity of 50% before captopril administration and a sensitivity of 81% and specificity of 32% after captopril administration. Significant predictors of a cure or reduction of hypertension after revascularization were low unilateral (p = 0.014) and bilateral resistive (p = 0.016) indexes on Doppler sonography before (p = 0.009) and after (p = 0.028) captopril administration. On multivariate analysis, the best predictors were a unilateral resistive index of less than 0.65 (odds ratio [OR] = 3.7) after captopril administration and a kidney longer than 93 mm (OR = 7.8). The two best combined criteria to predict the favorable therapeutic outcome were a bilateral resistive index of less than 0.75 before captopril administration combined with a unilateral resistive index of less than 0.70 after captopril administration (sensitivity, 76%; specificity, 58%) or a bilateral resistive index of less than 0.75 before captopril administration and a kidney measuring longer than 90 mm (sensitivity, 81%; specificity, 50%)., Conclusion: Measurements of kidney length and unilateral and bilateral resistive indexes before and after captopril administration were useful in predicting the outcome after renal angioplasty. Renal scintigraphy had no significant predictive value.

Sulpholipid immobilizing protein 1 (SLIP1) is an evolutionarily conserved 68 kDa plasma membrane protein, present selectively in germ cells. We have previously shown that mouse sperm SLIP1 is involved in sperm-zona pellucida (ZP) binding. In this report, we extended our study to the human system. Immunoblotting demonstrated that anti-SLIP1-reactive proteins (mol. wt 68 and 48 kDa) could be extracted from human spermatozoa by an ATP-containing solution, a result that is consistent with observations in other species. Direct immunofluorescence, using Cy3-conjugated anti-SLIP1 IgG, revealed SLIP1 staining over the acrosomal region, with higher intensity at the posterior area. Using the human sperm-ZP binding assay, we demonstrated that pretreatment of human spermatozoa from three donors with anti-SLIP1 IgG revealed lower numbers of zona-bound spermatozoa, as compared to the corresponding control spermatozoa treated with normal rabbit serum IgG. This decrease in zona pellucida binding was not from an antibody-induced decline in sperm motility or an increase in the premature acrosome reaction. The results strongly suggest that anti-SLIP-reactive proteins on human spermatozoa play an important role in ZP binding.

Objective: To evaluate the usefulness of morphology grading of the oocyte-corona-cumulus complex (OCCC) as a marker of oocyte nuclear maturity, fertilizability, embryo cleavage, and likelihood of pregnancy., Design: Prospective cohort study., Setting: Academic fertility center., Patient(s): Eighty-three infertile couples undergoing IVF-ET/intracytoplasmic sperm injection treatment., Intervention(s): All patients underwent a long stimulation protocol of GnRH agonist therapy followed by hMG administration and transvaginal oocyte recovery., Main Outcome Measure(s): All OCCCs, oocytes, and embryos were assessed. The relation among OCCC morphology and the nuclear maturity of denuded oocytes, the fertilization rate, and embryo development to the cleavage stage were analyzed., Result(s): Of 909 OCCCs collected from 92 cycles, 2.5%, 4.2%, 79.9%, and 13.4% were prophase I, metaphase I, metaphase II, and degenerating, respectively. No statistically significant differences were found in the percentage of intact metaphase II oocytes, the fertilization rate, or the cleavage rate among complexes with different morphologic grades. The morphologic grade of the OCCCs of transferred embryos in the pregnant group was not different from that in the nonpregnant group., Conclusion(s): Most oocytes were in metaphase II at the time of retrieval after ovarian stimulation. However, no relation was observed between the OCCC morphologic grade and oocyte nuclear maturity, the fertilization rate, or embryo cleavage. These observations suggest that OCCC morphology grading is a poor marker of oocyte quality.

Nitric oxide (NO) is a powerful vasoactive agent that contributes to the regulation of blood pressure (BP). However, the role of NO in uremic patients and during the course of hemodialysis is still debated. Blood L-arginine concentrations and exhaled NO concentrations were measured in 22 healthy controls and in 22 hemodialysis patients before and after dialysis. On the basis of their BP response during hemodialysis, the patients were divided into three groups: 6 of the 22 patients presented with a decrease in BP during dialysis (group 1), eight presented with a stable BP (group 2), and eight with an increase in BP (group 3). The exhaled NO concentration was higher in dialysis patients than in healthy controls (22.7 +/- 2.6 ppb in dialysis patients v 16.7 +/- 0.9 ppb in controls, mean +/- SEM, P = 0.044). The predialysis and postdialysis exhaled NO concentrations were inversely correlated with the change in BP during hemodialysis (r = -0.47, P = 0.013). Patients with a decrease in BP (group 1) had the highest NO concentrations; patients with an increase in BP (group 3) had the lowest values; and patients with a stable BP during the course of dialysis (group 2) had intermediary values (trend test, P = 0.0291). In addition, both the exhaled NO concentration and the blood L-arginine concentration decreased during dialysis in all patients (P = 0.005 and P = 0.001, respectively). These results provide several novel insights into NO metabolism and BP regulation during hemodialysis: (1) maintenance hemodialysis is associated with a chronic increase in NO concentrations; (2) changes in BP during hemodialysis are inversely correlated with exhaled NO concentrations, higher NO levels being associated with a decrease in BP and lower NO levels with an increase in BP during dialysis; (3) blood L-arginine levels decrease during hemodialysis, and this reduction may in turn influence NO production.

Purpose: A higher proportion of male offspring has been observed after transferring faster-developing embryos in a number of animal species. Therefore, we evaluated the correlation between the sex ratio of delivered babies and the cleavage stage of transferred embryos in a human IVF-ET program., Methods: The sex of infants born (n = 104) after transfer of exclusively slower-cleaving < or = 3 cell (n = 41) versus exclusively faster-cleaving > or = 4 cell (n = 63) embryos was compared. Furthermore, all boys and girls resulting from IVF-ET (n = 213) were compared with respect to: the average number of cells in the embryos that were transferred, the embryo with the greatest number of cells in the cohort transferred and the percentage of embryos that were faster cleaving., Results: Thirty seven percent (15/41) of infants resulting from the transfer of exclusively slower-growing embryos were girls and 38% (24/36) of the infants from the faster-growing embryos were girls (NS). The analysis all 213 babies born after 145 embryo transfer procedures did not suggest any differences in embryo cleavage rates in embryo transfers leading to male versus female infants., Conclusions: A greater number of boys born was not observed after transfer of faster-cleaving embryos as has been described in other animal species. The race to be male may not occur until later cleavage divisions or may not occur in the human embryo.

Human proteins normally used to supplement human in vitro fertilization-embryo transfer (IVF-ET) culture media were tested for their effects on mouse embryo development from the zygote stage. These proteins included follicular and luteal-phase maternal sera, fetal cord sera, and both human and bovine serum albumin. Our results revealed that both maternal and fetal cord sera did not permit mouse blastocyst formation. Furthermore, predialysis of the human maternal sera and removal of IgG by protein A column chromatography did not improve their support of mouse embryonic development to the blastocyst stage. Similar detrimental effects were observed with maternal sera from term-pregnant IVF-ET patients. Interestingly, these serum samples had supported the in vitro growth of the human zygotes which resulted in these patients' pregnancies. Only some batches of human serum albumin supported mouse blastocyst formation, whereas all sources of bovine serum albumin were effective in this regard. These results raise the question of the suitability of the mouse embryo culture system as a quality control for the testing of protein supplements for human IVF-ET.

Using an immunofluorescence technique on ovarian sections, zona-immunoreactive components were detected in the cytoplasm of the oocyte from the beginning of its growth, when it is surrounded by only a thin squamous follicular cell layer, up to the end of its growth. In parallel with oocyte growth, the staining intensity decreased in the ooplasm. No staining was observed in the cytoplasm of the granulosa cells during normal follicular development in adult cyclic females. However, staining of the granulosa cells was observed at some stages of follicular development in immature females. This staining was especially evident in the ovaries of immature females (22 or 26 days old) stimulated with PMSG. In addition, the staining of the granulosa cells was consistently observed in ovaries showing an abnormal histology. Increased staining of the zona at its outer and inner regions could be distinguished in normal follicles, but when staining occurred on the granulosa cells no such pattern was observed over the zona matrix. These studies indicate that the oocyte itself but not the granulosa cells elaborates the native immunogenic material of the zona pellucida. The administration of PMSG at particular stages of ovarian differentiation interferes with follicular development leading to an abnormal extracellular assembly of the zona and its degradation (phagocytosis) by the surrounding granulosa cells.

The effects of various substrates (15 mM glucose, 5 mM glucose, 20 mM acetate, or a combination of these substrates) on the coronary blood flow and on the energetic status of myocytes were studied in isolated perfused rat hearts. We demonstrate that low level glucose (5 mM) or high concentration of acetate (20 mM) leads to a simultaneous fall in tissue ATP, rise in tissue adenosine, and significant increment in coronary blood flow. The latter effect is especially marked with 20 mM acetate. Dipyridamole (10(-6) M) does not enhance the vasodilatation induced by acetate. The provision of 15 mM glucose together with 20 mM acetate fully prevents these changes, indicating that the vasodilatation induced by acetate is probably mediated by metabolic changes. The evidence supports the concept that a redistribution of blood flow together with a fall in tissue ATP may explain some of the adverse effects of acetate dialysis in man, and suggests that the provision of glucose may alleviate these changes.

The concentration of phosphate-containing metabolites in the resting gastrocnemius muscle of three patients in chronic renal insufficiency was monitored by NMR spectroscopy before and during their regular hemodialysis using a conventional acetate bath. During dialysis, the muscle ATP concentration did not change, and no significant pyrophosphate accumulation was seen. The intracellular and extracellular inorganic phosphate concentration fell as expected. It is concluded that the rapid metabolism of acetate induced by dialysis does not affect the concentration of high energy phosphate-metabolites in the resting muscle of acetate-tolerant patients.