Biomedical subjects

K H Chao

Publications and source records attributed to K H Chao.

At least 19 recordsLinked to original sources

PubMed1998

The simplified two-pipette technique is more efficient than the conventional three-pipette method for blastomere biopsy in human embryos.

OBJECTIVE: To evaluate the efficiency and efficacy of a simplified two-pipette technique in comparison to the conventional three-pipette method; in the two-pipette method, a single, larger drilling/biopsy pipette is used to perform zona pellucida (ZP) drilling and blastomere aspiration for embryo biopsy. DESIGN: A preclinical, prospective, randomized, in vitro experiment. SETTING: The reproductive unit of a university teaching hospital. PATIENT(S): Ninety-five excess embryos at the two- to four-cell stage were obtained from 35 patients undergoing IVF. INTERVENTION(S): At the six- to eight-cell stage, 88 embryos were allocated randomly to three groups: group I for the conventional method (n = 29), group II for the simplified technique (n = 30), and group III for controls (n = 29). The embryos then were cultured in vitro. The retrieved blastomeres were fixed and examined with fluorescence in situ hybridization using X and Y probes simultaneously. MAIN OUTCOME MEASURE(S): Biopsy time, successful retrieval of a blastomere, fixation of the cell, signals developed from fluorescence in situ hybridization, and growth potential and hatching capacity of the biopsied embryos were evaluated. RESULT(S): The mean time (+/- SD) for biopsy of each embryo in group I (435 +/- 137 seconds) was significantly longer than that in group II (126 +/- 32 seconds). The success rates for obtaining an intact blastomere were not different between group I (93%) and group II (97%). The growth capacity to the blastocyst stage was similar among the three groups (34%, 37%, and 38%, respectively). However, the ZP-drilled and biopsied embryos of groups I and II had higher percentages of hatching (34% and 37%, respectively) and complete hatching (17% and 20%, respectively) than did those of group III (10% and 0, respectively). The blastomeres obtained by biopsy in groups I and II were equally fixed (90% vs. 90%, respectively) and shown in fluorescence in situ hybridization (79% vs. 80%, respectively). CONCLUSION(S): Compared with the conventional method, the simplified technique is more efficient and equally efficacious for blastomere biopsy in preimplantation genetic diagnosis.

Biopsy↗

PubMed1998

Effects of repeated abdominal paracentesis on uterine and intraovarian haemodynamics and pregnancy outcome in severe ovarian hyperstimulation syndrome.

The aims of this study were to investigate the effects of paracentesis on uterine and intraovarian haemodynamics by colour Doppler ultrasound and the influences of repeated paracentesis on pregnancy outcome in severe ovarian hyperstimulation syndrome (OHSS). Forty-one abdominal paracenteses were performed on seven pregnant women with tense ascites and eight thoracocenteses were performed on three pregnant women with pleural effusion. Pulsatility index (PI) and maximum peak systolic velocity (MPSV) of uterine and intraovarian arteries were measured before and after each intervention. The mean PI of uterine arteries was decreased significantly after paracentesis, but not after thoracocentesis. Furthermore, uterine PI was decreased in 13 out of 14 (92.9%) paracenteses with <2500 ml ascites removed, compared with eight out of 13 (61.5%) with >2500 ml ascites removed. After paracentesis, there were no significant changes in the intraovarian PI and MPSV in either group. The 24-hour urine output increased significantly in the paracentesis group, but not in the thoracocentesis group. There were no significant changes in haematocrit and electrolytes as a result of paracentesis. However, gradual falls in serum total proteins and albumin concentrations were observed in all patients after repeated paracentesis, necessitating post-paracentesis albumin infusion. There was no significant difference in miscarriage rates between the two groups. We conclude that repeated abdominal paracentesis increases uterine perfusion and has no adverse effects on pregnancy outcome in severe OHSS. Extraction of 2500 ml of ascitic fluid did not impair uterine perfusion.

Adult↗

PubMed1998

Cornual pregnancy after IVF-ET. A report of three cases.

BACKGROUND: Ectopic pregnancy is a well-known complication of in vitro fertilization/embryo transfer (IVF-ET). Although ectopic pregnancy after IVF-ET in patients with prior bilateral salpingectomy is uncommon, it can occur and may be a cornual implantation. Because of the possibility of early rupture with severe hemorrhage, special attention to cornual pregnancy after IVF-ET is warranted. CASES: Three cases of cornual pregnancy occurred after IVF-ET. Two of these patients had prior bilateral salpingectomy, whereas another had prior tuboplasty for tubal disease. CONCLUSION: Patients who had prior salpingectomy or tuboplasty with cornual patency undergoing IVF-ET are at risk for cornual pregnancy. Close monitoring of pregnancies in these patients is important to prevent a deleterious delay in treatment of a cornual pregnancy.

Adult↗

PubMed1998

Controlled ovarian hyperstimulation and intrauterine insemination in subfertility. How many treatment cycles are sufficient?

OBJECTIVE: To examine the combined effects of factors influencing the pregnancy rates following controlled ovarian hyperstimulation (COH) and intrauterine insemination (IUI). STUDY DESIGN: After utilizing the couple as the unit of analysis, 416 couples undergoing COH and IUI with the husband's sperm were studied retrospectively. RESULTS: The woman's age, postwash total motile sperm counts and number of IUI treatment cycles were three major factors that affected the results of IUI. The predicted cumulative pregnancy rates under different values of the above three factors were therefore calculated using multiple logistic regression analysis and were compared to the pregnancy rate achieved by a cycle of in vitro fertilization and tubal embryo transfer. CONCLUSION: Three treatment cycles of IUI are optimal for women aged < or = 34 with postwash total motile sperm counts > 5 x 10(6). For women either aged > or = 35 or with postwash total motile sperm counts < 5 x 10(6), the cumulative pregnancy rates achieved by IUI are extremely low, and other means of assisted reproduction are recommended for couples in those categories. However, this conclusion pertains to our clinics, and other institutions should establish their own data.

Adult↗

PubMed1997

A simplified technique for embryo biopsy: use of the same micropipette for zona drilling and blastomere aspiration.

PURPOSE: Using different micropipettes for zona drilling and blastomere aspiration for embryo biopsy is prevalent at centers of preimplantation genetic diagnosis. The purpose of our study was to simplify the technique by using only one micropipette. METHODS: In this animal model, ICR mouse embryos at the four-cell stage (n = 446) were randomly allocated into two groups; a biopsied group (n = 224) for blastomere aspiration and a control group (n = 222) without micromanipulation. We used a drilling/biopsy micropipette to drill a hole in the zona by expulsion of acidified Tyrode's solution and to aspirate the blastomere by gentle suction with the same micropipette and pull it out of the zona. One blastomere was biopsied from each embryo. RESULTS: In all, 222 (99.1%) intact blastomeres were successfully biopsied from 224 embryos. Only two blastomeres were damaged during aspiration. The capacity for blastocyst development (92.4 vs 93.7%) was not different between the two groups, but the percentages of embryos hatching (51.8 vs 18.0%) and hatched (29.9 vs 8.1%) were significantly higher in the biopsied group than in the control group. CONCLUSIONS: This simplified technique of embryo biopsy is safe and highly efficient for obtaining blastomeres for preimplantation genetic diagnosis and may also facilitate hatching of the blastocysts.

Animals↗

PubMed1997

Assisted hatching increases the implantation and pregnancy rate of in vitro fertilization (IVF)-embryo transfer (ET), but not that of IVF-tubal ET in patients with repeated IVF failures.

OBJECTIVE: To assess the effect of augmenting IVF with assisted hatching in the treatment of patients with repeated IVF failures. DESIGN: Prospective randomized study. SETTING: Division of Reproductive Endocrinology and Infertility of National Taiwan University Hospital. PATIENT(S): From July 1993 to February 1996, 49 patients with repeatedly failed IVF were treated with assisted hatching and were compared with 51 control subjects without assisted hatching. INTERVENTION(S): Assisted hatching. MAIN OUTCOME MEASURE(S): Pregnancy rate and implantation rate per embryo after IVF-ET or IVF-tubal ET (TET) were measured. RESULT(S): The pregnancy rate (PR) in the assisted hatching group was found to be 36.7% compared with 19.6% in the control group, but the difference was not significant. When only patients receiving IVF-ET were considered, it was observed that the PR was significantly higher in the assisted hatching group than the control group (42.4% versus 16.1%). With IVF-TET however, the PR was found to be similar in both assisted hatching and control groups (25.0% and 25.0%, respectively). The rate of embryonic implantation in the IVF-ET patients was 11.0%, which was significantly higher than that of control embryos (3.7%). CONCLUSION(S): These results implied that IVF-ET, combined with assisted hatching, may improve the PR and implantation rate in patients with repeated IVF failures, but the same was not true in the case of IVF-TET.

Embryo Implantation↗

PubMed1997

Peritoneal interleukin-10 increases with decrease in activated CD4+ T lymphocytes in women with endometriosis.

This study was performed to determine whether peritoneal T cells are suppressed in the CD4+ or CD8+ T cell subpopulation and whether they are Th1 or Th2 predominant in women with endometriosis. Immune cells in the peritoneal fluid (PF) were obtained from women undergoing laparoscopy for endometriosis or tubal ligation. Three-colour flow cytometry was utilized for immunophenotyping of peritoneal fluid mononuclear cells (PFMC). Concentrations of interleukin (IL)-4, IL-5 and interferon-gamma (IFN-gamma) produced by PFMC with and without mitogen stimulation and concentrations of IL-10 and IL-12 were measured in PF. The peritoneal T lymphocytes were predominantly of the Th1 type that produced much more IFN-gamma but less IL-4 or IL-5 in women with or without endometriosis. The decrease in peritoneal lymphocytes was significant in the HLA-DR+ CD4+ CD3+ subpopulation and the concentrations of peritoneal IL-10 and IL-12 were significantly elevated in women with early stage endometriosis. There was impaired IL-5 production by PFMC after phytohaemagglutinin stimulation in women with advanced stage endometriosis. We concluded that the activated peritoneal CD4+ Th1 cells from the women with endometriosis were decreased in number. The suppression of these T cells may be due to the elevation of IL-10 and IL-12 in the peritoneal fluid.

CD4-Positive T-Lymphocytes↗

PubMed1997

Paired human chorionic gonadotrophin determinations for the prediction of pregnancy outcome in assisted reproduction.

The aim of this study was to determine the prognostic value of single and paired measurements of serum concentrations of human chorionic gonadotrophin (HCG) for successful pregnancy following in-vitro fertilization (IVF) and tubal embryo transfer (TET). We analysed serum HCG concentrations 15 and 22 days after IVF or TET in 198 conception cycles. Cut-off values of serum HCG were determined by a receiver operating characteristic (ROC) curve. On the basis of single HCG samples on day 15 (HCG15) after transfer, using a cut-off value of HCG15 = 150 mIU/ml, the sensitivity was 71% and the specificity was 77%. The positive predictive value (HCG15 > or = 150 mIU/ml indicating a normal pregnancy) was 89%, while the negative predictive rate (HCG15 < 150 mIU/ml indicating an abnormal pregnancy) was 51%. Patients with HCG15 < 150 mIU/ml but HCG22/HCG15 ratio > or = 15, still had a 90% chance of normal pregnancy. However, in patients with HCG15 < 150 mIU/ml and an HCG22/HCG15 ratio < 15, there was an 84% chance of an abnormal pregnancy. We conclude that a single HCG15 determination combined with the ratio of HCG22 to HCG15 has a higher diagnostic accuracy for prediction of pregnancy outcome than either analysis alone.

Chorionic Gonadotropin↗

PubMed1997

Total antioxidant status and nitric oxide do not increase in peritoneal fluids from women with endometriosis.

To explore the role of nitric oxide (NO) and oxidative stress in the pathogenesis of adhesion formation and in endometriosis-associated infertility, we examined the peritoneal total antioxidant status (TAS) and the concentrations of products of NO metabolism in women with endometriosis (early stage, n = 12; advanced stage, n = 12) and in fertile women without endometriosis (n = 10). Peritoneal CA 125 and oestrogen and progesterone concentrations were also measured to examine their contributions to TAS and the production of NO. We failed to demonstrate any significant difference in TAS and in the products of NO metabolism in peritoneal fluids among women with early and advanced stages of endometriosis compared with fertile women without endometriosis during the early follicular phase. TAS and the concentration of the products of NO metabolism were not related to concentrations of CA 125, oestrogen or progesterone. The concentration of CA 125 in serum, but not in peritoneal fluid, was positively correlated with the severity of endometriosis. The volume of peritoneal fluid and the progesterone concentration were significantly increased in the group with advanced endometriosis. TAS and the concentration of the products of NO metabolism did not increase in peritoneal fluids from women with endometriosis during the early follicular phase. Their role in the pathophysiology of endometriosis needs to be explored further.

Antioxidants↗

PubMed1997

KTP laser and neutral red phototherapy of human squamous cell carcinoma.

Neutral red (NR) is a cationic, nontoxic vital dye employed as a histologic stain for proliferating cells; it has been used clinically for photodynamic treatment of herpes simplex virus lesions. NR is selectively taken up and concentrated by mitotic cells, an important characteristic for more effective antineoplastic agents. In the present study, UCLA-SO-P3 human squamous carcinoma cells displayed minimal toxicity when incubated with up to 50 microg/ml NR in the absence of light. However, cells incubated with greater than 0.5 microg/ml NR followed by exposure to KTP laser light at 532 nm exhibited nearly 100% tumor cell death. The degree of cell toxicity was proportional to NR dose and laser light fluence. This study demonstrates that NR is an excellent cancer cell photosensitizer in vitro, and, after adding additional in vivo preclinical testing, may prove to be a useful agent in photodynamic destruction of head and neck tumors.

Antineoplastic Agents↗

PubMed1997

Serum estradiol level and oocyte number in predicting severe ovarian hyperstimulation syndrome.

Ovarian hyperstimulation syndrome (OHSS) is a relatively common and potentially life-threatening complication of ovarian stimulation, the pathogenesis of which remains unclear. To clarify the predictive values of serum estradiol levels and oocyte number in severe OHSS, and to investigate the impact of high serum estradiol levels on pregnancy outcome, we retrospectively analyzed clinical data from 431 cycles of ovarian stimulation for assisted reproduction performed from 1993 through 1995. Receiver operating characteristic plots were used to estimate the predictive power of the measured variables. The overall frequency of severe OHSS was 5.5%. Using a serum estradiol level of 3,600 pg/mL as the minimum cut-off value, the sensitivity was 58%, with a specificity of 92%, a positive predictive value of 29%, and a negative predictive value of 97%. The predictive power was similar when a cut-off point of 20 oocytes retrieved was used. The two criteria together gave a sensitivity of 33%, a specificity of 92%, a positive predictive value of 40%, and a negative predictive value of 98%. One of seven oocyte donors developed severe OHSS. The pregnancy rate was higher in patients with severe OHSS than in patients who did not develop this syndrome (73.9% vs 32.5%) but the pregnancy outcomes were not significantly different. We conclude that elevated estradiol concentrations and oocyte number appear to be helpful in predicting severe OHSS, but neither parameter by itself is predictive. This syndrome is rare in the absence of luteal hCG support, either exogenous or pregnancy-derived; when it occurs, there are usually extremely high preovulatory estradiol concentrations and numerous oocytes retrieved. High serum estradiol levels are unlikely to have adverse effects on pregnancy outcome in patients with severe OHSS.

Adult↗

PubMed1997

Long GnRH-agonist protocol in an IVF program. Is it appropriate for women with normal FSH levels and high FSH/LH ratios?

OBJECTIVE: To determine whether subjects with an elevated ratio of follicle-stimulating hormone (FSH) to luteinizing hormone (LH) but normal basal FSH levels should be regarded as poor responders to controlled ovarian hyperstimulation. STUDY DESIGN: One hundred twenty-five women undergoing in vitro fertilization (IVF) for the first time were recruited in this retrospective cohort study. Women over 40 years old or having serum basal FSH > 10 mIU/mL were excluded. RESULTS: Various cutoff values for the FSH/LH ratio were chosen, and the ratio demonstrated that pregnancy rates were apparently higher in patients with the long protocol than with the short one if they had an FSH/LH ratio < 3.0 (48.5% vs. 25.8%, P = .034), < 2.5 (53.3% vs. 28.6%, P = .030) or < 2.0 (57.8% vs. 21.7%, P = .005). Pregnancy rates were similar with the long and short protocols in patients with FSH/LH > or = 3.0 (57.1% vs. 70%, P = .521), FSH/LH > or = 2.5 (40% vs. 53.8%, P = .435) or FSH/LH > or = 2.0 (40% vs. 55.6%, P = .281). CONCLUSION: This study failed to demonstrate that FSH/LH was a useful parameter for predicting reproductive outcome in IVF programs and for patient selection for the long or short gonadotropin-releasing hormone agonist protocol.

Buserelin↗

PubMed1996

Activation status of T and NK cells in the endometrium throughout menstrual cycle and normal and abnormal early pregnancy.

Endometrial lymphocytes were studied at all stages throughout the menstrual cycle and early pregnancy by flow cytometry to examine different lymphocyte subpopulations and the expression of the T- and NK-cell activation markers. After pregnancy, CD8+CD3+ lymphocytes were decreased in the decidua. In both endometrium and decidua, more T cells expressed CD69, CD71, HLA-DR, and CD38 antigens than in peripheral blood. After pregnancy, CD71+CD3+ lymphocytes were further increased. CD25+CD3+ lymphocytes decreased significantly in the endometrium and decidua of ectopic pregnancies, but not in the decidua of normal pregnancies. These findings indicate that T cells are regionally activated in the first trimester, and it may be the result of the stimulation by fetal antigens. NK cells were the most abundant cell type in the decidua, which expressed the phenotype CD16- CD56+, and CD57-CD56+. The proportion of activated decidual NK cells was increased in anembryonic pregnancies more than in normal pregnancies, although the total NK subpopulation was similar in both groups. This might result in increased NK cytotoxicity in anembryonic pregnancies. In conclusion, T cells are activated, but NK cytotoxicity is decreased in the decidua of early normal pregnancies. This might be important in the control of trophoblast growth and placental development.

Abortion, Induced↗

PubMed1996

Decrease in interferon gamma production and impairment of T-lymphocyte proliferation in peritoneal fluid of women with endometriosis.

OBJECTIVE: Our purpose was to verify regional immune modulations and to test the effect of gonadotropin-releasing hormone agonist in women with endometriosis. STUDY DESIGN: Concentrations of peritoneal cytokines, including interleukin-1 beta, interleukin-2, soluble interleukin-2 receptor, interleukin-6, granulocyte-monocyte colony-stimulating factor, interferon gamma, and tumor necrosis factor-alpha were compared in women with and without endometriosis. Peritoneal cytokine and interleukin-2 production were examined by adding various mitogens to peritoneal fluid mononuclear cells of women with advanced endometriosis before and after gonadotropin-releasing hormone agonist treatment. RESULTS: A significant increase in peritoneal interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha and a decrease in interferon gamma were noted in women with endometriosis. After gonadotropin-releasing hormone agonist treatment interleukin-6 decreased and interferon gamma increased. A significant impairment of interleukin-2 production of peritoneal fluid mononuclear cells by phytohemagglutinin and pokeweed mitogen stimulation was demonstrated in endometriosis, and production could be restored after gonadotropin-releasing hormone agonist treatment. CONCLUSION: These results indicate that regional immunologic dysfunction might be invoked in the disease process of endometriosis.

Ascitic Fluid↗

PubMed1996

The value of human growth hormone as an adjuvant for ovarian stimulation in a human in vitro fertilization program.

The purpose of this study was to evaluate whether the combined treatment of growth hormone (GH) and gonadotropins can improve ovulation stimulation in previously poor responders. Twelve patients who, had suboptimal responses in previous in vitro fertilization cycle were enrolled. They underwent 1 cycle with gonadotropin-releasing hormone analogue (GnRH-a) and gonadotropins and another cycle with GnRH-a, gonadotropins, and GH. Serum gonadotropins, insulin-like growth factor-1 (IGF-1), and sex steroids, including estradiol (E2), progesterone (P4), testosterone, and androstenedione were measured on Day 2 and during ovulation induction. The serum IGF-1 level was higher in the GH cycle. There were no significant differences in the levels of the serum luteinizing hormone, E2, P4, testosterone, and androstenedione between the 2 cycles, so was IGF-1, E2 and P4 in follicular fluid. Co-treatment with GH did not improve the ovarian response. However, the GH cycles had better performance in terms of the number of oocytes fertilized and the pregnancy rate.

Androstenedione↗

PubMed1996

The suppression of peritoneal cellular immunity in women with endometriosis could be restored after gonadotropin releasing hormone agonist treatment.

PROBLEM: Our previous study reported that peritoneal natural killer (NK) cytotoxicity and CD3+CD25+ lymphocyte subpopulation were suppressed in women with advanced endometriosis. Whether these phenomena are general for all stages of endometriosis and whether these alterations could be restored by long-term use of gonadotropin releasing hormone agonist (GnRHa) are further tested in this study. METHOD: Lymphocyte subpopulations (B cells, NK cells, T cells, and T-cell activation markers such as CD69, HLA-DR, and CD25) and NK cell cytotoxicity of peripheral blood and peritoneal fluid by dual-color flow cytometry and 51Cr release assay in 30 cases of endometriosis were compared with those in 26 controls. We also compared these changes before and after 6-month treatment with GnRHa for advanced endometriosis. RESULTS: Compared with the controls, only those women with advanced endometriosis showed lower NK cytotoxicity in peritoneal fluid mononuclear cells (PFMC). The CD3+CD69+ lymphocyte subpopulation decreased in peripheral blood mononuclear cells (PBMC) of advanced endometriosis, while the CD3+CD25+ lymphocyte subpopulation decreased in both PBMC and PFMC of mild and advanced endometriosis. After GnRHa treatment, the CD3+CD69+ lymphocyte subpopulation increased in both PBMC and PFMC and the CD3+CD25+ lymphocyte subpopulation increased in PFMC, but not in PBMC. CONCLUSION: Impaired local immunological function in the PF of endometriosis was confirmed by this study and the impairments could be restored after long-term GnRHa therapy.

Ascitic Fluid↗

PubMed1996

How many embryos should be transferred in in vitro fertilization and tubal embryo transfer?

In order to achieve higher pregnancy rates, more than one embryo is usually transferred in in vitro fertilization (IVF) programs. Tubal embryo transfer (TET) produces an even higher pregnancy rate. However, the number of embryos that should be transferred in TET programs remains to be clarified. We studied a series of 241 consecutive TET cycles and analyzed their clinical characteristics, embryo numbers, cumulative embryo score (CES), and pregnancy outcomes. The results demonstrated that 1) four embryos was an adequate number to obtain a satisfactory pregnancy rate and fewer multiple pregnancies, 2) older patients and cases with male factor had less chance of pregnancy and more than four embryos could be transferred, and 3) CES values > 40 were preferred, but for young patients with unusually high anxiety about multiple pregnancies, a CES of 21 to 40 was optimal. A policy of transfer that limits transfer to a maximum of two, three or four embryos is not suitable in all cases and other factors (eg, repeated IVF failure or older age) should be individually considered. Therefore, two equations utilizing CES, age and failure of previous TET as the factors were developed to help practitioners to evaluate how many embryos should be transferred on an individual basis.

Adult↗

PubMed1995

Comparison between a two-layer discontinuous Percoll gradient and swim-up for sperm preparation on normal and abnormal semen samples.

PURPOSE: This work was to compare the effects of Percoll gradient and swim-up treatments for sperm preparation on the percentage of progressive motility, recovery of motile sperm, removal of debris, percentage of normal forms according to strict criteria, and movement characteristics of sperm using computer-assisted velocity analysis. RESULTS: In total, 50 semen samples from 50 patients were tested and divided into two groups: a normal group (n = 27) with normal parameters and an abnormal group (n = 23) with abnormal parameters. The results in both the normal and abnormal groups revealed that the sperm concentration in the Percoll samples was significantly greater than that in the swim-up samples. Although the percentage of progressive motility was greater in the swim-up samples than in the Percoll samples, the number of motile sperm, reflecting the percentage of motile sperm recovery, was till greater in the Percoll samples. The debris of semen was equally removed by both methods and the percentage of normal forms was also similar in the samples treated according to these two procedures. Both curvilinear velocity (VCL) and straight-line velocity (VSL) of sperm were significantly greater in the swim-up samples than in the Percoll samples. Sperm from the swim-up procedure also showed a greater mean amplitude of lateral head displacement than that from the Percoll gradient procedure, but the distinction was insignificant. CONCLUSION: The Percoll gradient technique, by recovering more motile sperm, may be applied to prepare oligospermic samples. The swim-up method may become the standard choice to prepare normal semen which could obtain sufficiently motile sperm, due to its simplicity and recovered sperm with superior motility.

Centrifugation, Density Gradient↗