Prostaglandins and post-abortion luteolysis in early pregnancy.

This study was undertaken to determine if post-abortion luteolysis in early pregnancy could be accelerated by the administration of 15(S)15-methyl-PGF2alpha(15-me-PGF2alpha) or delayed following pretreatment with indomethacin. Thirty-nine women were divided into four groups: 7 women were given 400mug 15-me-PGF2alpha extra-amniotically one hour prior to vacuum aspiration; 14 were pretreated with oral indomethacin (50 mg X4) over 24 hours; 7 were given indomethacin (50mg X 6) over 36 hours and 11 served as controls. Plasma progesterone and estradiol were measured at fixed intervals before and after abortion. There was a rapid drop in the plasma progesterone within the first hour after abortion followed by an exponential decline over the next 23 hours. The plasma estradiol fell rapidly duriing the same period. Under the experimental conditions of this study neither 15-me-PGF2alpha nor indomethacin exerted a significant effect on the decline in luteal function. These results are interpreted as suggesting that factors other than prostaglandins have a more significant role in post-abortion luteolysis.     

Prostaglandins and post-abortion luteolysis in early pregnancy

This study was undertaken to determine if post-abortion luteolysis in early pregnancy could be accelerated by the administration of 15(S)15-methyl-PGF_2α (15-me-PGF_2α) or delayed following pretreatment with indomethacin. Thirty-nine women were divided into four groups: 7 women were given 400μg 15-me-PGF_2α extra-amniotically one hour prior to vacuum aspiration; 14 were pretreated with oral indomethacin (50 mg X4) over 24 hours; 7 were given indomethacin (50mg X 6) over 36 hours and 11 served as controls. Plasma progesterone and estradiol were measured at fixed intervals before and after abortion. There was a rapid drop in the plasma progesterone within the first hour after abortion followed by an exponential decline over the next 23 hours. The plasma estradiol fell rapidly during the same period. Under the experimental conditions of this study, neither 15-me-PGF_2α nor indomethacin exerted a significant effect on the decline in luteal function. These results are interpreted as suggesting that factors other than prostaglandins have a more significant role in post-abortion luteolysis.     

Effect of intravenous infusion of 15-methyl-PGF2α on plasma hormone levels during early pregnancy

Ten pregnant women (7th–8th week of pregnancy) obtained an intravenous infusion of 15-methyl-prostaglandin-F_2α (2.5 μg/min) until clinical signs of abortion occurred or up to 7 hours. Surgical removal of the products of conception was performed 4–7 hours after the start of the infusion. Blood samples were taken prior to and during the infusion and then during the post-abortion period. The plasma levels of both progesterone and estradiol exhibited a significant decrease (p<0.001 and p<0.05, respectively) one hour after the beginning of infusion and remained reduced by approximately 35 and 45 per cent, respectively, during the entire infusion period. A drop in the levels of both steroids was seen after surgical interruption. This was followed by a gradual decrease to non-pregnancy levels. The levels of cortisol increased significantly (p<0.01) by approximately 60 per cent, starting with the second hour of infusion. Following surgical interruption, the levels dropped to pre-infusion values. 17-Hydroxyprogesterone levels increased (p<0.05) above the pretreatment levels by approximately 25 per cent, starting with the third hour of infusion. These levels were not correlated with those of cortisol during the infusion period. Following surgical interruption the plasma levels of 17-hydroxyprogesterone returned to non-pregnancy levels.     

Intra-amniotic prostaglandin F2α and urea for abortion

Prostaglandin F_2α (PGF_2α) 20 mg combined with urea 80 g was injected intra-amniotically in 20 patients to induce mid-trimester abortion. Abortion resulted in all subjects within 24 hours in a mean time of 12 hours 38 minutes (range 5 hours 50 minutes to 20 hours 45 minutes).Plasma sex steroids were evaluated before and hourly for 5 hours after the injection. A progressive decline in levels occurred with time. Decreases in plasma progesterone, estrone, estradiol and estriol were significant as soon as one hour after injection.Gastrointestinal side effects occurred with a greater frequency than when a comparable dose of PGF_2α is given alone and 2 patients had small cervical lacerations requiring suture. Further studies are indicated to establish whether a lower dose of PGF_2α will be associated with fewer side effects and be as effective.     

Abortifacient efficiency of 15(S)15-methyl-prostaglandin F2α-methyl ester administered vaginally during early pregnancy

Forty early pregnancies (menses delay 13 – 27 days) were terminated by administering four vaginal suppositories each containing 1.0 or 1.5 mg of 15(S)15-methyl-prostaglandin F_2α-methyl ester, one every third hour. In 14 cases serial measurements of serum estradiol and progesterone were performed during and after therapy. Uterine contractions and bleeding started 1 – 17 hours after administration of the first suppository. Abortion was complete after one week in five women (13 %), and after two weeks in 30 (75 %). A curettage was performed on eight women, residual placentral fragments were found in seven and pregnancy continued in one woman. Mild diarrhoea (65 %) and vomiting (40 %) were the major side-effects, despite premedication. Estradiol and progesterone levels fell progressively during the therapy. Self-administration of 4 or 6 mg of the methyl ester caused too low a rate of complete abortion for use in practice, but it may be a valuable and practical agent for preoperative dilation of the cervix.     

Estrogen-induced luteolysis in the rhesus monkey: Reversal with indomethacin

Normally cycling Rhesus monkeys were treated with diethylstilbestrol (25 mg/day) alone or in combination with indomethacin (25 mg/day) for five consecutive days beginning in the early luteal and mid-luteal phase of the menstrual cycle. Blood specimens were obtained daily to monitor corpus luteum function (progesterone), and the length of each menstrual cycle was recorded. Diethylstilbestrol alone cause premature luteolysis as indicated by decreasing plasma progesterone and shortened menstrual cycle, and indomethacin effectively blocked the luteolytic action of diethylstilbestrol. These results suggest that the probable mechanism of diethylstilbestrol action in causing luteolysis is mediated via the prostaglandins.     

环境激素睾丸酮和雌二醇对卜氏晶囊轮虫繁殖影响

采用生态学实验方法, 研究睾丸酮(Testoterone)和雌二醇(Estradiol)单一及组合对卜氏晶囊轮虫生命周期中主要发育阶段历时和种群增长率的影响。结果显示: 睾丸酮(T)的48h LC₅₀为9.56 mg/L, 直线回归方程为Y=1.98X+3.06 (R2=0.92); 雌二醇(E) 48h LC₅₀为8.18 mg/L, 直线回归方程为Y=3.53X+1.78 (R2=0.92)。除0.5 mg/L浓度组外, 睾丸酮和雌二醇均显著缩短轮虫平均寿命、减少后代幼体个数以及降低种群增长率(r)。其中8 mg/L睾丸酮与对照组相比显著延长轮虫生殖前期11.71%、平均寿命缩短52.22%, 后代个数减少82.20%; 8 mg/L雌二醇与对照组相比, 生殖前期显著延长7.10%, 平均寿命缩短49.75%, 后代个数减少83.33%。在2种激素定量组合(8 mg/L)实验中, 与对照组相比, 各实验组的生殖前期显著延长34.17%—48.01%, 平均寿命显著缩短31.56%—42.12%, 后代个数明显减少24.44%—80.33%; 第5天, 处理组T₆E₂、T₄E₄、T₈E₀、T₀E₈和T₂E₆的r值比对照组分别降低30.00%、37.14%、41.43%、60.00% 和65.71%。研究结果显示睾丸酮和雌二醇对卜氏晶囊轮虫生长发育及种群增长率有明显影响, 并且卜氏晶囊轮虫对雌二醇表现更为敏感, 2种激素呈现出协同作用。     

Effect of systemic or intrauterine injections of indomethacin on plasma oxytocin-neurophysin concentrations in ewes over the time of normal corpus luteum regression

This study was undertaken to investigate the effect of systemic or intrauterine injections of indomethacin, a known prostaglandin (PG) synthetase inhibitor, on peripheral plasma oxytocin-associated neurophysin (OT-N) concentrations in ewes over the time of expected luteolysis. In the first experiment, 9 ewes were given i.m. injections of indomethacin (4 mg/kg live weight, n = 4) or vehicle (n = 5) 3 times/day over Days 13-15 of the estrous cycle. Blood samples were collected at hourly intervals from 0700 h on Day 13 to 1800 h on Day 15 post-estrus. In the second experiment, indomethacin (20 mg, n = 5) or the injection vehicle (n = 4) was given twice daily into the uterine horn over Days 12-14 post-estrus. Blood samples were collected at hourly intervals from Day 12 to 14. In the third experiment, 4 additional ewes were bled at 5-min intervals from 1200 to 1600 h on Day 13 of the estrous cycle. Plasma samples were analyzed for OT-N and 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) to provide an indirect index for ovarian oxytocin and uterine prostaglandin F2 alpha release, respectively. Results from the first experiment indicated that surges in plasma OT-N concentrations occurred in the vehicle-treated ewes but were suppressed in ewes given systemic injections of indomethacin. Intrauterine indomethacin injections did not cause a significant reduction in the maximum peak height or number of peaks when compared with the control ewes. In the third experiment, there was a marked increase in plasma OT-N concentrations, but no significant rise in plasma PGFM concentrations in one ewe.(ABSTRACT TRUNCATED AT 250 WORDS)     

Termination of early gestation with vaginal prostaglandin F2α tablets

The abortifacient activity of prostaglandin F_2α was investigated by placing one or two 50 mg tablets of prostaglandin F_2α in THAM salt into the vagina of nine women less than 4 weeks pregnant at intervals of 2 to 4 hours for a 24 hour period. Serum levels of HCG, estradiol (E₂), progesterone and 17α-hydroxyprogesterone were measured by radioimmunoassay prior to starting therapy and at frequent intervals thereafter for 48 hours. All but two patients had significant side-effects, mainly diarrhea and vomiting, indicating that systemic absorption took place. Although bleeding was induced in 8 of 9 women, only 3 had complete abortions. A D&C was performed on all patients 48 hours after starting therapy. A significant fall in HCG levels was noted only in the patients who aborted. Only 3 of the 9 women had significant changes in steroid levels. A fall in progesterone and 17α-hydroxyprogesterone occurred in the 3 women who aborted and took place following the fall in HCG. Estradiol levels remained in the same range in all subjects. These findings indicate that prostaglandin F_2α when administered in this vehicle and this dosage is relatively ineffective as an abortifacient. When effective, its action would appear to be due to contractions of uterine muscle and not secondarily to luteolysis.     

Treatment with high-dose estrogen (diethylstilbestrol) significantly decreases plasma estrogen and androgen levels but does not influence in vivo aromatization in postmenopausal breast cancer patients

While growth factors and hormones are known to influence aromatase expression in experimental systems, little is know about potential factors influencing peripheral aromatization in postmenopausal women. The fact that peripheral aromatase activity is higher in old compared to young women and the finding of relatively high tissue estradiol (E₂) concentrations after the menopause suggests peripheral aromatization could be influenced by estrogen concentration. To test this hypothesis, we determined plasma hormone levels (n = 9) and in vivo aromatization (n = 3) in postmenopausal women suffering from advanced breast cancer before and during treatment (4 weeks) with diethylstilbestrol (DES) 5 mg three times daily. Plasma levels of cortisol (C), corticosteroid-binding globulin (CBG), and sex hormone binding globulin (SHBG) were significantly increased in all patients (P < 0.05 for all). While we found no change in total body aromatization and plasma estrone (E₁) levels, estradiol (E₂) and estrone sulfate (E₁S) were suppressed by a mean of 48.8 and 68.2%, respectively (P = 0.043 and 0.008). Surprisingly, plasma levels of androstenedione (A), testosterone (T), dehydroepiandrosterone (DHEA) and its sulfate (DHEAS) were also suppressed by a mean in the range of 32.1 to 52.6% (P < 0.05 for all androgens). In contrast, no change in plasma progesterone or 17-hydroxyprogesterone was found. Thus, one possible explanation to our findings could be that DES administered in high doses reduces 17,20-lyase activity in the adrenal gland.     

The efficacy and acceptability of the "prostaglandin impact" in inducing complete abortion during the second week after the missed menstrual period

Clinical research was undertaken using PGF2a (prostaglandin) to induce abortion in 22 pregnant women at 12 +/- 1 days following their missed menstrual period. The PG was administered in a 5 mg single intrauterine dose through the cervix for a 10-minute period. The PG administration caused increased uterine contraction within 20 minutes and raised intrauterine pressure which was sustained for 2 hours. During the initiation of the intrauterine pressure, bleeding started and progesterone and estradiol levels decreased and continued to do so. In those patients who had been sedated, side effects were minimal. At 24 hours following the PG administration, progesterone had been withdrawn at a rate of 44%, bleeding was continuing, and cervical dilatation was at approximately 1 cm. Complete abortion was achieved in 20 out of the 22 women. It is believed that the abortion was effected through the action of endogenous PG, due to the withdrawal effect on progesterone of the exogenously-administered PG.     

Effect of continuous infusion of a GnRH agonist (Buserelin) on ovarian hormone secretion and estrous cycle length in cows

The importance of the ovarian steroid hormones estradiol and progesterone in the control of luteolysis in domestic ruminants is well established. However, there is a lack of studies specifically investigating the effect of stimulating "physiological" changes in endogenous estradiol or progesterone secretion on subsequent luteolysis. In this study we have stimulated endogenous ovarian hormone secretion by infusion of the GnRH analogue, Buserelin, and have assessed the effect of these changes on the timing of luteolysis. Concentrations of estradiol and progesterone were monitored in plasma samples collected from 6 mature, cyclic, lactating, Friesian cows during a control cycle and during a cycle in which Buserelin was infused via osmotic minipump (8.6 microg/h) for 28 days starting on Day 2 of the cycle. Buserelin infusion had little effect on progesterone secretion but did result in a marked stimulation of estradiol secretion from Days 6 to 10 of the cycle (treated cycle 4.3+/-0.2 pg/mL; control cycle 1.8+/-0.3 pg/mL; P<0.001). In addition, there was a significant advancement in the timing of luteolysis during the Buserelin -infused cycle (Day 19.3+/-0.3 compared with Day 21.3+/-0.4; P<0.01). In this study, we have found that infusion of buserelin caused both a significant stimulation of estradiol secretion from the first follicle and a significant advancement in the timing of luteolysis. We hypothesise that the increased secretion of estradiol may have been involved in causing this advancement of luteolysis.     

Concentrations of 13, 14-dihydro-15-keto prostaglandin F2 alpha after pulsatile progesterone administration at the time of luteolysis of heifers

Progesterone was administered in pulses to 12 dairy heifers from days 17.5 to 22.5 post-estrus in order to determine its ability to modify secretion of PGF2 alpha around the time of luteolysis. Control heifers exhibited pulses of PGFM concomitant with a sharp decline in progesterone concentrations and thus these pulses were temporally associated with luteolysis. Additional pulses of PGFM were observed in heifers receiving exogenous progesterone, but these were not statistically predictable by either dose of progesterone (50 or 100 micrograms) or time of administration (3 or 6 hour intervals). However, all heifers (4/4) treated with progesterone at 3 hour intervals had additional pulses of PGFM as compared to only one heifer (1/4) treated at 6 hour intervals. When pulses of PGFM were induced by exogenous progesterone there was a substantial lag time between the initiation of progesterone treatment and their occurrence. The limited response to progesterone administration and the lack of synchrony is not consistent with an ability of exogenous progesterone to directly stimulate secretion of PGF2 alpha at the time of luteolysis.     

Abortifacient and endocrine effects of metergoline in beagle bitches during the second half of gestation

The purpose of this study was to investigate the abortifacient effects of high doses of metergoline when administered to pregnant beagle bitches during the second half of gestation and to define the endocrine effects of this treatment as represented by plasma progesterone and estradiol concentrations. Previously, metergoline had been shown to be incompletely luteolytic and induced abortion in only one of eight pregnant bitches when 0.4-0.5 mg/kg were administered twice daily for 5 days from Days 18 to 20 of diestrus. Nine pregnancies in six beagle bitches were used for the present study. Three bitches were treated in each of two consecutive pregnant cycles. Metergoline was administered at a dose of 0.6 mg/kg per os twice daily, starting on Day 28 after the cytological onset of diestrus. Abortion was induced in eight of the nine treated pregnancies and started after 3-23 days of treatment (mean 12.5 days, S.D. 6.4 days). The abortions were completed within 0.5-8 days (mean 2.2 days, S.D. 2.7 days). There were no side effects associated with metergoline treatment and none of the abortions was associated with complications that required intervention. In the single bitch that did not abort, an ovarian granulosa cell tumor was discovered when the single fetus had to be removed surgically at term. Plasma progesterone concentrations declined after the start of metergoline administration in all pregnancies but levels below 4.8 nmol/l were required for successful abortions. Plasma estradiol concentrations showed a tendency to decline and fluctuate concurrently with the plasma progesterone levels. However, suppression of plasma estradiol concentrations by metergoline was not as complete as the suppression of progesterone and did not seem a prerequisite for abortion. The hormone profiles and treatment period required for abortion tended to be similar for both cycles of the three bitches that were treated during two consecutive pregnancies. This suggests a bitch effect on the factors that determine the efficacy of metergoline to induce abortion. The large variation and length of the treatment period that was required until abortion commenced was probably related to the relatively early start of treatment compared to other studies. The results of this investigation suggest that, similar to other prolactin suppressing ergot derivatives, metergoline causes complete luteolysis and can be used to reliably induce abortion only during the last 3 weeks of gestation.     

Changes in estrogen levels in maternal venous plasma after intra-amniotic infusion of prostaglandin F2 for therapeutic abortion

Peripheral plasma levels of estrone, estradiol-17beta and estriol were measured by the method of Shutt and Cox in 10 women following intra-amniotic infusion of prostaglandin F2alpha (PGF2a) for therapeutic abortion. Initial dose was 30 mg, followed if necessary, by doses of 15 mg at 24 hours and 42 hours. Gestational age of pregnancies ranged from 14 to 19 weeks, with a mean of 16 weeks. All 10 women completely aborted. Mean induction-abortion interval was 24 + or - 12 hours. The mean estrone, estradiol 17beta and estriol levels declined to about half of the pre-infusion levels after 80% of the induction-abortion interval had elapsed. The main decline in estrogen levels occurred in individual women either during the 1st quarter or during the last quarter of the induction-abortion interval. There were no significant relationships between changes in estrogen levels and the interval from 1st administration of PGF2a to subsequent abortion.     

Interrelationship between plasma estradiol concentration and oxytocin-induced PGF2alpha release in heifers

The objective of this study was to determine if the increase in responsiveness to oxytocin toward the time of luteolysis was correlated with an increase in plasma estradiol in the cow. Six heifers each had a cannula placed in the jugular vein on Day 14 of the estrous cycle. Then, beginning on Day 15, growth of the largest follicles was determined by ultrasonography, and a blood sample was taken via the cannula for the measurement of progesterone and estradiol by radioimmunoassay (RIA). After the first blood sample, 3 more samples were taken at 10-min intervals, 100 IU oxytocin were injected into the vein, and a further 3 blood samples were taken at 15, 30 and 60 min after injection. The concentration of 13,14-dihydro-15-keto prostaglandin F2alpha (PGFM) was measured in these frequent samplings and was used to determine the ability of oxytocin to stimulate PGF2alpha release from the uterus. This procedure was repeated daily for at least 7 d. The results showed that the response to oxytocin increased before luteolysis and that there was a significant increase in the response to oxytocin (P<0.05) before any changes in plasma estradiol or progesterone were detected. These data show that an increase in estradiol secretion from the ovulatory follicle does not appear to initiate luteolysis.     

Associations among progesterone, estradiol-17 beta, oxytocin and prostaglandin in cattle treated with hCG during diestrus to extend corpus luteum function

Treatment of cattle during the middle of the luteal phase with appropriate doses of human chorionic gonadotropin (hCG) causes a 5 d extension of the estrous cycle. Three experiments were conducted to determine how treatment with hCG affected the pattern of secretion of prostaglandin F2 alpha, as indicated by blood levels of 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM). In experiment 1, Holstein cows were given saline (Sal) or hCG (10,000 IU, im) on d 10 of the estrous cycle and blood samples were collected over a 6 h period on d 14 and 18 during which oxytocin (10 and 100 IU, iv) was given at 2 and 4 h. Concentrations of PGFM before and after oxytocin were similar between Sal and hCG-cycles, but PGFM was higher on d 18 than d 14 (P less than 0.05). In experiment 2, episodic PGFM was measured from d 16 to 20 in cows given Sal or hCG on d 10. There was tendency for hCG to reduce PGFM baseline and pulse amplitude (P = 0.22). In experiments 1 and 2, estradiol increased during d 16 to 20 of Sal-cycles, but did not change during this period of hCG-cycles. Therefore, in experiment 3, Holstein heifers were given Sal or hCG (5000 IU, im) on d 10, followed by corn oil (Oil) or estradiol benzoate (EB; 200 micrograms, im, 2X/day) on d 15 to 18. No difference in progesterone secretion was observed between Sal-Oil and Sal-EB heifers; however, EB hastened luteolysis in hCG-treated heifers (P less than 0.05), without causing an increase in PGFM. Although subtle differences were seen in pulsatile PGFM, we conclude that hCG altered the pattern of estrogen secretion, and this led to delayed luteolysis.     

Stimulation of a pulse of LH and reduction in PRL concentration by a physiologic dose of GnRH before, during, and after luteolysis in heifers

A single physiologic dose (5.0 μg) of GnRH was given to 9 heifers each day (Hour 0) beginning on Day 15 postovulation until regression of the corpus luteum. Blood samples were taken each day for Hours -3, -2, -1, 0, 0.25, 0.5, 0.75, 1, 1.25, 1.50, 1.75, 2, 3, 4, and 5. Based on daily progesterone concentrations, data were grouped into phases of before (n=4), during (n=8), and after (n=7) luteolysis. The number of LH pulses with a peak at pretreatment Hours -2 or -1 (0.35 ± 0.12 pulses/sampling session) was less (P<0.0001) than for a pulse peak at posttreatment Hours 1 or 2 (1.0 ± 0.0 pulses/session). The characteristics and effects of LH pulses on progesterone and estradiol were similar between natural (pretreatment) and primarily induced (posttreatment) LH pulses. The same dose of GnRH stimulated an LH pulse with greater (P<0.05) amplitude after luteolysis than during luteolysis. Concentrations of PRL and number and prominence of PRL pulses decreased (P<0.05) between Hours 0 and 2 within each of the phases of before, during, and after luteolysis. The hypothesis that a physiologic dose of GnRH increases the concentration of PRL was not supported; instead, GnRH reduced the concentration of PRL. Results supported the hypotheses that an appropriate dose of GnRH stimulates an LH pulse during luteolysis that is similar to a natural pulse in characteristics and in the effects on progesterone and estradiol.     

Hormonal changes around bovine luteolysis

Plasma concentrations of estradiol-17β, progesterone, LH and 13,14-dihydro-15-keto-PGF₂ were determined by radioimmunoassay on 2-hourly samples obtained around luteolysis and estrus in three dairy cows. The decline in progesterone occurred before the pre-estrous rise in estrogen and no pre-estrous peak of progesterone could be detected. The major activity of prostaglandin coincided with declining progesterone levels and the active stage of estrogen secretion.     

Temporal associations among pulses of 13,14-dihydro-15-keto-PGF2alpha, luteal blood flow, and luteolysis in cattle

Luteal blood flow was studied in heifers by transrectal color-Doppler ultrasound. Data were normalized to the decrease in plasma progesterone to <1 ng/ml (Day 0 or Hour 0). Blood flow in the corpus luteum (CL) was estimated by the percentage of CL area with color flow signals. Systemic prostaglandin F2alpha (PGF) treatment (25 mg; n=4) resulted in a transient increase in CL blood flow during the initial portion of the induced decrease in progesterone. Intrauterine treatment (1 or 2 mg) was done to preclude hypothetical secondary effects of systemic treatment. Heifers were grouped into responders (luteolysis; n=3) and nonresponders (n=5). Blood flow increased transiently in both groups; induction of increased blood flow did not assure the occurrence of luteolysis. A transient increase in CL blood flow was not detected in association with spontaneous luteolysis when examinations were done every 12 h (n=6) or 24 h (n=10). The role of PGF pulses was studied by examinations every hour during a 12-h window each day during expected spontaneous luteolysis. At least one pulse of 13,14-dihydro-15-keto-PGF2alpha (PGFM) was identified in each of six heifers during the luteolytic period (Hours -48 to -1). Blood flow increased (P<0.02) during the 3-h ascending portion of the PGFM pulse, remained elevated for 2 h after the PGFM peak, and then decreased (P<0.03) to baseline. Results supported the hypothesis that CL blood flow increased and decreased with individual PGFM pulses during spontaneous luteolysis.