Endometrial Estradiol Research Articles

P-474 State-Trait Anxiety Inventory (STAI) punctuations as a non-invasive method for evaluating endometrial prognosis associated with cortisol and estradiol endometrial levels in IVF patients

Abstract Study question Are STAI State punctuations a potential non-invasive marker of endometrial prognosis in IVF patients? Summary answer Being stressed (determined by STAI State punctuations) is positively and negatively correlated with endometrial cortisol and estradiol levels, respectively, and highly associated with pregnancy consecution. What is known already Despite it is well known IVF treatments are a source of stress, this factor is frequently overlooked mainly due to the difficulty of stress evaluation and the lack of accurate biomarkers, not being standardized in the clinical setting. Steroid hormones are essential for reproductive physiology and highly related to well-known stress biomarkers such as cortisol. The lack of correlation between endometrial and serum steroid levels creates the need to look for non-invasive tools reflecting the endometrial microenvironment. In this study, by molecularly and psychologically characterizing patients, we aim to unveil non-invasive potential markers of stress associated to pregnancy consecution. Study design, size, duration This prospective cohort study included a total of 74 IVF patients (<45 years old, no uterine or systemic pathologies and good quality embryos) who underwent endometrial biopsy collection in mid-secretory phase for metabolites measurement between 2019 and 2023. These patients also underwent psychological evaluation by stress related questionnaires. Participants/materials, setting, methods The endometrial concentration of eleven steroid metabolites was measured by ultra-performance liquid chromatography-tandem mass spectrometry. Patients also underwent stress and anxiety evaluation (STAI), and reproductive outcomes in the first embryo transfer after biopsy collection were followed up. Wilcoxon and Barnard’s test were applied to compare the mean and proportion of stressed/unstressed, pregnant/not pregnant patients and metabolites levels. Correlations between metabolites’ levels and the proportion of stressed patients were evaluated by Pearson’s correlation coefficient. Main results and the role of chance Patients with STAI State punctuations > 60 presented higher mean endometrial cortisol levels (8.01 ng/g (n = 23) vs 5.08 ng/g (n = 35), p-value = 0.05) and lower mean estrone levels (54.97 ng/g (n = 23) vs 56.31 ng/g (n = 35), p-value = 0.049) than those patients with STAI State punctuations < 60. Also, in patients with high endometrial estradiol levels (>2 ng/g) a higher significant proportion of unstressed patients (STAI State < 60) was observed (88.9% vs 52% of unstressed patients in > 2 ng/g and < =2 ng/g estradiol concentrations, respectively) (n = 34, p-value = 0.036). As expected, a significant positive correlation between endometrial cortisol levels and stress (measured by STAI State questionnaire) (cor = 0.977, p-value = 0.023) and a negative correlation between endometrial estradiol levels and stress (cor = -0.9945, p-value = 0.0055) was evidenced. Additionally, a higher proportion of pregnant patients was observed when endometrial estradiol concentrations were high (>1 ng/g) (78.9% vs 50% pregnant in > 1 ng/g vs < =1 ng/g estradiol respectively, p-value = 0.035) and a higher proportion of not pregnant patients was observed when endometrial cortisol concentrations were high ( > =13.9 ng/g) (80% vs 34.4% not pregnant in > =13.9 ng/g and <13.9 ng/g cortisol respectively, p-value = 0.029). Limitations, reasons for caution Although we have employed validated psychological questionnaires and stress biomarkers, a causal relationship between stress and reproductive outcomes should be further explored. Wider implications of the findings This study demonstrated a significant correlation between stress and cortisol and estradiol levels. Since both metabolites’ levels were also related to pregnancy chances, STAI State psychological evaluation could be implemented in the clinical practice as a non-invasive method to prevent endometrial failure through psychological counseling. Trial registration number Not applicable

P-571 Endometrial cortisol and estradiol levels as indicators of pregnancy prognosis and their association with State-Trait Anxiety Inventory (STAI) psychological questionnaire punctuations in IVF patients

Abstract Study question How anxiety and stress questionnaires punctuations and endometrial steroid biosynthesis metabolites levels are associated with embryo implantation and pregnancy outcomes in IVF patients? Summary answer Those patients with high cortisol and low estradiol endometrial levels, were stressed according to STAI State punctuations and showed lower rates of implantation and pregnancy. What is known already Although IVF treatments are known to be a source of stress, its relevance in reproductive success remains controversial, mainly due to the lack of accurate biomarkers. Steroid hormones, such as progesterone and estrogens, are essential for reproductive physiology, and directly related to common stress biomarkers such as cortisol. Nowadays, clinical stress evaluation is exclusively performed via highly subjective psychological questionnaires, not being standardized in the clinical setting. In this study, we measured endometrial levels of main steroid hormones -including cortisol-, stress psychological questionnaires, and reproductive outcomes for determining the relationship among potential stress biomarkers and their influence on fertility. Study design, size, duration In this prospective cohort study, a total of 55 IVF patients were included (<45 years old, no uterine or systemic pathologies and good quality embryos). All endometrial biopsies were collected in mid-secretory phase for metabolites measurement between 2019 and 2021. Also, these patients were psychologically evaluated by stress related questionnaires the same day of biopsy collection or days later in the same cycle. Reproductive outcomes were compared between groups established according to stress biomarkers thresholds. Participants/materials, setting, methods Concentration of eleven selected steroid metabolites was measured by ultra-performance liquid chromatography-tandem mass spectrometry. A psychological questionnaire was also assessed to measure patients’ stress and anxiety (STAI). According to metabolites’ concentration distribution, Barnard’s test was applied to compare the proportion of patients with successful and unsuccessful treatments at different thresholds. A Wilcoxon test was performed for mean metabolites’ levels comparison between stressed and unstressed patients. A p-value of 0.05 was stablished as significant. Main results and the role of chance All patients with high endometrial cortisol levels (>12.5 ng/g; n = 3) failed to implant (100% vs 45.8% non-implanted in > 12.5 ng/g and <12.5 ng/g patients respectively, p-value=0.047), while the totality of patients with high endometrial estradiol levels (>0.9 ng/g; n = 5) achieved embryo implantation and on-going pregnancy (100% vs 50% implanted in > 0.9 ng/g and <0.9 ng/g patients respectively, p-value=0.038). Those patients with high cortisol levels, all not pregnant, had also low estradiol levels and high stress state punctuation (STAI State punctuations ≥ 60). Conversely, patients with high estradiol levels, all pregnant, had also low cortisol levels and were not stressed (STAI State punctuations < 60). Stratification of patients into stressed (STAI State>60) and unstressed (STAI State<60) independently of their implantation results, revealed that 27% of stressed women presented high cortisol levels, while 100% of unstressed patients had low cortisol levels. In the same way, 42% of unstressed patients had high estradiol levels, while 100% of stressed patients presented low estradiol values. Hence, mean values differences of cortisol (10.54 vs 3.05 ng/g) and estradiol (0.55 vs 4.10 ng/g) between stressed and unstressed patients were not significant (p-value=0.08862 and p-value=0.2327, respectively). No relevant associations were observed with stress as a trait. Limitations, reasons for caution Due to the heterogeneous and non-normal metabolites’ concentration distribution among patients, this study would need a largest sample size for having a representative population in the different subgroups. Although we have used molecular and psychological validated stress biomarkers, a causal relationship between stress and reproductive outcomes should be further explored. Wider implications of the findings This study highlighted cortisol and estradiol as potential endometrial biomarkers that better reflect pregnancy prognosis. Further investigation is needed to find if other psychological questionnaires molecularly recognize stressed patients as a noninvasive diagnosis associated to reproductive outcomes. These patients would benefit from psychological counseling to improve their reproductive success. Trial registration number Not applicable

Association of serum progesterone level and endometrial estradiol & progesterone receptors in women with unexplained recurrent miscarriage

Abstract This study investigated the endometrial tissue to study the fluctuation of estrogen receptors (ERs) and progesterone receptors (PRs) in women with recurrent miscarriage compared with normal endometrium of fertile women. Forty women were divided into two groups: 20 non-pregnant women with history of recurrent miscarriage (who had three or more first- trimester miscarriages) and 20 controls. Both groups had regular ovulatory menses and proven fertility. Endometrial biopsy was taken in the two groups during the luteal phase of the menstrual cycle, between the 8th and the 10th postovulatory days using an endometrial biopsy curette for determination of estrogen (E2) and progesterone (P4) hormones levels and their receptors. On the same day as the biopsy, a blood sample was taken for determination of estradiol (E2) and progesterone (P4) levels. The obtained results showed no significant differences between both groups regarding age, BMI, menarche, menstrual cycle & menstruation, significant decrease in E2 and P4 levels in the serum and endometrial biopsy of recurrent miscarriage women versus control. In control and recurrent miscarriage; ER levels in cytoplasm and salt extracted nucleus were higher than PR levels. ER and PR values were higher in the nuclear compartment than in the cytoplasmic compartment. The women with early recurrent miscarriage showed lower levels of both ER and PR significantly. All types of endometrial receptors (ER &PR) and hormones (E2 &P4) in serum and endometrium showed correlation relating to number of previous miscarriages.

Letrozole as an adjunct in GnRH antagonist cycles improves IVF outcome in poor responders

Letrozole as an adjunct in GnRH antagonist cycles improves IVF outcome in poor responders

The role of progesterone in endometrial estradiol- and progesterone-receptor synthesis in women with menstrual disorders and habitual abortion

The objective of this comparative study was to determine the influence of changes in estradiol and progesterone during ovulatory vs. anovulatory cycles on levels of estradiol receptor (ER) and progesterone receptor (PgR) in endometrium. Thirty women (range age 20–35 years) were divided into three groups: women with a history of habitual abortion, obese women with menstrual disorders, and women with regular ovulatory cycles as well as proven fertility. A single venous blood sample and an endometrial sample were simultaneously obtained during the secretory phase of the menstrual cycle, in order to measure estradiol and progesterone levels and ER and PgR concentrations in cytosol and salt-extracted nucleosol. Plasma estradiol levels were not different between groups. Plasma progesterone was two times higher in fertile women than in habitual aborters. In endometrial tissue, progesterone content was 200 times higher in fertile women than in habitual aborters. ER and PgR were lower in the cytosol than in the nuclear fraction in fertile and obese women. Both receptors were at their lowest level in the cytosol and nuclear compartment of women with recurrent miscarriage. Fluctuations mainly in the sex hormone progesterone, in plasma and endometrium tissue, could interfere with ER and PgR levels.

Identification of elevated concentrations of estradiol in bovine uterine endometrium

We have investigated the estradiol content of bovine endometrium and related this to circulating plasma estradiol content. In 9 heifers, mean ± S.E.M. plasma estradiol concentration was 0.64 ± 0.25 pg/ml while the mean ± S.E.M. endometrial estradiol content was 43.0 ± 14.7 pg/g tissue; there was a close relationship between plasma and tissue estradiol levels ( R 2 = 0.81; P < 0.001). During culture of endometrial tissue there was a progressive transfer of estradiol from tissue to culture media but no change in total estradiol. Culture of endometrium from 4 heifers with 5 ng/ml testosterone for 72 h resulted in no increase in estradiol. Furthermore, immunohistochemistry revealed no aromatase protein in uterine endometrium. These results confirm high stored tissue concentrations of estradiol in bovine endometrium while providing no evidence for estradiol synthesis by this tissue. The mechanism(s) through which this sequestration of estradiol into uterine tissue occurs remains to be determined.

Local levonorgestrel regulation of androgen receptor and 17beta-hydroxysteroid dehydrogenase type 2 expression in human endometrium.

The levonorgestrel-releasing intrauterine system (LNG-IUS) is a highly effective contraceptive. However, unscheduled breakthrough bleeding (BTB), leads to discontinuation in a proportion of users. The LNG-IUS down-regulates endometrial progesterone and estrogen receptors and this may play a role in the mechanism responsible for BTB. LNG is an androgenic progestogen and so we examined the regulation of the androgen receptor (AR) in endometrium exposed to intrauterine LNG. Furthermore, as the enzyme 17beta-hydroxysteroid dehydrogenase type 2 (17betaHSD2) regulates intracellular levels of estrogens, progestins and androgens, we evaluated the changes in expression of 17betaHSD2 in the same tissue endometrial samples. Immunohistochemistry and real time quantitative RT-PCR were used to compare protein and mRNA expression of AR and 17betaHSD2 in endometrial biopsies from women with normal menstrual cycles and those using a LNG-IUS. Immunohistochemistry showed that AR and 17betaHSD2, which were immunolocalized to the stroma and glands of endometrium respectively, were both suppressed by LNG-IUS treatment, though moderate staining of 17betaHSD2 was evident 1 month after insertion of the LNG-IUS. AR mRNA expression was down-regulated in LNG-exposed endometrium when compared with the proliferative phase of the menstrual cycle. 17betaHSD2 mRNA was significantly increased 3 months (but not 6-12 months) after LNG-IUS insertion. Endometrial intracellular estradiol levels would have been suppressed by 17betaHSD2 during the first few, but not the later, months of LNG-IUS action, and the lowered endometrial estradiol level may contribute to the frequent BTB evident in the early months of LNG-IUS use. The subsequent decline in 17betaHSD2 would lead to elevated local intracellular estradiol in the later months, when the BTB tends to subside. The suppression of AR by the LNG-IUS may also play a role in BTB, as elevated AR has been associated with amenorrhoea.

Serum and tissue hormone levels of vaginally and orally administered estradiol

Objective: Our purpose was to determine serum and endometrial estradiol levels when micronized estradiol is administered vaginally and orally. Study Design: Five subjects were given oral estradiol (2 mg twice daily), during an artificial luteal phase, and another group of 5 subjects were given the same dose of estradiol by the vaginal route. Endometrial biopsies and blood samples were obtained on day 21 of the cycle, 2 hours after the last dose was administered. Tissue and blood samples were assayed for estradiol. Results: Serum estradiol levels were significantly higher with vaginally administered estradiol than with orally administered estradiol (2344 ± 398 vs 279 ± 76 pg/mL, P < .005). Endometrial estradiol concentrations were also significantly higher with vaginally administered estradiol than with the oral preparation (918 ± 412 vs 13 ± 2 pg/mg protein, P < .05). Conclusions: Vaginal administration of estradiol is more effective in increasing serum and endometrial levels of estradiol than the oral route and may represent the optimal route of administration for recipients of egg donation. If the vaginal route of estradiol administration is considered for menopausal replacement therapy, much lower doses of the standard oral quantities should be used. Furthermore, if the uterus is present, a progestin must be used to compensate for the high tissue levels of estradiol. (Am J Obstet Gynecol 1999;180:1480-3.)

Effect of labor induction on the expression of oxytocin receptor, cytochrome P450 aromatase, and estradiol receptor in the reproductive tract of the late-pregnant ewe.

In this study, we investigated the timing of changes in aromatase, estradiol receptor, and oxytocin receptor expression in ovine uterine and placental tissues before parturition. Labor was induced by betamethasone injection into the fetus on Days 130-132 of pregnancy. Tissue samples were collected at injection and then every 14 h until labor (56 h) from four ewes at each time point. Samples were analyzed for aromatase, estradiol receptor, and oxytocin receptor expression by in situ hybridization; for oxytocin binding to its receptor using a specific antagonist; and for estradiol receptor quantitation by immunocytochemistry. Aromatase mRNA expression increased by 14 h postinjection (p < 0.02) in the fetal villi and remained high until labor. Expression of estradiol and oxytocin receptor mRNAs was unchanged in myometrium but increased in the endometrial luminal epithelium by 28 h (p < 0.05) and remained high until labor. Estradiol receptor protein concentration increased modestly at labor while oxytocin receptor binding in the luminal epithelium changed in parallel to the mRNA concentration. 1) induction of aromatase may facilitate the expression of endometrial estradiol and oxytocin receptors in the placentome, 2) changes in endometrial rather than myometrial oxytocin receptor may be important in inducing parturition, and 3) the transcription of estradiol receptor and oxytocin receptor in the uterine epithelium are positively correlated during parturition.

Estradiol and progesterone endometrial receptors and body fat distribution in obese women.

The content of endometrial receptors for estradiol (ER) and progesterone (PR) was determined in three groups (10 women each) classified with respect to body fat distribution: (1) normal, nonobese; (2) lower body segment obesity and (3) upper body segment obesity. The ER in group 3 were significantly (p < 0.05) higher than those of group 1. As regards PR, no statistical differences were found among the three groups. These findings demonstrate different endometrial ER concentrations in obese women according to body fat distribution, greater in upper segment obesity. Probably the lack of progesterone as well as other factors in the endocrine milieu of these patients may determine the differences found; therefore, future research on this matter should be carried out.

Tamoxifen increases plasma estrogen-binding equivalents and has an estradiol agonistic effect on histologically normal premenopausal and postmenopausal endometrium

To determine the effects of single dose tamoxifen on plasma estrogen (E)-binding equivalents and endometrial estradiol (E2) and progesterone (P) receptors. Controlled clinical study. Normal human volunteers were studied in an academic research environment. Premenopausal and postmenopausal women with histologically normal endometrium undergoing curettage or hysterectomy were selected. Tamoxifen was administered orally; blood and endometrial samples were collected 4 to 96 hours after tamoxifen administration. Plasma E-binding equivalents, endometrial cytosolic and nuclear E2 and P receptors. (1) Plasma E-binding equivalents increased eightfold at 4 to 24 hours of tamoxifen administration and declined exponentially thereafter, reaching control levels at 73 to 96 hours. Plasma E-binding equivalents were not affected by endogenous E2 levels. (2) Endometrial total E2 and P receptor levels increased in all women 2.9 to 19.2-fold after tamoxifen. (3) Tamoxifen resulted in an increase in the fraction of the E2 receptor measured in the nuclear extract 2.1 to 7.5-fold in midcycle, secretory, and menopausal endometria but not in proliferative endometrium. (1) Tamoxifen has an E2 agonistic effect on histologically normal human endometrium. (2) Irrespective of the total level of the endometrial E2 receptor, the nuclear capacity of that receptor in vivo is limited (approximately 75% to 80% of the total level).

Effects of clomiphene citrate on cytosolic estradiol and progesterone receptor concentrations in secretory endometrium

Cytosolic estradiol and progesterone receptor concentrations were measured in luteal phase andometrial biopsy samples obtained from 12 anovulatory or oligoovulatory women on clomiphene citrate therapy and from 40 normal control subjects. Clomiphene citrate treatment (250 to 750 mg per cycle) decreased both endometrial estradiol and progesterone receptor concentrations. Estradiol receptor levels were nondetectable in seven tissue samples and progesterone receptor levels in four samples from the 12 subjects given clomiphene citrate compared to nondetectable estradiol receptor concentrations in one tissue sample and progesterone receptor concentrations in two samples from the 40 normal control subjects. In histologically dated endometrium, mean estradiol receptor concentrations on days 20 to 23 and progesterone receptor levels on days to 24 to 27 were lower than the values observed in the comparable dated endometrium of normal ovulatory women. Steroid receptor concentrations correlated negatively with the duration of clomiphene citrate therapy, which implies a time-dependent suppressive effect of clomiphene citrate on measurable cytosolic estradiol and progesterone receptor concentrations during the luteal phase of the cycle.

Endometrial estradiol and progesterone receptors in patients with luteal phase defects and endometriosis

Endometrial cytosol estrogen receptors (ERs) and progesterone receptors (PgRs) were quantitated in postovulatory endometrial biopsy samples of patients with luteal phase defect (LPD), those with endometriosis, and normal control subjects. Serum levels of estradiol (E2) and progesterone (P), obtained on the day of the biopsy, were also measured. No significant differences among endometrial ER, PgR, serum E2, and P levels were detected between the patients with endometriosis and normal control subjects. Although ER concentrations in the luteal defect group did not differ from those of control subjects, the PgR levels in day 20 to 23 endometrium were significantly higher. Mean serum E2 and P levels in the group with luteal insufficiency were significantly lower than those of the control subjects. Our data suggest that in patients with LPD the increase in PgR levels during the midluteal days is compatible with a relative deficiency of P secretion by the corpus luteum.

Stimulation of arylsulfotransferase activity by progestins in human endometrium in vitro.

The effect of progestin on the rate of estradiol (E2) sulfurylation in human endometrium was studied in two culture systems: 1) in intact endometrial tissue fragments and 2) in isolated endometrial epithelial glands and stromal cells. Human endometrial tissue fragments were cultured in medium in the presence and absence of progesterone (P) for 24-48 h. The arylsulfotransferase activity was determined in the cytosol of the tissue homogenate by measuring the rate of conversion of E2 to E2-3 sulfate (E2S) in the presence of an excess amount of substrate and cofactor (adenosine-3'-phosphate-5'-phosphosulfate). The enzyme activity was found to be stimulated in the sample cultured with P. The increase in activity correlated with the dose of P and period of culture. Stimulation of endometrial estradiol dehydrogenase activity by P, which has been reported in a series of previous publications, was also evident in the current study. Human endometrial glandular epithelial and stromal cells were cultured separately in medium in the presence and absence of medroxyprogesterone acetate (MPA). The cultured cells were incubated with E2 (0.4-0.6 nM) for 2 h. The formation of estrogen sulfates, [estrone sulfate (E1S) and E2S] was greatly increased in all the proliferative glandular epithelial cells cultured with MPA. However, the rate of sulfurylation was not appreciably affected by MPA in stromal cells and in glandular epithelial cells from secretory endometrium. The oxidation of E2 in these cultured cells was also influenced by P but not to the extent of sulfurylation. These results indicate that arylsulfotransferase in endometrium originates from glandular epithelial cells and can be stimulated by progestin.

Steroid specificity in the stimulation of human endometrial estradiol dehydrogenase.

Progesterone (P) stimulates estradiol dehydrogenase (E2DH) activity in human proliferative endometrium. The potencies of several progestins were studied in this system. Endometrium was incubated for 2–3 days in a culture medium containing various concentrations of steroids. The E2DH activity was measured at the end of the incubation. Steroid specificity was studied by examining the dose-response characteristics for each progestin in the stimulation of the enzyme activity. In this system, the most potent progestins, medroxyprogesterone acetate, R-5020, and P, were able to stimulate endometrial E2DH at a concentration as low as 3 nM after 2 days of incubation. 17α-Hydroxyprogesterone (17-OHP) caproate was effective at 30 nM, whereas high concentrations (>100 nM) of medroxyprogesterone and 17-OHP were required in order to produce a detectable increase in activity. All of these compounds, except 17-OHP, produced a dose-dependent increase in E2DH activity. Based on the relative stimulation observed, it appears that a hydroxyl group, especially at the 17α position, greatly reduces the ability of a progestin to stimulate the enzyme. However, this capacity is restored by esterification of the hydroxyl group. In addition, corticosterone (B), but not cortisol, was found to be able to increase E2DH activity. However, the doseresponse curve was entirely different from that of the progestins. At least 200 nM B was required before an increase in enzyme activity could be detected. Kinetic analysis of the stimulation of E2DH revealed that the maximum enzyme activity stimulated was similar when using either P or B. However, the concentrations required to reach the half-maximum enzyme activity were very different (10 nM for P and 1400 nM for B). These results suggest that B may not be physiologically significant as a stimulator. (Endocrinology102: 1398, 1978)

Induction of Human Endometrial Estradiol Dehydrogenase by Progestins

Estradiol-17beta dehydrogenase activity in proliferative human endometrium (average of 1.5 nmole of estrone formed from estradiol/mg protein/h) was stimulated as much as as 6-fold during incubations of tissue slices in culture medium containing progesterone. Stimulation was already detectable at 7 h and the highest activity values were reached at 48-72 h of incubation in the presence of excess progesterone. Maximal stimulation was achieved with concentrations of the hormone of 0.25 mug/ml or higher. At concentrations approximately equal to midluteal plasma levels (20 ng/ml) more than 50% of the maximal response was observed. Norgestrel (17alpha-ethynyl-18-methyl-19-nortestosterone) was also effective in inducing enzymatic activity. The similarity of the effects obtained with progesterone (a possible substrate for estradiol dehydrogenase) and the synthetic progestin indicates that the stimulation of enzymatic activity was not due to substrate induction. Addition of estradiol to the culture medium had no influence on the activity of the enzyme. The induction of estradiol dehydrogenase by progesterone was inhibited by puromycin or actinomycin D. These observations indicate that progestational agents increase the rate of de novo synthesis of the enzyme. Stimulation of endometrial estradiol dehydrogenase was also observed after 2-3 day oral administration of medroxyprogesterone acetate to women in the follicular phase. In contrast, the enzymatic activity in endometrium obtained from women taking estrogens was found to be as low as in normal proliferative tissue. These in vitro and in vivo results point to progesterone as the agent responsible for the 10-fold increase in endometrial estradiol dehydrogenase activity observed during the luteal phase in menstruating women. Data obtained from superfusion studies of estrogen dynamics in endometrium indicate that changes in enzyme concentrations may play a physiologic role in the regulation of tissue levels of estradiol.