Therapeutic effects of surgical debulking of metastatic lymph nodes in cervical cancer IIICr: a trial protocol for a phase III, multicenter, randomized controlled study (KGOG1047/DEBULK trial).

BACKGROUND: Bulky or multiple lymph node (LN) metastases are associated with poor prognosis in cervical cancer, and the size or number of LN metastases is not yet reflected in the staging system and therapeutic strategy. Although the therapeutic effects of surgical resection of bulky LNs before standard treatment have been reported in several retrospective studies, well-planned randomized clinical studies are lacking. Therefore, the aim of the Korean Gynecologic Oncology Group (KGOG) 1047/DEBULK trial is to investigate whether the debulking surgery of bulky or multiple LNs prior to concurrent chemoradiation therapy (CCRT) improves the survival rate of patients with cervical cancer IIICr diagnosed by imaging tests. METHODS: The KGOG 1047/DEBULK trial is a phase III, multicenter, randomized clinical trial involving patients with bulky or multiple LN metastases in cervical cancer IIICr. This study will include patients with a short-axis diameter of a pelvic or para-aortic LN ≥2 cm or ≥3 LNs with a short-axis diameter ≥1 cm and for whom CCRT is planned. The treatment arms will be randomly allocated in a 1:1 ratio to either receive CCRT (control arm) or undergo surgical debulking of bulky or multiple LNs before CCRT (experimental arm). CCRT consists of extended-field external beam radiotherapy/pelvic radiotherapy, brachytherapy and LN boost, and weekly chemotherapy with cisplatin (40 mg/m²), 4-6 times administered intravenously. The primary endpoint will be 3-year progression-free survival rate. The secondary endpoints will be 3-year overall survival rate, treatment-related complications, and accuracy of radiological diagnosis of bulky or multiple LNs. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT05421650; Clinical Research Information Service Identifier: KCT0007137.

Diagnostic Clues for Women with Acute Surgical Abdomen Associated with Ruptured Endometrioma.

(1) Background: An investigation of the preoperative diagnostic clues used to identify ruptured endometrioma by comparing the ruptured and unruptured states in patients who underwent laparoscopic operations due to endometrioma. (2) Methods: Patients with ruptured endometriomas (14 patients) and unruptured endometriomas (60 patients) were included, and clinical symptoms, laboratory findings, and radiological findings were analyzed. (3) Results: There were no significant differences in age, parity, last menstrual cycle days, or median size of endometrioma between two groups (group A: ruptured; group B: unruptured). The median serum level of CA 125 was 345.1 U/mL in group A and 49.8 U/mL in group B (p = 0.000). The median serum levels of CA 19-9 in group A and B were 46.0 U/mL and 19.1 U/mL, respectively (p = 0.005). The median serum level of CRP in group A was 1.2 g/dL, whereas it was 0.3 in group B (p = 0.000). ROC analysis showed that the optimal CA 125 cutoff value was 100.9 U/mL; the optimal CA 19-9 cutoff value was 27.7 U/mL; and the optimal CRP cutoff value was 1.0 g/dL. (4) Conclusions: Ruptured endometrioma can be diagnosed preoperatively using a combination of clinical symptoms, laboratory findings, and radiological findings. If a physician suspects a ruptured endometrioma, surgery should be performed to ensure optimal prognosis.

Pretreatment CT differential diagnosis of tuberculous peritonitis from peritoneal carcinomatosis of advanced epithelial ovarian cancer.

Delayed diagnosis of female genital tuberculosis (FGTB) can lead to inappropriate treatment and unnecessary surgical procedures rather than standard anti-TB medication. We tried to evaluate the use of computed tomography (CT) imaging to differentiate TB peritonitis from peritoneal carcinomatosis of advanced epithelial ovarian cancer (AEOC). We investigated women who underwent CT to distinguish between TB peritonitis and peritoneal carcinomatosis of AEOC. We evaluated various CT imaging features to identify differences between the two diseases. In addition, we performed univariate and multivariate logistic regression analyses to identify the independent imaging parameters associated with TB peritonitis and evaluated the diagnostic performance of the combined imaging parameters. We also performed the histopathological analysis of the available salpinx specimens of TB peritonitis. We included 25 women with TB peritonitis and 34 women with peritoneal carcinomatosis of AEOC. A multivariate analysis of the discriminant CT imaging features between the two diseases revealed that changes in fallopian tubes and peritoneal micronodules were independent parameters associated with TB peritonitis (p ≤ 0.012). Combining the two imaging parameters showed an area under the receiver operating characteristic curve of 0.855, a sensitivity of 88.0%, and a specificity of 67.7% for differentiating TB peritonitis from peritoneal carcinomatosis. Furthermore, changes in fallopian tubes were correlated with histopathological abnormalities in salpinx specimens. Pretreatment CT evaluation with useful imaging features could help differentiate TB peritonitis from peritoneal carcinomatosis of AEOC.

Laparoscopic systemic restaging surgery for women with unexpected uterine malignancy.

OBJECTIVE: We investigated the feasibility of laparoscopic restaging surgery in patients with unexpected uterine cancer. METHODS: This retrospective study included eight patients who underwent laparoscopic restaging surgery for Iran University uterine cancer after a prior hysterectomy or myomectomy. RESULTS: The median age of the patients and their body mass index were 55 years (range, 44-78) and 23.8 kg/m2 (range, 20.75- 31.89), respectively. The median interval between the prior hysterectomy and the restaging surgery was 21 days (range, 10-35). The median operating time and time for the return of bowel activity were 325 minutes (range, 200-475) and 35 hours (range, 18-50), respectively. The median numbers of harvested pelvic and para-aortic lymph nodes were 17.5 (range, 14-29) and 20.5 (range, 7-36), respectively. In seven of the eight patients, uterine extraction was performed with vaginal or electronic morcellation. The final International Federation of Gynecology and Obstetrics stage was IA in all patients. Intraoperative and postoperative complications did not occur in any of the patients, except for the need for transfusion. Patient 4 had synchronous primary cancer (stage IA) of the endometrium and left ovary. Two of the eight patients with clear cell carcinoma received chemotherapy, and none received radiotherapy. All patients survived without disease recurrence. CONCLUSION: Restaging surgery might be necessary for highly selective patients with unexpected uterine malignancies. This would be an alternative surgical modality for complete staging and planning tailored adjuvant treatments. However, lymphadenectomy might not be performed in patients with early uterine cancer.

Telomere shortening and expression of TRF1 and TRF2 in uterine leiomyoma.

Uterine leiomyoma is a benign smooth muscle tumor of the uterus that can exhibit histopathological traits that mimic malignancy. Telomere shortening is an early event in tumorigenesis and telomerase activation facilitates tumor progression later in the course of carcinogenesis. Telomeric repeat­binding factor (TRF)1 and TRF2 protect telomeres, and their gene expression levels are dysregulated in various cancer types. However, the roles of telomeres and telomere protection proteins in uterine leiomyoma remain largely unknown. In this study, telomere length and the mRNA levels of various telomere­related genes in normal tissues and leiomyoma were determined, and their relationships were evaluated. Uterine leiomyoma and normal myometrium were surgically obtained from 18 and 13 patients, respectively. Telomere length and gene expression were determined by Southern blot analysis and reverse transcription­quantitative PCR, respectively. In matched samples, telomeres were consistently shorter in leiomyoma tissue than in adjacent normal tissue. TRF1, TRF2, PIN2­interacting telomerase inhibitor 1 (PINX1), and telomerase RNA component were expressed at comparable levels in both leiomyoma and normal tissues. None of these genes were associated with telomere length in leiomyoma. All tested tissues were negative for telomerase reverse transcriptase, which encodes the catalytic component of telomerase, indicating that cells in uterine leiomyoma were not immortalized. In summary, telomere erosion, which reflects active proliferation during tumor evolution, was evident in uterine leiomyoma. Steady­state expression of TRF1, TRF2 and PINX1 may be important for maintenance of telomere integrity in leiomyoma, where telomere length is shortened.

Nitration of protein phosphatase 2A increases via Epac1/PLCε/CaMKII/HDAC5/iNOS cascade in human endometrial stromal cell decidualization.

Decidualization of the endometrial stroma is an essential differentiation process for embryo implantation and maintenance of pregnancy. We previously reported that protein phosphatase 2A (PP2A) acts as a key mediator during cAMP-induced decidualization of human endometrial stromal cells (hESCs). However, the mechanism underlying its activation has remained obscure in hESCs. In the present study, we aimed to reveal the mechanism that induces the nitration of PP2A catalytic subunit (PP2Ac) during cAMP-induced decidualization of hESCs. First, cAMP-induced PP2Ac nitration was significantly repressed using L-NAME, an inhibitor of nitric oxide synthase (NOS). Among several NOS isoforms, only inducible NOS (iNOS) was highly expressed in hESCs, indicating that iNOS directly induces the nitration of PP2Ac. Second, cAMP-induced iNOS expression and PP2Ac nitration were decreased by treatment with TSA, an inhibitor of histone deacetylase 5 (HDAC5). cAMP-induced phosphorylation of CaMKII and HDAC5 was suppressed by treatment with U73122 (an inhibitor of phospholipase C) or transfection of PLCε siRNA. Finally, small G protein Rap1 and its guanine nucleotide exchange factor Epac1 were found to be involved in cAMP-induced PP2A activation. Taken together, our results suggest that PP2Ac nitration during cAMP-induced decidualization of hESCs is induced through the Epac1-Rap1-PLCε-CaMKII-HDAC5-iNOS signaling pathway.

Increased amounts and stability of telomeric repeat-containing RNA (TERRA) following DNA damage induced by etoposide.

Telomeric repeat-containing RNAs (TERRAs) are long noncoding RNAs transcribed from subtelomeres toward telomeric repeat tracts, which have been implicated in telomere protection and heterochromatin formation. Genotoxic stress leads to upregulation of TERRAs. However, the mechanism of DNA damage-mediated TERRA induction remains elusive. Here, we treated HeLa cells with etoposide, a DNA double-strand break-generating agent, for various times and monitored the levels of TERRAs. Etoposide treatment led to a gradual time-dependent increase in TERRAs. Etoposide-mediated induction was evident in many TERRAs arising from various chromosome loci, including 20q and XpYp. Chromatin immunoprecipitation assays revealed no significant changes in the occupancy of RNA polymerase II at telomeres upon etoposide treatment. Interestingly, TERRAs arising from 20q, XpYp, 10q, and 13q degraded at slower rates in cells treated with etoposide, while degradation rates of TERRAs from many loci tested were nearly identical in both etoposide- and mock-treated cells. Telomere damage occurred from early time points of etoposide treatment, but telomere lengths and abundance of telomeric repeat-binding factor 2 (TRF2) at telomeres remained unchanged. In summary, etoposide treatment led to telomere damage and TERRA accumulation, but telomere lengths and TRF2-mediated telomere integrity were maintained. Etoposide-mediated TERRA accumulation could be attributed partly to RNA stabilization. These findings may provide insight into the post-transcriptional regulation of TERRAs in response to DNA damage.

Clinical characteristics of patients with leiomyoma who undergo surgery after high intensity focused ultrasound (HIFU).

OBJECTIVE: We evaluated the clinical characteristics of patients who underwent surgery after high intensity focused ultrasound (HIFU) to treat uterine leiomyoma. METHODS: From June 2016 to September 2017, patients at our hospital who underwent HIFU to treat uterine leiomyoma prior to surgery were enrolled. All patients underwent pelvic magnetic resonance imaging (MRI) before and after HIFU. If 6 months had passed since the last pelvic MRI was performed, imaging was performed again before the operation. RESULTS: A total of 12 patients were analyzed. The median age was 45 (range, 28-51) years. The median body mass index was 24.9 (range, 18.1-29.2) kg/m2. The median size of the leiomyoma was 10.1 (range, 7.8-14.0) cm before HIFU, which changed to 8.75 (range, 5.9-14.8) cm after HIFU. The median size increased to 9.1 (range, 5.9-18.0) cm before the operation. Surgery was planned for several reasons, including an increase in the leiomyoma size (n=6), persistent symptoms (n=4), and newly developed lesion (n=2). The median interval between HIFU and surgery was 7 (range, 3-32) months. Ten of the 12 patients underwent laparoscopic surgery, while the others underwent laparotomy; 6 patients also underwent laparoscopic myomectomy, and 4 underwent hysterectomy. Histopathologic findings showed infarction-type necrosis surrounded by granulation tissue with the infiltration of lymphocytes and macrophages in all patients. CONCLUSION: Treatment of leiomyoma with operative procedures should be considered in selected patients with tumor size greater than 10 cm, multiple tumors, and persistent symptoms after HIFU treatment.

Systemic Laparoscopic Para-Aortic Lymphadenectomy to the Left Renal Vein.

BACKGROUND: No large-scale clinical study has been done to show the standard surgical boundary and efficacy of laparoscopic para-aortic lymphadenectomy (LPAL). OBJECTIVES: Therfore, this study aimed to evaluate the feasibility, efficacy, and standard surgical boundary of LPAL performed up to the left renal vein level in gynecological malignancies. METHODS: Medical records of 333 patients were retrospectively reviewed. All cases had gynecologic malignancies and had an operation including LPAL by a single surgical team between November 2003 and May 2018. RESULTS: Three hundred twenty-six patients underwent LPAL as part of their staging, restaging, or debulking surgery. Seven patients with isolated para-aortic lymph node recurrence underwent a repeat LPAL. The median age and body mass index were 54 years (range, 28-81 years) and 26.0 kg/m2 (range, 20.3-37.2 kg/m2), respectively. The median operating time was 60 minutes (range, 24-135 minutes), and the median number of harvested para-aortic lymph nodes was 12 (range, 6-49). There were 11 cases of complications: 5 of major vessel injuries (3 inferior vena cava, 1 aorta, and 1 common iliac vein), 2 lymphocysts, 2 cases of chylous ascites, a cisterna chyli rupture, and 1 case of ureteric injury. There were 2 conversions to laparotomy: 1 left common iliac vein laceration that needed to be repaired and removal of an enlarged para-aortic lymph node completely. CONCLUSION: It is feasible and efficient to perform LPAL to the left renal vein level for women with gynecologic malignancies by well-trained gynecologic oncology surgeons according to our suggested standard surgical boundary.

Comparison of vaginal hysterectomy and laparoscopic hysterectomy: a systematic review and meta-analysis.

BACKGROUND: There are various surgical approaches of hysterectomy for benign indications. This study aimed to compare vaginal hysterectomy (VH) and laparoscopic hysterectomy (LH) with respect to their complications and operative outcomes. METHODS: We selected randomised controlled trials that compared VH with LH for benign gynaecological indications. We included studies published after January 2000 in the following databases: Medline, EMBASE, and CENTRAL (The Cochrane Library). The primary outcome was comparison of the complication rate. The secondary outcomes were comparisons of operating time, blood loss, intraoperative conversion, postoperative pain, length of hospital stay and duration of recuperation. We used Review Manager 5.3 software to perform the meta-analysis. RESULTS: Eighteen studies of 1618 patients met the inclusion criteria. The meta-analysis showed no differences in overall complications, intraoperative conversion, postoperative pain on the day of surgery and at 48 h, length of hospital stay and recuperation time between VH and LH. VH was associated with a shorter operating time and lower postoperative pain at 24 h than LH. CONCLUSIONS: When both surgical approaches are feasible, VH should remain the surgery of choice for benign hysterectomy.

Coexisting ovarian and tubal pregnancies on opposite sides after intrauterine insemination: a case report.

BACKGROUND: Ovarian pregnancy is very rare, and contralateral tubal pregnancy coexisting with ovarian pregnancy must be even rarer. CASE PRESENTATION: A 33-year-old Korean nulliparous woman was referred to our hospital because she suffered lower abdominal pain and had missed her periods after controlled ovarian hyperstimulation and intrauterine insemination. We could not identify any normal gestational sac in the endometrium, or specific ectopic pregnancies, on an initial ultrasound scan. However, there was a large hematoma in the cul-de-sac and free fluid in the right paracolic gutter. We decided to perform emergent laparoscopic surgery. We found contralateral tubal and ovarian ectopic pregnancies. CONCLUSION: To the best of our knowledge, this is the first report of a case in which a patient underwent laparoscopic right salpingectomy and left ovarian ectopic mass excision due to contralateral tubal and ovarian ectopic pregnancies after assisted reproductive technology.

Encountering the Accessory Polar Renal Artery during Laparoscopic Para-Aortic Lymphadenectomy.

A 60-year-old Korean woman underwent laparoscopic bilateral salpingo-oophorectomy and was confirmed to have high-grade serous carcinoma of both ovaries with a huge omental cake, extensive agglutinated intra-abdominal metastatic masses, extensive serosa invasion of the intestines, and mesenterial deposits. She underwent 3 cycles of neoadjuvant chemotherapy followed by laparoscopic interval debulking surgery, including hysterectomy, pelvic and para-aortic lymphadenectomy, appendectomy, partial peritonectomy, and omentectomy. We encountered the right accessory polar renal artery (APRA) during the surgery and carefully preserved the right APRA from the abdominal aorta to the right kidney (Fig. 1). Postoperative computed tomography angiography showed an intact right APRA and normal-appearing kidney (Fig. 2). The patient had adjuvant chemotherapy and is alive without disease recurrence. Because APRA is a functional end artery, it is important to preserve it during surgery to prevent ischemic damage and renal failure [1]. It is very important for the gynecologic-oncologist to have knowledge of the retroperitoneal vascular anatomy, experience in laparoscopic surgery, and an accurate surgical technique to avoid vascular injury during laparoscopic para-aortic lymphadenectomy.

Preoperative diagnostic clues to ovarian pregnancy: retrospective chart review of women with ovarian and tubal pregnancy.

OBJECTIVE: To analyze the preoperative diagnostic clues to ovarian pregnancy (OP). METHODS: This study conducted a retrospective chart review of 23 patients with OP and 46 patients with tubal pregnancy (TP) from October 1, 2003 to September 31, 2016 in Hanyang University Hospital. RESULTS: There were no significant differences in demographic and clinical characteristics between the two groups. The presence of an ectopic gestational sac and hemoperitoneum was significantly higher in the TP group (13.0% vs. 95.7%, P=0.000; 13.0% vs. 54.3%, P=0.001, respectively) in preoperative ultrasonogram. The OP group had more ruptured ectopic gestational sacs than the TP group (73.9% vs. 45.7%, P=0.039) in surgical findings. CONCLUSION: For the patients in whom a gestational sac is not detected in the uterus or the fallopian tubes, it is important to be aware of the possibility of OP and rupture of an ovarian gestational sac to promote early diagnosis and surgical intervention.

Variable TERRA abundance and stability in cervical cancer cells.

Telomeres are transcribed into long non-coding RNA, referred to as telomeric repeat-containing RNA (TERRA), which plays important roles in maintaining telomere integrity and heterochromatin formation. TERRA has been well characterized in HeLa cells, a type of cervical cancer cell. However, TERRA abundance and stability have not been examined in other cervical cancer cells, at least to the best of our knowledge. Thus, in this study, we measured TERRA levels and stability, as well as telomere length in 6 cervical cancer cell lines, HeLa, SiHa, CaSki, HeLa S3, C-33A and SNU-17. We also examined the association between the TERRA level and its stability and telomere length. We found that the TERRA level was several fold greater in the SiHa, CaSki, HeLa S3, C-33A and SNU-17 cells, than in the HeLa cells. An RNA stability assay of actinomycin D-treated cells revealed that TERRA had a short half-life of ~4 h in HeLa cells, which was consistent with previous studies, but was more stable with a longer half-life (>8 h) in the other 5 cell lines. Telomere length varied from 4 to 9 kb in the cells and did not correlate significantly with the TERRA level. On the whole, our data indicate that TERRA abundance and stability vary between different types of cervical cancer cells. TERRA degrades rapidly in HeLa cells, but is maintained stably in other cervical cancer cells that accumulate higher levels of TERRA. TERRA abundance is associated with the stability of RNA in cervical cancer cells, but is unlikely associated with telomere length.

Phosphatidic acid induces decidualization by stimulating Akt-PP2A binding in human endometrial stromal cells.

Decidualization of human endometrial stromal cells (hESCs) is crucial for successful uterine implantation and maintaining pregnancy. We previously reported that phospholipase D1 (PLD1) is required for cAMP-induced decidualization of hESCs. However, the mechanism by which phosphatidic acid (PA), the product of PLD1 action, might regulate decidualization is not known. We confirmed that PA induced decidualization of hESCs by observing morphological changes and measuring increased levels of decidualization markers such as IGFBP1 and prolactin transcripts (P < 0.05). Treatment with PA reduced phosphorylation of Akt and consequently that of FoxO1, which led to the increased IGFBP1 and prolactin mRNA levels (P < 0.05). Conversely, PLD1 knockdown rescued Akt phosphorylation. Binding of PP2A and Akt increased in response to cAMP or PA, suggesting that their binding is directly responsible for the inactivation of Akt during decidualization. Consistent with this observation, treatment with okadaic acid, a PP2A inhibitor, also inhibited cAMP-induced decidualization by blocking Akt dephosphorylation.

Safety of laparoscopically assisted vaginal hysterectomy for women with anterior wall adherence after cesarean section.

OBJECTIVE: To evaluate the safety and surgical outcomes of laparoscopically assisted vaginal hysterectomy (LAVH) for women with anterior wall adherence after cesarean section. METHODS: We conducted a retrospective study of 328 women with prior cesarean section history who underwent LAVH from March 2003 to July 2013. The subjects were classified into two groups: group A, with anterior wall adherence (n=49); group B, without anterior wall adherence (n=279). We compared the demographic, clinical characteristics, and surgical outcomes of two groups. RESULTS: The median age and parity of the patients were 46 years (range, 34 to 70 years) and 2 (1 to 6). Patients with anterior wall adherence had longer operating times (175 vs. 130 minutes, P<0.05). There were no significant differences in age, parity, number of cesarean section, body mass index, specimen weight, postoperative change in hemoglobin concentration, or length of hospital stay between the two groups. There was one case from each group who sustained bladder laceration during the vaginal portion of the procedure, both repaired vaginally. There was no conversion to abdominal hysterectomy in either group. CONCLUSION: LAVH is effective and safe for women with anterior wall adherence after cesarean section.

Feasibility and efficacy of laparoscopic restaging surgery for women with unexpected ovarian malignancy.

OBJECTIVE: To evaluate the feasibility, surgical outcomes and complications of laparoscopic restaging surgery for women with unexpected ovarian malignancy. STUDY DESIGN: We conducted a retrospective chart review of 14 women with unexpected ovarian malignancy who underwent laparoscopic restaging surgery including peritoneal washing cytology, laparoscopic pelvic and paraaortic lymphadenectomy up to the left renal vein level, omentectomy, and multiple peritoneal biopsies, and hysterectomy except three fertility saving surgery. RESULTS: The median age and median body mass index women were 49 years (range, 22-63) and 24.2m/kg(2) (range, 18.9-25.3), respectively. The median operating time was 230min (range, 155-370). The median numbers of harvested pelvic and paraaortic lymph nodes were 26 (range, 6-41) and 18 (range, 2-40), respectively. The median return of bowel activity was 28h (range, 21-79). Four of the women were upstaged from the initial presumed stage. There were two intraoperative complications, laceration of the inferior vena cava and cisterna chyli rupture. There was one postoperative complication, port-site metastasis. There was no conversion to laparotomic surgery. The median follow-up period was 33 months. Thirteen of the patients have no evidence of recurrences, however one patient died after 22 months after the surgery. CONCLUSION: Laparoscopic restaging surgery, performed by a specialized laparoscopic oncologist with sufficient laparoscopic experience and a well-trained operating team, is both feasible and effective in the management of unexpected ovarian malignancies.

Increased Stability of Nucleolar PinX1 in the Presence of TERT.

PinX1, a nucleolar protein of 328 amino acids, inhibits telomerase activity, which leads to the shortening of telomeres. The C-terminal region of PinX1 is responsible for its nucleolar localization and binding with TERT, a catalytic component of telomerase. A fraction of TERT localizes to the nucleolus, but the role of TERT in the nucleolus is largely unknown. Here, we report a functional connection between PinX1 and TERT regarding PinX1 stability. The C-terminal of PinX1(205-328), a nucleolar fragment, was much more stable than the N-terminal of PinX1(1-204), a nuclear fragment. Interestingly, PinX1 was less stable in TERT-depleted cells and more stable in TERT-myc expressing cells. Stability assays for PinX1 truncation forms showed that both PinX1(1-328) and PinX1(205-328), nucleolar forms, were more rapidly degraded in TERT-depleted cells, while they were more stably maintained in TERT-overexpressing cells, compared to each of the controls. However, PinX1(1-204) was degraded regardless of the TERT status. These results reveal that the stability of PinX1 is maintained in nucleolus in the presence of TERT and suggest a role of TERT in the regulation of PinX1 steady-state levels.