Cytological Evaluation and REBA HPV-ID HPV Testing of Newly Developed Liquid-Based Cytology, EASYPREP: Comparison with SurePath

BACKGROUND: The objective of this study was to evaluate a newly-developed EASYPREP liquid-based cytology method in cervicovaginal specimens and compare it with SurePath. METHODS: Cervicovaginal specimens were prospectively collected from 1,000 patients with EASYPREP and SurePath. The specimens were first collected by brushing for SurePath and second for EASYPREP. The specimens of both methods were diagnosed according to the Bethesda System. Additionally, we performed to REBA HPV-ID genotyping and sequencing analysis for human papillomavirus (HPV) on 249 specimens. RESULTS: EASYPREP and SurePath showed even distribution of cells and were equal in cellularity and staining quality. The diagnostic agreement between the two methods was 96.5%. Based on the standard of SurePath, the sensitivity, specificity, positive predictive value, and negative predictive value of EASYPREP were 90.7%, 99.2%, 94.8%, and 98.5%, respectively. The positivity of REBA HPV-ID was 49.4% and 95.1% in normal and abnormal cytological samples, respectively. The result of REBA HPV-ID had high concordance with sequencing analysis. CONCLUSIONS: EASYPREP provided comparable results to SurePath in the diagnosis and staining quality of cytology examinations and in HPV testing with REBA HPV-ID. EASYPREP could be another LBC method choice for the cervicovaginal specimens. Additionally, REBA HPV-ID may be a useful method for HPV genotyping.

Human papillomavirus 16/18 AS04-adjuvanted cervical cancer vaccine: immunogenicity and safety in 15-25 years old healthy Korean women / 부인종양

OBJECTIVE: The study assessed the immunogenicity and safety of human papillomavirus (HPV)-16/18 AS04-adjuvanted cervical cancer vaccine in healthy Korean women aged 15-25 years. METHODS: Phase IIIB, double-blind, randomised (2:1), multi-centre trial was conducted in Korea from June 2007 to March 2008. The study enrolled 225 women in the HPV (N=149) and placebo (N=76) groups who received three doses of HPV-16/18 AS04-adjuvanted vaccine or placebo (aluminium hydroxide) administered intramuscularly at 0, 1, and 6 months and were followed until one month post-dose 3. Serum samples were collected pre-vaccination and one month post-dose 3. Safety and reactogenicity data were collected throughout. RESULTS: In this trial, 208 women completed the study (141 in HPV group; 67 in placebo group). At month 7, all initially seronegative women had seroconverted for HPV-16 and HPV-18 antibodies with anti-HPV-16 and anti-HPV-18 geometric mean titres of 9,351.4 El.U/mL (95% CI, 8,145.5 to 10,735.8) and 4204.1 El.U/mL (95% CI, 3,626.5 to 4,873.6), respectively. Initially seropositive women showed similar increase in geometric mean titre levels. Compliance to the three dose vaccination course was 95.3% in HPV and 89.5% in placebo group. Solicited local (pain) and general (fatigue, myalgia or headache) symptoms were commonly reported in both groups. Three serious adverse events were reported (two in HPV group; one in placebo group), all unrelated to vaccination by the investigator; all recovered. CONCLUSION: The HPV-16/18 AS04-adjuvanted vaccine was highly immunogenic with a clinically acceptable safety profile in Korean women. This study was in line with previous global studies in Europe, North America, and Brazil. (ClinicalTrials.gov number, NCT 00485732.)

Measurement of apoptosis using M30 in culture media of cell lines treated with anti-cancer agents / 대한산부인과학회지

OBJECTIVE: We investigated a possible use of the induced apoptosis as a biomarker in the cells and their media treated with commonly used anti-cancer agents in gynecologic malignancies. METHODS: After treatments with low and high concentrations of paclitaxel, cisplatin, and camptothecin in HeLa and OVCAR-3 cells, the levels of M30 antigen were detected in the cells and their media by immunofluorescence staining and ELISA methods, respectively. RESULTS: The percentages of M30-fluoresein isothiocyanate (FITC) positive cells in HeLa and OVCAR-3 cells treated with paclitaxel, cisplatin, and camptothecin were 4.3% vs 18.1% vs 34.87% and 4.07% vs 18.6% vs 32.63%, 4.3% vs 17.87% vs 32.38% and 4.07% vs 16.83% vs 32%, and 4.3% vs 16.75% vs 31.3% and 4.07% vs 15.18% vs 29.9% in control, low dose, and hight dose groups, respectively (P<0.001). M30 antigen levels (U/L) measured in culture media of HeLa and OVCAR-3 cells treated with paclitaxel, cisplatin, and camptothecin were 53.03 vs 101.53 vs 355.59 and 86 vs 114.41 vs 412.04, 53.03 vs 79.84 vs 327.64 and 86 vs 125.44 vs 385.09, and 53.03 vs 88.41 vs 295.005 and 86 vs 108.42 vs 263.1 in control, low dose, and hight dose groups, respectively (P<0.001). CONCLUSION: Our results obtained in this preclinical study suggests that measurement of the levels of M30 antigen may help to predict the clinical responses and to select the effective anti-cancer agents in clinical settings, rapidly and quantitatively.

Evaluation of 110 cases of single-port access laparoscopically assisted vaginal hysterectomy (SPA-LAVH) and comparison with multi-port access / 대한산부인과학회지

OBJECTIVE: To evaluate the safety and feasibility of single-port access laparoscopically assisted vaginal hysterectomy (SPA-LAVH) using conventional laparoscopic instruments compared to multi-port access laparoscopically assisted vaginal hysterectomy (MPA-LAVH). METHODS: We reviewed the medical records of 220 patients with uterine leiomyoma or adenomyosis who underwent 110 SPA-LAVH and 110 MPA-LAVH in Incheon St. Mary's Hospital between April 2007 and November 2009. We performed SPA-LAVH with conventional rigid straight laparoscopic instruments in all cases. We also performed a new vaginal cuff closure method, Kim's Vaginal Vault Suspension Method, named after the operator (Kim, YW) in both SPA-LAVH and MPA-LAVH. RESULTS: There was no significant difference in patients' age, operating time, uterine weight, hemoglobin change, frequency of blood transfusion, and incidence of postoperative fever between the two groups. The patients' mean age was 46.1+/-7.0 years (SPA-LAVH) and 45.5+/-6.3 years (MPA-LAVH). The mean operating time was 87.2+/-21.0 minutes (SPA-LAVH) and 83.3+/-20.3 minutes (MPA-LAVH). The mean uterine weight was 261.4+/-139.7 g (SPA-LAVH) and 257.8+/-132.9 g (MPA-LAVH). The mean hemoglobin change was 1.1+/-0.7 g/dL (SPA-LAVH) and 1.2+/-0.6 g/dL (MPA-LAVH). Neither bowel injury nor urinary tract injury occurred during the operation in the two groups. One of the SPA-LAVH and one of the MPA-LAVH cases were converted to abdominal total hysterectomy. The mean hospital stay time was shorter with SPA-LAVH (2.6+/-0.6 days [SPA-LAVH] and 3.3+/-0.7 days [MPA-LAVH], P<0.05). CONCLUSION: SPA-LAVH using conventional rigid straight laparoscopic instruments can be offered as a safe and feasible alternative to MPA-LAVH.

Detection of chemosensitivity using K18-Asp(396) (M30) antibody in HeLa and OVCAR-3 cell lines treated with anticancer agents / 대한산부인과학회지

OBJECTIVE: The aim of this study was to detect the levels of M30-antigens as a biomarker of apoptosis in cells and their culture media after treatments with anticancer drugs as a preclinical study. METHODS: After HeLa and OVCAR-3 cells were treated respectively with paclitaxel, cisplatin, and camptothecin, the harvested cells were stained sequentially with M30 monoclonal antibodies and propidium iodide (PI). Afterwards, they were analyzed using a FACScan flow cytometer and observed under an immunofluorescence microscope for M30-FITC immunofluorescences. Levels of M30 antigens were also detected in their culture media using M30-Apoptosense ELISA kit. RESULTS: The levels of M30-FITC immunofluorescences were elevated in both cell lines after each drug treatments compared with those of control cells. The levels of M30 antigens detected by ELISA in media culturing each cell line treated with each of drugs were elevated compared with those of control cells. CONCLUSION: This study suggests that M30-antigens representing chemotherapy induced apoptosis may be a useful biomarker for predicting and monitoring the response of neoadjuvant chemotherapy in patients with gynecologic cancers.

A Case of Fetal Cardiac Rhabdomyoma with Tuberous Sclerosis / 대한주산의학회잡지

Fetal cardiac tumors are a rare condition and are histologically benign. Cardiac rabdomyoma is the most common cardiac tumor and may be associated with tuberous sclerosis. Clinical follow-up including monitoring of fetal well-being is warranted as long as the fetus remains asymptomatic and there is no evidence for hydrops fetalis. If obstruction to blood flow and/or early fetal compromise is noted, then the decision of whether to deliver early must be made. We report a case of fetal cardiac tumor diagnosed by antenatal ultrasonography and diagnosed as tuberous sclerosis with brain MRI scanning after birth.

Myxoid neurofibroma of the labia majora: A case report / 대한산부인과학회지

Myxoid neurofibroma is a benign tumor of perineural cell origin, which is demonstrated with a positive immunohistochemical stainig for S-100. The most common locations of the myxoid neurofibroma are face, shoulder, arm, periungual and foot. We experienced an unusual case of myxoid neurofibroma which originated from labia majora area then reported it. To our knowledge, this is the first time that a labia majora location is reported.

Myxoid neurofibroma of the labia majora: A case report / 대한산부인과학회지

Myxoid neurofibroma is a benign tumor of perineural cell origin, which is demonstrated with a positive immunohistochemical stainig for S-100. The most common locations of the myxoid neurofibroma are face, shoulder, arm, periungual and foot. We experienced an unusual case of myxoid neurofibroma which originated from labia majora area then reported it. To our knowledge, this is the first time that a labia majora location is reported.

Fertility-sparing radical abdominal trachelectomy for cervical clear cell adenocarcinoma unrelated to diethylstilbestrol exposure : A case report / 대한산부인과학회지

Clear cell adenocarcinoma (CCAC) is a rare cancer that comprises less than 9% of the cervical adenocarcinoma cases. We experienced a case of fertility-sparing radical abdominal trachelectomy for cervical clear cell adenocarcinoma (CCAC). Thus, reported it. A 27 year old female was diagnosed with clinical stage Ib cervical CCAC. She had no history of maternal exposure to diethylstilbestrol and had negative PAP cytology and HPV tests. She was treated with neoadjuvant chemotherapy followed by radical abdominal trachelectomy. After 2 cycles of postoperative adjuvant chemotherapy, the lesion disappeared completely in an imaging study, and potential fertility was preserved. Radical abdominal trachelectomy with chemotherapy may be a valuable approach for treating stage Ib cervical CCAC in women that wish to preserve potential fertility.

Development of Korean Academy of Medical Sciences Guideline Rating the Physical Impairment; Kidney, Bladder, Urethra, Male and Female Reproductive Systems (Preliminary Report)

For the evaluation of the kidney impairment, serum creatinine concentrations or glomerular filtration rates are mainly used, and the conditions of solitary or transplanted kidney and chronic dialysis are also taken into the considerations. Some symptoms and signs of the chronic renal disability in spite of adequate treatment add one additional grade. For evaluating bladder and urethral impairment, the criteria include voiding symptoms and signs. The patients with urinary diversions have impairment grades depending on the alteration of upper urinary tract function. For penile impairment, the degrees are evaluated using the international index of erectile function, nocturnal penile tumescence and color doppler ultrasonography. For evaluating impairment of other male reproductive organs, functional and anatomical changes of these organs, analysis of the semen or hormones and the state of solitary testis are used as the criteria. For evaluating impairment of female reproductive organs, pregnancy potential, requirement of continuous treatment and the ability of sexual intercourse are used. Also, degree of impairment is modified according to the ages in evaluating female reproductive systems. We have tried to make this evaluation system objective, scientific, and convenient, but still find it leaving much to be desired.

Antiproliferative Effects of Mullerian Inhibiting Substance on Human Ovarian Cancer Cell Lines / 대한산부인과학회지

OBJECTIVE: In order to explore Mullerian inhibiting substance (MIS) effects on the ovarian neoplasia, the expression and localization of the MIS type II receptor (MISR II), the growth inhibitory effects of MIS, and the underlying molecular mechanisms were investigated in the ovarian cancer cell lines. METHODS: Expression of MISR II were studied in SKOV-3, OVCAR-3, and OVCAR-8 cell lines by immunohistochemical staining. The antiproliferative effects of MIS in these cell lines were investigated by methylthiazoletetrazolium (MTT) assay, fluorescence-activated cell sorting (FACS) analysis, annexin-V-FITC binding, and western blot analysis. RESULTS: All cell lines showed strong specific staining for MISR II, although staining in OVCAR-8 cells was more intense than that in SKOV-3 and OVCAR-3. Treatment of OVCAR-8 cells with MIS led to a dose- and time-dependent inhibition of cell growth and survival was determined use by MTT assay. But OVCAR-3 cells exhibited growth inhibition at higher doses after 48 hours of treatment and SKOV-3 cells did not demonstrate response. Using FACS analysis, exposure of OVCAR-8 cells to MIS (71 nM) resulted in G1 arrest after 24 hours of treatment. This pattern was changed by time-dependent increase in the percentage of cells with a sub G0G1 DNA content, suggesting apoptosis, after 48 hours of treatment. These results suggested that cell death be preceded by cell cycle arrest. Time-related induction of apoptosis was also observed in this cell line as measured by annexin-V-FITC binding. In OVCAR-8 cells, the growth inhibitory effects of MIS were mediated through specific induction of CDKI p16 protein expression and via regulation of E2F1 in the absence of detectable levels of pRb. We estimated that OVCAR-3 cells were affected by MIS through p16-independent, alternative mechanistic pathways, since the growth inhibitory effects of MIS were minimal. SKOV-3 cells did not express p16 protein. CONCLUSION: We have demonstrated that ovarian cancer cells express the MISR II. Epithelial ovarian cancer cells respond to MIS by growth inhibition. Although the precise mechanisms of MIS mediated inhibition of ovarian cancer cell growth have not been fully defined, these data suggest that MIS has activity against ovarian cancers in vitro and may also be an effective targeted therapy for ovarian cancer.

Time Trend of Survival and Treatment Modalities of Endometrial Cancer in Korea (1990-2005) / 대한산부인과학회지

OBJECTIVE: This study was performed to evaluate the overall survival and the change in treatment modalities in patients with uterine endometrial cancer in Korea. METHODS: From January 1990 to March 2005, medical records of 740 patients with endometrial cancer in nine hospitals were reviewed. The overall survival was determined supported by the death statistics of Korea National Statistical Office. RESULTS: The mean age of patients was 51.5 years (range: 21-82 years). The mean gravidity and parity were 3.3 and 2.1 (range: 0-18, 0-9), respectively. The most common stage, grade and histological type at diagnosis were FIGO stage I, grade 1 and endometrioid adenocarcinoma (76.5%, 56.4% and 87.2%), respectively. The main treatment modalities was surgery on stage I (59.8%), surgery-adjuvant radiotherapy on stage II (55.1%), surgery-adjuvant radiotherapy or surgery-adjuvant chemoradiation on stage III (38.3%) and surgery-adjuvant chemotherapy on stage IV (55.6%). The preferred treatment modality was surgery only on grade 1 (69.0%) and surgery-adjuvant radiotherapy on grade 2-3 (43.4% and 53.2%). Surgery had been the most common method of therapy before 1998 but its prevalence gradually decreased. As a result, surgery-adjuvant radiotherapy and surgery-adjuvant chemotherapy were most widely performed in 2004-2005 and surgery-adjuvant chemoradiation also increased more than a twofold. The overall 5 years survival rate (5YSR) for all 740 patients was 81.3%. The overall 5YSR of stage I was 89.0%. The overall 5YSR of grade 1, grade 2, grade 3 were 96.0%, 92.0%, 80.0%. Before 1998, the survival rate was 77.0%. Since then it increased to 83-88% and in 2000-2001 it increased to 88.0%, and it was the highest survival rate. CONCLUSION: The survival rate of endometrial cancer has been improving for the past 15 years and the method of treatment is also being changed currently. In order to improve the survival rate of endometrial cancer, a close investigation including genetic and environmental factors of the pathophysiology of endometrial cancer along with the epidemiology of risk factors, should be carried out.

A case of protruded subserosal myoma through pelvic floor in pregnancy / 대한산부인과학회지

Myoma is the most common tumor in gynecologic field. As ultrasonography because popular in antenatal care, the more cases of myoma and those adverse effects during pregnancy are more frequently detected. The management of myoma during pregnancy is conservative, but in rare circumstances, surgical intervention including myomectomy may be required. We have experienced a case of protruded subserosal myoma with the uterine cervix in midtrimester of pregnancy. The patient was managed surgically by transvaginal myomectomy and had successfully maintained pregnancy. We report a case of protruded subserosal myoma through pelvic floor in pregnancy with brief review of literatures.

Secondary acute myeloid leukemia after platinum based chemotherapy for ovarian cancer / 대한산부인과학회지

Among chemotherapeutic regimens used for advanced ovarian cancer, platinum-based combination chemotherapy remains a mainstay of the treatment of advanced ovarian cancer, providing significant response rates and survival benefits. However, with widespread use of long-term chemotherapy in treating ovarian cancer, emergence of secondary leukemia has become medical concern as one of the most unfavorable late complications. Depending upon the type, duration, and dosage of previous chemotherapy, the risk of developing acute myeloid leukemia has been estimated to be between 2% and 10%. Moreover, the frequency of this complication might increase as the survival in patients with ovarian cancer undergoing chemotherapy continues to increase with developing therapeutic options. Recently, we experienced a case of secondary acute myeloid leukemia developing 3.5 years after platinum-based chemotherapy. In this report, clinical course of the patient and contributing factors for the secondary leukemia were presented.

Trousseau's syndrome in association with ovarian clear cell carcinoma / 대한산부인과학회지

The association of neoplastic disease and thromboembolic disorder was first recognized by Trousseau in 1865. Since then, thromboembolic events have often been reported in a variety of malignant conditions. Recently, we experienced an unusual case of a 67-year-old ovarian cancer patient with extensive thromboembolic events involving lung, brain, extremity and abdominal cavity. In this report, we describe a systemic coagulopathy fluctuating in accordance with anticoagulant therapy, and have special regard for a role of anticoagulants for the treatment of this syndrome.

The prognostic factors and survival analysis of primary peritoneal carcinoma / 대한산부인과학회지

OBJECTIVE: To date, few attempts have been made at clinical features and prognostic factors of primary peritoneal carcinoma (PPC) because of low prevalence. The aim of this study is to evaluate the clinical characteristcs and determine the prognosis factors of PPC. METHODS: From March 1996 to March 2004, a total of 23 women newly diagnosed with PPC were recruited into the study. Overall survival and prognostic factors were evaluated using Kaplan-Meier method and Cox regression model. RESULTS: The mean age of patients was 58.7+/-7.6 years and the FIGO stage was advanced disease; stage IIIc (73%) and IV (27%). The mean survival time for patients enrolled was 26.0 months. By univariate analysis, tumor state (p=0.028), performance status (p=0.045), the presence of initial debulking operation (p=0.035), and normalization of CA125 at 3 months of treatment (p=0.003) were significantly correlated with survival. On multivariate analysis, only the normalization of CA125 at 3 months of treatment remained as the independent factor for survival (Odds ratio, 6.896; 95% Confidence interval, 1.504-31.623; p=0.013). CONCLUSION: The mean survival time for patients with PPC was 26.0 months, and the normalization of CA125 at 3 months of treatment was identified as the independent prognostic factor. From this study, we analysis the clinical characteristics of PPC and provide more precise understanding of this disease.

Diagnosis of Cervical Neoplasia Using Immunohistochemical Staining of p16(INK4A) / 부인종양

OBJECTIVE: There is need for more objective diagnostic parameters to identify cervical dysplastic or neoplastic cells. So, we examined the p16(INK4A) expression in the cervical tissues to evaluate the value of p16(INK4A) as a diagnostic parameter. METHODS: We examined the p16(INK4A) expression by immunohistochemical staining in normal cervical tissues (n=3), preneoplastic lesions (n=6), carcinoma in situ (CIS, n=5), and invasive carcinomas (n=5) of the cervix, which were selected randomly by H and E staining from the archives of formalin-fixed and paraffin-embedded tissues and we also examined the status of human papillomavirus (HPV) infection in the same tissues. RESULTS: The positive rates of p16(INK4A) expression was significantly higher in all abnormal cervical tissues including subclinical papillomavirus infection (SPI), dysplasia, CIS, and invasive carcinoma than in normal cervical epithelium (p=0.001). Despite the strong expression of p16(INK4A) in the area of CIS, no expression of p16(INK4A) was observed in the area of normal epithelium in the vicinity of CIS. 11 cases among 19 cases of examined tissue samples were tested for HPV infection. Seven of them showed positivity for HPV DNA. CONCLUSION: We herein demonstrated that p16(INK4A) would be a sensitive and specific marker for the abnormal cervical cells in tissue sections. This approach will help to reduce interobserver variations in the histopathologic interpretation of cervical biopsy specimens.

Expression of mullerian inhibiting substance and its receptor in ovarian neoplsia / 대한산부인과학회지

Mullerian inhibiting substance (MIS) is a glycoprotein hormone produced by fetal Sertoli cells that causes regression of the Mullerian ducts in males during sexual differentiation. Cell lines derived from human ovarian epithelium and rodent Leydig cell tumors, which respond to MIS in growth inhibition assays and express the MIS type II receptors (MISR II). But the pathophysiological role of MIS in human ovarian neoplasia development has not yet been fully established. In order to understand its role in pathogenesis of ovarian neoplasia, the expression and localization of the MIS and MISR II were studied in 5 normal ovaries, 11 benign tumors, 9 borderline ovarian malignancies, 40 ovarian malignancies in paraffin embedded tissue and tissue microarrays by using immunohistochemical stain. The results were as follows; 1. The first staining for MIS and MISR II were detected in granulosa cells in primary follicles of normal ovary. Among the growing follicles, larger developing follicles stained more intensely than smaller follicles. 2. In benign ovarian tumors, 8 (72.73%) in MIS and 5 (45.45%) in MISR II out of 11 cases were stained. The intensity scores of staining were 1.18 in MIS and 0.64 in MISR II. 3. In borderline malignancies, 6 (66.67%) in MIS and 7 (77.78%) in MISR II out of 9 cases were stained. The intensity scores of staining were 0.89 in MIS and 1.22 in MISR II. 4. In ovarian malignancies, the expression of MIS and MISR II were 50% (9/18) and 50% (9/18) in epithelial, 92.30% (12/13) and 76.72% (10/13) in germ cell, and 88.9% (8/9) and 100% (9/9) in sex-cord stromal tumors. The intensity scores of MIS and MISR II expression were 0.72 and 0.72 in epithelial, 1.45 and 1.62 in germ cell, and 1.78 and 1.67 in sex-cord stromal tumors. 5. There was significant high expression of MIS and MISR II in non-epithelial (90.91%, 86.36%) than epithelial ovarian cancers (50%, 50%). The scores of expression intensity was also higher in non-elithelial cancers (MIS: 1.67 +/- 0.16 vs 0.72 +/- 0.20, p=0.003, MISR II: 1.64 +/- 0.20 vs 0.72 +/- 0.21, p=0.022). In conlusion, the expression of MIS and MISR II were not different according to the differentiation, but tissue type specific. The frequency of MIS and MISR II expression was higher in non-epithelial cancers, especially in sex-cord stromal tumors. The results of this experiment could be utilized as scientific basis of researches, furthermore clinical applications in diagnosis and treatment of non-epithelial ovarian malignancies.

Expression of Mullerian Inhibiting Substance and Its Receptor in the Human Ovary During Menstrual Cycle / 대한산부인과학회잡지

In this study, in order to further understanding of function of Mullerian inhibiting substance (MIS) and the ontogeny of the production profile of biologically active MIS and MIS type II receptor (MISR II), the patterns of their localization according to the follicular development in 21 ovarian specimens from women in reproductive age were studied by immunohistochemical staining. The flattened granulosa cells in primordial follicles failed to stain for MIS and MISR II, but the first staining was detected in the cuboidal granulosa cells in primary follicles. MIS and MISR II were detected specifically and exclusively in the cytoplasm of granulosa cells. The granulosa cells of both single and multiple layered growing preantral follicles showed strong specific staining for MIS and MISR II. Among the growing follicles, large follicle stained more intensely than small one. Within the multiple layers of granulosa cells, the innermost cells, closer to the oocyte, stained more intensely for MIS than those near the basement membrane, but MISR II was evenly distributed. In antral follicles, expression of the MIS was only seen in the granulosa cells, but MISR II was seen in the granulosa cells and theca cells. In large antral follicles, cumulus cells and periantral granulosa cells stained more intensely for MIS than those in the periphery. MIS staining waned in the mature follicles just before ovulation and could not be found in atretic follicles, corpus luteum, and corpus albicans. The expression levels of MISR II in mature follicles was lower than those in growing follicles and were even further reduced, but still detectable, in corpus luteum. There was a decreased level of MISR II expression when follicles become atretic and eventually lost from atretic follicles. The MIS and MISR II staining were not found in primordial follicles, oocytes, interstitial cells, ovarian epithelium, and corpus albicans. It is concluded that actions of MIS via MISR II are autocrine and paracrine in nature. The pattern of MIS and MISR II expression according to the menstrual cycles and development suggest that MIS may act as an intraovarian regulator of follicle maturation, selection and ovulation during the adult reproductive cycle.

Detection of Apoptosis by the Stainings of Annexin V, Propidium Iodide and Cytokeratin in OVCAR-3 Ovarian Cancer Cell Line / 대한산부인과학회잡지

OBJECTIVE: This study was designed to estimate the chemosensitivity by a quantitative evaluation of the apoptotic cell fractions using flow cytometry. METHODS: The OVCAR-3 cells were exposed to 20 nM or 30 nM taxol for 0 (control), 24 and 48 hours, then removed the taxol contained media, and cultured further with fresh media without taxol. (1) Fluorescein isothiocyanate-conjugated Annexin V (Annexin V-FITC) and propidium iodide (PI) were added to one test tube to detect the apoptotic cell fractions and at the same time, PI was added to the other tube to stain the DNA. (2) Annexin V-FITC and cytokeratin (clone CAM5.2 and MNF116) were added to the test tube. They were fixed and permeabilized with 1% paraformaldehyde solution and 100% methanol. They were then incubated with phycoerythrin (PE)-conjugated goat anti-mouse immunoglobulin G (GAM IgG1-PE or GAM IgG2a-PE) and sequentially stained with PI for DNA. All the stained cells were analyzed by a FACScan flow cytometer. RESULTS: (1) After treatment of 20 nM or 30 nM of taxol, G2M arrest was observed in both of treatment groups, which increased with time. (2) The G0G1 sub-fraction indicative of apoptosis increased with increase of culturing time from 24 hrs to 48 hrs. (3) The early apoptotic cell fraction with positive annexin V-FITC and negative PI increased with increase of culturing time. (4) In cells stained sequentailly with annexin V-FITC, cytokeratin (CAM5.2 and MNF116), and PI after 30 nM taxol treatment, the early apoptotic cell fractions increased with increase of culturing time. However, their extent was somewhat lower than those observed by positive annexin V-FITC and negative PI in cells treated with 20 nM of taxol. CONCLUSION: The results of sequential stainings with annexin V-FITC, cytokeratin, and PI were consistent with the those of annexin V-FITC and PI with parallel DNA staining. Our results suggested that the level of apoptosis detected by flow cytometry could be a marker of chemosensitivity which could select the sensitive anti-cancer agents before administration to gynecologic cancer patients.