Study on the level of serum Mullerian inhibiting substance in children with cryptorchidism / 国际检验医学杂志

Objective To analyze the difference of serum levels of anti-Mullenan hormone (AMH) in chil-dren with different ages and different types of cryptorchidism,and to explore its role in the evaluation of tes-ticular development.Methods 60 children with simple cryptorchidism were selected as case group and 52 healthy children were selected as control group.The levels of serum AMH in two groups of children were measured and the differences were compared.Results (1)The level of AMH in the case group was lower than that in control group (P < 0.05),and there was no statistical significance between two subgroups of >6 to 11 years old children with cryptorchidism and healthy children (P>0.05).(2)The level of AMH in bi-lateral cryptorchidism group was lower than that in unilateral cryptorchidism group (P<0.05),and there was no significant difference between two subgroups of >6 to 11 years old children with bilateral cryptorchidism and unilateral cryptorchidism (P>0.05).(3)The level of AMH in the high level cryptorchidism group was lower than that of the low level cryptorchidism group (P<0.05),and there was no statistical difference be-tween between two subgroups of 3~11 year old children with cryptorchidism and low level cryptorchidism (P>0.05).(4)AMH level was negatively correlated with age,and positively correlated with testicular devel-opment.Conclusion AMH can be used as an important indicator of testicular development in children with cryptorchidism.

Anti-Proliferative and Apoptotic Activities of Mullerian Inhibiting Substance Combined with Calcitriol in Ovarian Cancer Cell Lines

PURPOSE: This study aimed to investigate whether Mullerian inhibiting substance (MIS) in combination with calcitriol modulates proliferation and apoptosis of human ovarian cancer (OCa) cell lines (SKOV3, OVCAR3, and OVCA433) and identify the signaling pathway by which MIS mediates apoptosis. MATERIALS AND METHODS: OCa cell lines were treated with MIS in the absence or presence of calcitriol. Cell viability and proliferation were evaluated using the Cell Counting Kit-8 assay and apoptosis was evaluated by DNA fragmentation assay. Western blot and enzyme-linked immunosorbent assay were used to determine the signaling pathway. RESULTS: The cells showed specific staining for the MIS type II receptor. Treatment of OCa cells with MIS and calcitriol led to dose- and time-dependent inhibition of cell growth and survival. The combination treatment significantly suppressed cell growth, down-regulated the expression of B-cell lymphoma 2 (Bcl-2), and up-regulated the expressions of Bcl-2 associated X protein, caspase-3, and caspase-9 through the extracellular signal-regulated kinase signaling pathway. CONCLUSION: These results, coupled with a much-needed decrease in the toxic side effects of currently employed therapeutic agents, provide a strong rationale for testing the therapeutic potential of MIS, alone or in combination with calcitriol, in the treatment of OCa.

Mullerian inhibiting substance/anti-Mullerian hormone: A novel treatment for gynecologic tumors

Mullerian inhibiting substance (MIS), also called anti-Mullerian hormone (AMH), is a member of the transforming growth factor-beta super-family of growth and differentiation response modifiers. It is produced in immature Sertoli cells in male embryos and binds to MIS/AMH receptors in primordial Mullerian ducts to cause regression of female reproductive structures that are the precursors to the fallopian tubes, the surface epithelium of the ovaries, the uterus, the cervix, and the upper third of the vagina. Because most gynecologic tumors originate from Mullerian duct-derived tissues, and since MIS/AMH causes regression of the Mullerian duct in male embryos, it is expected to inhibit the growth of gynecologic tumors. Purified recombinant human MIS/AMH causes growth inhibition of epithelial ovarian cancer cells and cell lines in vitro and in vitro via MIS receptor-mediated mechanism. Furthermore, several lines of evidence suggest that MIS/AMH inhibits proliferation in tissues and cell lines of other MIS/AMH receptor-expressing gynecologic tumors such as cervical, endometrial, breast, and in endometriosis as well. These findings indicate that bioactive MIS/AMH recombinant protein should be tested in patients against tumors expressing the MIS/AMH receptor complex, perhaps beginning with ovarian cancer because it has the worst prognosis. The molecular tools to identify MIS/AMH receptor expressing ovarian and other cancers are in place, thus, it is possible to select patients for treatment. An MIS/AMH ELISA exists to follow administered doses of MIS/AMH, as well. Clinical trials await the production of sufficient supplies of qualified recombinant human MIS/AMH for this purpose.

Serum anti-Mullerian hormone is a better predictor of ovarian response than FSH and age in IVF patients with endometriosis / 대한생식의학회지

OBJECTIVE: To evaluate the ability of serum anti-Mullerian hormone (AMH), FSH, and age to clinically predict ovarian response to controlled ovarian hyperstimulation (COH) in IVF patients with endometriosis. METHODS: We evaluated 91 COH cycles, including 43 cycles with endometriosis (group I) and 48 cycles with male factor infertility (group II) from January to December, 2010. Patients were classified into study groups based on their surgical history of endometriosis-group Ia (without surgical history, n=16), group Ib (with a surgical history, n=27). RESULTS: The mean age was not significantly different between group I and group II. However, AMH and FSH were significantly different between group I and group II (1.9+/-1.9 ng/mL vs. 4.1+/-2.9 ng/mL, p<0.01; 13.1+/-7.2 mIU/mL vs. 8.6+/-3.3 mIU/mL, p<0.01). Furthermore, the number of retrieved oocytes and the number of matured oocytes were significantly lower in group I than in group II. In group II, AMH and FSH as well as age were significant predictors of retrieved oocytes on univariate analysis. Only the serum AMH level was a significant predictor of poor ovarian response in women with endometriosis. CONCLUSION: Serum AMH may be a better predictor of the ovarian response of COH in patients with endometriosis than basal FSH or age. AMH level can be considered a useful clinical predictor of poor ovarian response in endometriosis patients.

Inhibitory effects of recombinant human Mullerian inhibiting substance on cell proliferation in two human ovarian carcinoma cell lines / 中国病理生理杂志

AIM:To investigate the inhibitory effects of recombinant human Mullerian inhibiting substance on cell proliferation in human ovarian carcinoma cells (OVCAR8 and SKOV3 cell lines). METHODS:The expression of MISIIR protein and the localization of MISIIR protein were analyzed by Western blotting and confocal spectral microscopy,respectively. Cell apoptosis and cell cycle were detected by flow cytometry (FCM). Cell viability was determined via MTT method. Clone formation test was used to detect oncogenicity in vitro.RESULTS:The MISIIR protein expression in OVCAR8 cells but not in SKOV3 cells was observed. MISIIR expression was seen on the OVCAR8 cell surface and in the cytoplasm with both antibodies. After treated with rhMIS for 48 h,the cell viability was significantly decreased in OVCAR8 cells. rhMIS inhibited the oncogenicity of OVCAR8 cells greatly. The cell apoptosis of OVCAR8 cell exposed to 10 mg/L rhMIS was (31.3±2.1)%,and OVCAR8 cells in the G_1 phase were increased by (70.4±3.0)%. Compared to SKOV3 cells the differences were significant (P<0.01). CONCLUSION:Recombinant human Mullerian inhibiting substance suppresses the growth of MISIIR-positive ovarian cancer cells by inducing apoptosis and cell cycle arrest. We predict that rhMIS might be a new target to treat human ovarian malignancies.

Mullerian inhibiting substance as a predictive marker of menopausal transition / 대한산부인과학회지

OBJECTIVE: To identified whether serum Mullerian inhibiting substance (MIS) level may be used as a predictive marker of menopausal transition. METHODS: Serum MIS level was measured in reproductive women (n=87), in menopausal transition women (n=58), and in menopausal women (n=5) by ELISA. And we examined the immunohistochemical staining of the MIS in the ovarian tissues of 15 reproductive, 15 menopausal transition, and 5 menopausal women. RESULTS: 1. In the reproductive women, mean serum MIS level was 1.73+/-1.07 ng/ml. In the menopausal transition women, mean serum MIS level was 0.18+/-0.11 ng/ml. Serum MIS level did not show any significant fluctuation patterns according to follicular development. In menopausal transition women, serum MIS level was significantly lower than that of reproductive women (P<0.001). The cutoff value of serum MIS level for menopausal transition was 0.5 ng/mg. In the menopausal women, serum MIS level was not detected. 2. Serum MIS level was significantly decreased as patient age was increased. 3. In the reproductive group, the immunohistochemical staining demonstrated strong expression of MIS in the granulosa cells of the primary follicles and the growing follicles, but not in corpus luteum, preovulatory mature follicle, atretic follicle, and corpus luteum. In the menopausal transition women, immunohistochemical staining for MIS was observed in the nearly same pattern as that of thereproductive women, but with weaker expression. In the menopausal women, immunohistochemical staining of the MIS was not observed. CONCLUSION: MIS is a good candidate for predictive marker for ovarian aging and perimenopausal transition.

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.

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.

Expression of Mullerian Inhibiting Substance Type II Receptor during Follicular Development in the Human Ovary / 대한산부인과학회잡지

OBJECTIVES: This study was aimed to obtain information on the ontogeny of the production profile of MIS type II receptor (MISR II) and the pattern of its localization according to follicular development METHODS: Expression of MISR II were studied in 21 ovarian specimens from adult normal cycling women by RT-PCR and in situ hybridization of the MISR II mRNA and immunohistochemical staining of the MISR II. RESULTS: The first staining for MISR II and MISR II mRNA were detected in the granulosa cells in primary follicles. The granulosa cells of multiple layered growing follicles showed strong specific staining for MISR II and MISR II mRNA. Among the growing follicles, large follicle stained more intensely than small one. Expression of the MISR II and MISR II mRNA were also seen in the granulosa cells and theca cells of antral follicles. The expression levels of MISR II and MISR II mRNA in mature follicles were 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 and MISR II mRNA expression when follicles become atretic. Both expressions were eventually lost from atretic follicles. And the MISR II and MISR II mRNA staining were not found in primordial follicles, oocytes, interstitial cells, ovarian epithelium, and corpus albicans. CONCLUSION: The production and localization of MISR II in granulosa cells, theca cells, and corpus luteum in normal reproductive ovary indicate that actions of MIS via MISR II are autocrine and paracrine in nature. The pattern of MISR II and MISR II mRNA expression according to follicular development indicate that MIS function in the ovary is turned on in primary follicles, increases to maximal levels in large growing follicles, and decreases just before ovulation. These experiments suggest that MIS may play an important role in follicle maturation and follicle selection during the adult reproductive cycle. And this study may yield important information to direct the development of newer contraceptive strategies.

Serum levels and expression of Mullerian inhibiting substance in the human ovary during menstrual cycle / 대한산부인과학회잡지

OBJECTIVE: This study was aimed to obtain information on normal MIS serum levels according to menstrual cycles of adult normal cycling women . It was also designed to obtain information on the ontogeny of the production profile of MIS and the pattern of its localization in ovary from adult normal cycling women. METHODS: Between January 1998 and January 1999, normal MIS serum levels were measured according to menstrual cycles using 160 serum samples from adult normal cycling women by ELISA. The ontogeny of the production profile of MIS and the pattern of its localization were also studied by immunohistochemical staining using the rabbit polyclonal antibody against human recombinant MIS in 35 ovarian specimens from adult normal cycling women. RESULT: The MIS levels were gradually increased through the follicular phase, reaching at its maximum at the ovulatory phase(4.2+/-2.6 ng/ml), and sharply decreased at the beginning of the luteal phase being minimized at the premenstrual phase(0.5+/-0.2 ng/ml). In average, the MIS levels of the follicular phase(3.7+/-1.9 ng/ml) were significantly higher than those of the luteal phase(1.8+/-2.4 ng/ml)(P<0.05). The MIS levels of the preovulatory and ovulatory phase were significantly higher than those of the other cycle days(P<0.05). Even the early follicular phase(2.9+/-1.6 ng/ml) showed higher MIS levels than the advanced luteal phase(0.9+/-0.7 ng/ml) and the premenstrual phase(0.5+/-0.2 ng/ml)(P<0.05 and P<0.05, respectively). The first staining for MIS was detected in the cytoplasm of granulosa cells when the flattened granulosa cells changed to the cuboidal cells in primordial follicles. The granulosa cells of both single and multiple layered growing follicles showed strong specific staining for MIS. but the MIS staining was not found not in the mature follicle just before ovulation, atretic follicles, corpus luteum, and corpus albicans. MIS staining waned in the mature follicles just before ovulation. CONCLUSION: These experiments demonstrate that the MIS is produced by ovarian granulosa cells in normal reproductive females. The MIS may play an important role as a hormone of follicular development and oocyte maturation through interactions with female steroid hormones, gonadotropins, and growth factors during the adult reproductive cycle.

The Significance of Measurement of Mullerian Inhibiting Substance in Neonates with Respiratory Distress Syndrome / 대한주산의학회잡지

OBJECTIVE: The objectives of this study were to obtain information on MIS levels in normal and RDS neonates and to investigate the relationship between the RDS prevalence and MIS level in preterm and term neonates. METHODS: Total 131 male neonates were selected randomly and they were consisted of 50 term normal neonates, 15 term neonates with RDS, 50 prematurely born normal neonates, and 16 prematurely born neonates with RDS. Total 131 female neonates were also selected like male neonates. The venous blood was collected from all subjects and measured the level of MIS using ELISA. The ANCOVA was conducted to evaluate any influence of adjusted value of gestational age and body weight on MIS level between normal neonates and neonates with RDS. RESULTS: 1) The MIS levels of female neonates were significantly lower than those of male neonates with no overlap. 2) The MIS levels of normal female neonates were not significantly different from those of female neonates with RDS. 3) There were significant negative relationships between MIS concentration and gestational age (r=-0.777, p<0.001), and birth weight(r=-0.728, p<0.001) in normal rnale neonates. 4) There were significant negative relationships between MIS concentration and gestational age (r=-0.726, p<0.001), and birth weight(r=-0.725, p<0.001) in male neonates with RDS. 5) After adjusting the value of gestational age, the MIS level of male neonates with RDS was significantly higher than that of normal male neonates(p<0.001). 6) After adjusting the value of body weight, the MIS level of male neonates with RDS was significantly higher than that of normal male neonates(p<0.001). CONCLUSION: Male neonates with RDS had higher MIS levels than normal male neonates of the same body weight or same calculated gestational age. The results of this study suggest that MIS may play a causative or important ancillary role in the sexual dimorphism that characterizes the neonatal RDS and may be used as a predictive marker of RDS in male neonates.

Expession of Mullerian Inhibiting Substance in the Human Ovary / 대한산부인과학회잡지

Mullerian inhibiting substance(MIS) has been known as a non-steroidal testicular Sertolicell product responsible for the regression of Mullerian duct in male embryos. More recently MIS was also found to be present in an bioactive form in the bovine and rat ovaries but the function of MIS in the ovary has not been fully delineated. In this study, in order to understand its function in the ovary the ontogeny of the production profile of MIS and the pattern of its localization in ovaries from adult normal cycling women were studied by immunohistochemical staining using the rabbit polyclonal antibody against human recombinant MIS that almost completely blocks its biological activity. MIS was detected specifically and exclusively in the cytoplasm of granulosa cells. The flattened granulosa cells in primordial follicles failed to stain for MIS, but the cuboidal cells of growing follicles stained intensely. The granulosa cells of both single and multiple layered growing follicles showed strong specific staining for MIS. Within the multiple layers of granulosa cells, closer to the oocyte, stained more intensely than those near the basement membrane. Similarly, in antral follicles, cumulus cells and periantral granulosa cells stained more intensely than those in the periphery. MIS staining waned in the mature follicles just before ovulation and could not be found in atretic follicles, corpus albicans. In conclusion, this specific localization suggest that MIS may act as an intraovarian regulator of follicular development and oocyte maturation during the adult reproductive cycle.