Biomedical subjects
Publications and source records attributed to Chii-Ruey Tzeng.
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BACKGROUND: Hatching is an important process for early embryo development, differentiation and implantation. However, little is known about its regulatory mechanisms. By integrating the technologies of RNA amplification and cDNA microarrays, it has become possible to study the gene expression profile at this critical stage. METHODS: Pre-hatched and hatched ICR mouse embryos (25 blastocysts in each group were used in the triplicate experiments) were collected for RNA extraction, amplification, and microarray analysis (the mouse cDNA microarray, 6144 genes, including expressed sequence tags). RESULTS: According to cDNA microarray data, we have identified 85 genes that were expressed at a higher level in hatched blastocyst than in pre-hatched blastocysts. In this study, 47 hatching-related candidate genes were verified via re-sequencing. Some of these genes have been selected and confirmed by real-time quantitative RT-PCR. These hatching-specific genes were also expressed at a lower level in the delayed growth embryos (morula or blastocyst without hatching at day 6 post hCG). These genes included: cell adhesion and migration molecules [E-cadherin, neuronal cell adhesion molecule (NCAM), lectin, galactose binding, soluble 7 (Lgals7), vanin 3 and biglycan], epigenetic regulators (Dnmt1, and SIN3 yeast homolog A), stress response regulators (heme oxygenase 1) and immunoresponse regulators [interleukin (IL)-2-inducible T-cell kinase, IL-4R, interferon-gamma receptor 2, and neurotrophin]. The immunostaining of E-cadherin and NCAM showed strong and specific localization in hatched blastocyst. CONCLUSIONS: This work provides important information for studying the mechanisms of blastocyst hatching and implantation. These hatching-specific genes may have potential as new drug targets for controlling fertility.
BACKGROUND: This study compares the fertilization rate and embryonic development of oocytes randomly inseminated by conventional IVF or ICSI in patients with polycystic ovarian syndrome (PCOS) and normozoospermic semen during IVF cycles. METHODS: Sibling oocytes were randomized to be inseminated either by ICSI or IVF. Fertilization rate (two pronuclei/COC), day 2 embryonic morphology and rate of development were assessed. RESULTS: A total of 1089 cumulus-oocyte complexes (COC) were collected in 60 cycles (mean+/-SD, 18.2 +/- 7.2). Totals of 541 and 548 COC were inseminated by IVF and ICSI respectively, with a significantly higher fertilization rate in the ICSI group (ICSI versus IVF, 72.3 +/- 15.5 versus 44.8 +/- 25.1%). No fertilization failure occurred in the group of oocytes inseminated by ICSI, whereas the COC in nine patients (15%) inseminated by IVF had complete fertilization failure. The day 2 embryonic morphology and rate of development were not different regardless of the insemination method. CONCLUSIONS: Our results suggested that another randomized controlled study, randomizing patients instead of sibling oocytes, should be undertaken to compare the pregnancy rate per started cycle and to see whether ICSI should be performed on all, or at least on a portion of, oocytes for patients with PCOS undergoing IVF cycles.
Fallopian tube carcinoma is the least common of gynecological malignancies. We report the case of a 56-year-old woman who presented with a 2 1/2-year history of intermittent vaginal bleeding and lower abdominal pain. Transvaginal sonography revealed a cystic lesion of the fallopian tube with papillary projections, distinct from the ovary and uterus. Doppler examination showed low vascular impedance (resistance index 0.50, pulsatility index 0.80). The suspicion of tubal malignancy was confirmed at surgery.
OBJECTIVE: To evaluate the transcription and translation ability of mitochondria in terminally differentiated granulosa cells, these cells were incubated with ionic calcium. DESIGN: Prospective laboratory research. SETTING: In vitro fertilization laboratory in a university hospital. PATIENT(S): Granulosa cells were harvested from 50 female patients undergoing IVF. INTERVENTION(S): Analysis of mitochondrial gene expression by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and of mitochondrial-encoded proteins by Western blot. MAIN OUTCOME MEASURE(S): Comparison of the RNA expression levels of genes including cytochrome c oxidase subunit I (COX I), adenosine triphosphate synthase 6 (ATPase 6), flavoprotein, and succinate-ubiquinone oxidoreductase, and protein levels of COX I and flavoprotein in different calcium ion treatment groups. RESULT(S): There were dose-dependent increases in RNA expressions of the four genes analyzed from granulosa cells cultured in a serial concentration of calcium ions. This effect was abolished when cells were preincubated with the extracellular calcium-chelating agent, Ethylene glycol-bis (2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). The effect of ionic calcium on both the nuclear- and mitochondrial-encoded subunits also was determined. Expression levels of mitochondrial transcription factor A in RNA significantly increased in granulosa cells that were exposed for 24 and 48 hours to 0.5 and 1 microM A23187. CONCLUSION(S): The present study is the first report to present calcium-dependent increases in the transcription and translation levels of both nuclear-encoded and mitochondrial-encoded mitochondrial respiratory enzyme subunits and also indicates that mitochondrial transcription factor A is involved in mitochondrial biogenesis.
Malignant mixed mullerian tumors (MMMTs), also known as carcinosarcoma because they contain both carcinomatous and sarcomatous elements are aggressive tumors, which usually arise in the uterus and ovary. Extragenital carcinosarcomas are extremely rare and most cases develop from the peritoneum. To our knowledge, only 29 cases have been described in English literature. Here we report a case of a primary carcinosarcoma of the pelvic peritoneum with five-year disease-free survival after managing the patient with surgery, chemotherapy and radiotherapy.
The rearrangement of mitochondrial DNA in luteinized granulosa cells was determined in order to evaluate the fertilization capacity of oocytes and the development of embryos. Multiple deletions of mtDNA were found in luteinized granulosa cells from in vitro fertilization (IVF) patients. The 4977-base pair (bp) deletion was the most frequent deletion found in human granulosa cells. No significant difference was noted between mtDNA deletions of granulosa cells based on the fertilization capacity of oocytes and the development of embryos. To determine the relationship of proportions of mtDNA rearrangements with the aging process, granulosa cells were grouped into three different cohorts according to maternal age: younger than 32 years, between 32 and 37 years, and older than 37 years. No statistical correlation was noted between patient age and the frequency of occurrence of multiple mtDNA deletions. However, an increase in granulosa cell apoptosis was associated with an increase in mtDNA deletions. Accumulation of mtDNA deletions may contribute to mitochondrial dysfunction and impaired ATP production. We concluded that the accumulation of rearranged mtDNA in granulosa cells might not interfere with fertilization of human oocytes and further embryonic development; it was, however, associated with apoptosis processes.
Ovarian granulosa cells are known to play a key role in regulating ovarian physiology. Age increases apoptosis in follicular granulosa cells and subsequently decreases ovarian fecundity. The aging ovary also contains fewer follicles that possess fewer granulosa cells. The viability of follicular granulosa cells may be essential for development of the oocyte. Calcium ion plays an important role in a variety of biological processes, including gene expression, cell cycle regulation, and cell death. To study the ability of mitochondrial biogenesis in human granulosa cells, we determined the mitochondrial marker proteins, including the nuclear-encoded NADH-ubiquinone oxidoreductase alpha subunit 9 (NDUFA9) and mitochondrial-encoded COX I, after treatment of the cells with the calcium ionophore A23187. We showed that the expression of these mitochondrial marker proteins in human granulosa cells increased with changes in cytosolic Ca2+ using the ionophore A23187. Treatment of granulosa cells with 0.5 microM of A23187 for 120 h increased the levels of NDUFA 9 and COX I subunit by up to 2.6- and 2.4-fold, respectively. Raising Ca2+ by exposing granulosa cells to 1 microM of A23187 for 48 h significantly increased mitochondrial transcription factor (mtTFA) gene expression by up to 2.9-fold. Our results indicate that the adaptive responses of granulosa cells to increased Ca2+ may include upregulation of mitochondrial proteins and that mtTFA may be involved in such a mitochondrial biogenesis pathway.
To clarify the relationship between mitochondria and embryo development, we collected human unfertilized oocytes, early embryos, and arrested embryos. Unfertilized oocytes and poor-quality embryos were collected, and the ultrastructure of mitochondria was determined by transmission electron micrography. Four criteria for determining the mitochondrial state were mitochondrial morphology, cristae shape, location, and number of mitochondria. In mature oocytes, mitochondria were rounded with arched cristae and a dense matrix and were distributed evenly in the ooplasm. In pronuclear zygotes, the size and shape of mitochondria were similar to those in mature oocytes; however, mitochondria appeared to migrate and concentrate around pronuclei. In this study, 67% of examined unfertilized oocytes had fewer mitochondria in the cytoplasm. A decreased number of mitochondria located near the nucleus was also demonstrated in 60% of arrested embryos. Fewer differentiated cristae were determined in all three arrested blastocyst stages of embryos. The relative expressions of oxidative phosphorylation genes in oocytes and embryos were also determined. These data imply that inadequate redistribution of mitochondria, unsuccessful mitochondrial differentiation, or decreased mitochondrial transcription may result in poor oocyte fertilization and compromised embryo development.
Granulosa cells are known to contribute to maturation of oocytes, and most of the growth factors exert their action via granulosa cells. It has been established that granulosa cell death during follicular atresia and luteolysis results from apoptosis. However, the precise mechanistic pathways of granulosa cell apoptosis have not yet been defined. In this study, we determined the proportions of apoptosis in granulosa cells treated with two kinds of gonadotrophin-releasing hormone agonists (GnRHa): buserelin and leuprorelin depot. The incidences of DNA fragmentation of human granulosa cells treated with buserelin and leuprorelin were 54.33% and 39.02%, respectively. The proportions of apoptotic bodies were 6.04% and 4.29%, respectively. There was a significant difference in the proportions of DNA fragmentation between the two kinds of GnRHa-treated granulosa cells. The apoptosis pathway and associated protein expression in granulosa cells treated with GnRHa were also determined. The Bax molecule, a pro-apoptosis protein, was expressed in granulosa cells undergoing apoptosis. In contrast, Bcl-2, an anti-apoptosis protein, could not be detected in the same group of granulosa cells. The distribution of cytochrome c determined via immunostaining showed a diffuse pattern, which most likely indicated that cytochrome c was translocated from mitochondria into the cytoplasm. Western blotting showed the expressions of caspase-9 and caspase-3 in patients' granulosa cells. The GnRHa effects on granulosa cells indicated a higher incidence of DNA fragmentation and apoptotic bodies in the buserelin-treated than in the leuprorelin depot-treated group. The granulosa cells go through the mitochondria-dependent apoptosis pathway; the indicated pro-apoptosis protein Bax was expressed and induced cytochrome c release from mitochondria, which then activated caspase-9 and caspase-3 until cell death occurred.
Adenosine triphosphate (ATP) has been shown to induce programmed cell death in various systems. However, little is known about the effect of ATP on human granulosa-luteal cells (hGLCs). The present study was designed to examine the effect of ATP on the activation of the caspase signaling pathway and its role in inducing programmed cell death. Human GLCs were collected from patients undergoing in vitro fertilization programs, and then were cultured in FBS-supplemented DMEM for 3 days prior to our studies. To examine the dose-response relationship, hGLCs were treated with increasing concentrations of ATP (10 microM, 100 microM, 1 mM or 10 mM) for 24 hours. For time-course experiments, hGLCs were treated with 10 mM ATP for 6, 12, or 24 hours. Western blot analysis was performed using antibodies against the pro- and active forms of caspase-3, -9, or PARP. To quantify the induction of apoptosis, DNA fragmentation was measured using the cell death detection enzyme-linked immunosorbent assay. To examine the effect of human chorionic gonadotropin (hCG) in protecting cells from apoptosis, hGLCs were treated with 10 IU hCG in the presence of 10 mM ATP for 12 hours. It was demonstrated that ATP was capable of inducing DNA fragmentation in a dose- and time-dependent manner. Furthermore, Western blot analysis, which detected the pro- and active forms of caspase-3, or PARP, demonstrated that ATP activated the caspase-signaling pathway, leading to the proteolytic conversion of pro-caspase-3 to active caspase-3, and the subsequent cleavage of the caspase substrate PARP. Based on our observation, caspase-9 was not triggered by ATP. Interestingly, hCG attenuated the effect of ATP in activating the caspase signaling pathway. To our knowledge, this is the first demonstration of the ATP-induced activation of the caspase signaling pathway in the human ovary. These results support the notion that the caspase-signaling pathway is involved in mediating ATP actions in the human ovary.
PURPOSE: To define the prevalence rate of cervical human papilloma virus (HPV) using DNA oligonucleotide microarray and its correlation with risk factors in Taiwanese women in metropolitan Taipei. METHODS: Thirteen hundred and twenty healthy women, aged 21 - 65 yr without history of cervical intraepithelial neoplasia (CIN) or carcinoma were included in this prospective study. Pap smear and HPV typing using oligonucleotide microarray were performed for each woman. They were given a standardized questionnaire to obtain information about the risk factors of cervical cancer in Taiwan. RESULTS: The overall HPV positivity was 19.85% and multiple infections were found in 35.84% of the infected group, 7.92% of the whole study population. The younger the subject, the higher was the infection rate and multiple infection rates. The most common HPV types were 16, 18, 58, 52, 51 and 56, which is different from the western world. The sensitivity of the HPV DNA chip in detecting CIN and cervical carcinoma is 97.06%, and 100% in detecting CIN 2 or more lesions. Risk factors for HPV infection include earlier coitarche (P < 0.01), multiple sexual partners (P < 0.05), history of sexually transmitted disease (P < 0.05), two or more vaginal deliveries (P < 0.05) and infrequent use of condoms (P < 0.05). The association between oral contraception or cigarette smoking and HPV infection could not be determined because few women smoke or used oral contraception. There was no relationship between induced abortion and HPV infection. CONCLUSIONS: About one-fifth of adult women in metropolitan Taipei were cervical HPV positive. The popular HPV types and the risk factors of HPV infection in metropolitan Taipei are not the same as those in the western world. The sensitivity of the HPV DNA chip in detecting cervical neoplasia is very high.
OBJECTIVE: To examine changes in sexuality after total vaginal hysterectomy (TVH) or transvaginal sacrospinous uterine suspension (SSUS)for uterine prolapse. STUDY DESIGN: One hundred fifty-eight women with moderate to severe uterine prolapse undergoing TVH (78) or SSUS (80) were included in a prospective study from January 2001 to June 2002. All women were <50 years old and sexually active within the last 6 months before surgical intervention. None had major medical disorders. Sexual functioning before and 6 months after surgery was examined via a face-to-face questionnaire. Sexual interest, sexual satisfaction, frequency of sexual intercourse and frequency of orgasm were measured using an analogue scale. RESULTS: Of women undergoing TVH, 5.1% had decreased sexual interest, and 21% had less frequent orgasms postoperatively. For women undergoing SSUS, 13% had decreased sexual interest, and 20% had less frequent orgasms postoperatively. Frequency of orgasm was the only parameter that changed significantly after surgery in the 2 groups. All women with less frequent orgasms said that they were afraid of wound disruption or disease recurrence and so refrained from vigorous or exciting sexual intercourse. About four-fifths of the women in both groups accepted or were satisfied with their sexuality after surgery. For women undergoing TVH, 2.6% had increased frequency of orgasm, and 5% had better overall sexual satisfaction postoperatively. For women undergoing SSUS, 10% had increased sexual satisfaction postoperatively. There was a 2.5% and 2.6% increase in postoperative sexual interest in the SSUS and TVH groups, respectively. The sexual functioning scores were not different before or after surgical intervention in either group. CONCLUSION: There is a decrease in thefrequency of orgasm after both TVH and SSUS. However, there is no significant difference in postoperative sexual functioning between women with and without preservation of the uterus in correcting uterine prolapse.
BACKGROUND: Patients with polycystic ovary syndrome (PCOS) may need a longer period of pituitary downregulation to suppress the elevated serum LH and androgen levels effectively during IVF treatment using the GnRH agonist long protocol. We proposed a stimulation protocol incorporating Diane-35 and GnRH antagonist (Diane/cetrorelix protocol) and compared it with the GnRH agonist long protocol for PCOS patients. METHODS: Part I of the study was an observational pilot study to evaluate the hormonal change as a result of the Diane/cetrorelix protocol (n = 26). Part II of the study was a prospective randomized study comparing the Diane/cetrorelix protocol (n = 25) and the GnRH agonist long protocol (n = 24). In the Diane/cetrorelix protocol, patients were pre-treated with three cycles of Diane-35, followed by 0.25 mg of cetrorelix on cycle day 3. From day 4, cetrorelix and gonadotrophin were administered concomitantly until the day of HCG injection. RESULTS: Serum LH, estradiol and testosterone levels were suppressed comparably in both protocols at the start of gonadotrophin administration. Serum LH was suppressed at constant levels without a premature LH surge in the Diane/cetrorelix protocol. The clinical results for both protocols were comparable, with significantly fewer days of injection, lower amounts of gonadotrophin used and lower estradiol levels on the day of HCG injection following the Diane/cetrorelix protocol. Furthermore, there was no significant difference in clinical pregnancy outcome between the two stimulation protocols. CONCLUSIONS: The Diane/cetrorelix protocol has a similar pregnancy outcome to the GnRH agonist long protocol for women with PCOS undergoing IVF treatment.
Endometriosis is a benign but aggressive disease. It occurs when shed endometrium from the female reproductive tract grows at a site outside the uterus. The physiological changes in endometriosis-abnormal tissue growth, invasion, and adhesion phenomena-are similar to those seen in tumorous tissues. Although the etiology of endometriosis is not well understood, the disease is widely accepted to result from the ectopic implantation of refluxed menstrual tissues. In addition, immunologic changes, genetic factors, and environmental factors might also affect a woman's susceptibility to develop endometriosis. Thus far, laparoscopic examination is required to confirm the presence of endometriosis; there is no reliable marker for its diagnosis. Many studies are therefore focusing on identifying markers for the diagnosis and follow-up of endometriosis. This chapter provides a systematic review of these studies, including recent findings from our group on the identification of molecules, in serum and/or endometrium, which are associated with the development of endometriosis at different stages. From this research, we hope to be able to suggest how to approach the potential markers. The identification of highly sensitive and specific markers of endometriosis should facilitate the development of accurate and non-invasive techniques for diagnosis and prognosis.
OBJECTIVE: To evaluate the relationship between mitochondrial gene expression of oocytes/embryos and their fertilizability in unfertilized oocytes, arrested embryos, and tripronucleate zygotes, because both nuclear and cytoplasmic factors contribute to oocyte activation, fertilization, and subsequent development. DESIGN: Prospective laboratory research. SETTING: In vitro fertilization (IVF) laboratory in a university hospital. PATIENT(S): Seventy-five unfertilized oocytes, 45 arrested embryos, and 24 tripronucleate (3PN) embryos from 45 female patients undergoing IVF. INTERVENTION(S): Analysis of mitochondrial gene expression by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). MAIN OUTCOME MEASURE(S): Comparison of the expression levels of mitochondrial genes including ND2, CO I, CO II, ATPase 6, CO III, ND3, ND6, and Cyt b in three groups. RESULT(S): Significantly decreased transcription levels were expressed in unfertilized oocytes and arrested embryos. The average expression levels of the eight determined genes compared with the control (GAPDH) was 4.4 +/- 0.7, 6.4 +/- 1.1, and 13.2 +/- 1.1 in unfertilized oocytes, arrested embryos, and 3PN embryos, respectively. Significantly decreased expressions of the ATPase 6, CO III, and ND3 genes were detected from samples with 4977-bp common deletion in the mitochondrial DNA (mtDNA) compared with the non-deletion group. CONCLUSION(S): The present study is the first report to present globally decreased mitochondrial gene expression levels in human compromised oocytes and embryos. These data support the notion that the down-regulation of mitochondrial RNA by defective oxidative phosphorylation genes possibly affects oocyte quality including fertilization and further embryo development.
OBJECTIVE: To evaluate the therapeutic results of premyomectomy uterine depletion for the treatment of symptomatic fibroids, compared with myomectomy only. DESIGN: Controlled, clinical study without randomization. SETTING: University-affiliated tertiary referral center. PATIENT(S): Four hundred eighty-six women with symptomatic fibroids warranting surgical treatment and who wished to retain their uteri. INTERVENTION(S): Ligation of the uterine arteries was performed by either an abdominal or a laparoscopic approach before myomectomy. MAIN OUTCOME MEASURE(S): Operation time, intraoperative blood loss, postoperative improvement of symptoms, and recurrence rates of fibroids. RESULT(S): Of 342 women with pathology-confirmed fibroids who were included in the study, 108 received myomectomy only (group I), and 234 underwent the uterine depletion procedure followed by myomectomy (group II). Average blood loss was 250 +/- 132.5 mL for group I and 50 +/- 26.9 mL for group II. For patients with menorrhagia, 79 (84%) of 94 women in group I experienced complete resolution; all of the 194 women (100%) in group II had resolution within 2 months of surgery. The recurrence rate of ultrasound-confirmed fibroids was 19.4% (21 of 108) in group I and 0% in group II. Of the sexually active patients who were not using contraception, 50% (49 of 98) in group I and 37.5% (15 of 40) in group II had a live birth. CONCLUSION(S): This study demonstrates the value of uterine depletion before myomectomy for the management of patients with symptomatic fibroids. The procedure reduced blood loss during the operation, resulted in complete resolution of fibroid-related menorrhagia, and has the potential to prevent fibroid recurrence. Fertility capacity was apparently not compromised by this new treatment approach.