Gene amplification and expression of the DNA repair enzyme, N-methylpurine-DNA glycosylase (MPG) in HPV-infected cervical neoplasias.

BACKGROUND: Lethal and mutagenic damages of DNA is caused by a variety of agents including viruses. It is known that HPV is one of the major causes of cervical carcinogenesis and that cells eliminate DNA lesions with DNA repair enzymes. However, the role of N-methylpurine-DNA glycosylase (MPG) is not known in the development of cervical cancer. MATERIALS AND METHODS: Multiplex polymerase chain reaction (PCR) was used for the detection and typing of HPV in the biopsy. Gene amplification of MPG was measured by a PCR-based assay. The mRNA levels of MPG were determined by reverse transcription-PCR using hypoxanthine-guanine phosphoribosyl transferase as the reference gene. An immunohistochemical technique was used to examine the distribution of MPG in the tissues. RESULTS: Of 68 Korean cervical neoplasia patients, 86.8% showed HPV infection. High-risk HPV 16/18 were the most prevalent but positive only in 47.3% of the invasive cancer patients. Gene amplification of MPG was significantly increased in high-risk HPV-infected tissues as compared to low-risk HPV-infected and normal tissues (p < 0.05). The mRNA levels of MPG were higher in HPV-infected invasive carcinoma than normal cervical tissues. Immunohistochemical staining revealed that the intracellular expression and distribution (localization) of MPG altered in the cervical neoplasia. Interestingly, MPG expression in CIN III and invasive carcinoma (IC) was much higher than normal and CIN I. Granular positivity of MPG was notable in the perinuclear regions of the cytoplasm in HPV-infected invasive cancer. CONCLUSION: This is the first report on MPG expression in cervical neoplasia. Our results indicate that the gene amplification and expression of MPG were increased in high-risk HPV-infected cervical neoplasias and the intracellular distribution of MPG protein was altered, suggesting a role of MPG in carcinogenesis.

Does prayer influence the success of in vitro fertilization-embryo transfer? Report of a masked, randomized trial.

OBJECTIVE: To assess the potential effect of intercessory prayer (IP) on pregnancy rates in women being treated with in vitro fertilization-embryo transfer (IVF-ET). STUDY DESIGN: Prospective, double-blind, randomized clinical trial in which patients and providers were not informed about the intervention. Statisticians and investigators were masked until all the data had been collected and clinical outcomes were known. The setting was an IVF-ET program at Cha Hospital, Seoul, Korea. IP was carried out by prayer groups in the United States, Canada and Australia. The investigators were at a tertiary medical center in the United States. The patients were 219 women aged 26-46 years who were consecutively treated with IVF-ET over a four-month period. Randomization was performed after stratification of variables in two groups: distant IP vs. no IP. The clinical pregnancy rates in the two groups were the main outcome measure. RESULTS: After clinical pregnancies were known, the data were unmasked to assess the effects of IP after assessment of multiple comparisons in a log-linear model. The IP group had a higher pregnancy rate as compared to the no-IP rate (50% vs. 26%, P = .0013). The IP group showed a higher implantation rate (16.3% vs. 8%, P = .0005). Observed effects were independent of clinical or laboratory providers and clinical variables. CONCLUSION: A statistically significant difference was observed for the effect of IP on the outcome of IVF-ET, though the data should be interpreted as preliminary.

Fertility outcome after tubal anastomosis by laparoscopy and laparotomy.

STUDY OBJECTIVE: To evaluate fertility outcome and benefit of laparoscopic tubal anastomosis compared with laparotomy. DESIGN: Retrospective study (Canadian Task Force classification II-2). SETTING: University hospital. PATIENTS: Eighty-one women requesting reversal of sterilization. Fertility outcome was analyzed in 76 patients for a minimum of 6 months. INTERVENTION: Laparoscopic tubal anastomosis in 37 women and abdominal tubal anastomosis in 44. MEASUREMENTS AND MAIN RESULTS: In both groups anastomosis was performed in two layers with four stitches using microsurgical technique. Overall pregnancy rates were 80.5% in the laparoscopy and 80.0% in the laparotomy group. The mean interval from operation to pregnancy was similar in the two groups (p = 0.9). Mean operating time was significantly longer for laparoscopy (201.9 +/- 33.8 min) than for laparotomy (148.7 +/- 32.5 min), including diagnostic laparoscopy. However, mean hospital stay was shorter for laparoscopy than for laparotomy (3.3 +/- 2.0 vs 6.1 +/- 0.6 days, p <0.05). CONCLUSION: Laparoscopic tubal anastomosis is less invasive and could be an alternative to laparotomy for reversal of tubal sterilization. Advanced laparoscopic equipment and much experience could enhance the pregnancy rate and reduce operating time.

Establishment of an animal embryo culture system containing various embryotropins and its efficacy for culturing ICR mouse one-cell embryos derived in vivo or in vitro.

OBJECTIVE: To develop an effective ICR mouse embryo culture medium. DESIGN: In vitro model study. SETTING: University-affiliated hospital. ANIMALS: Four-week-old, superovulated mice. INTERVENTION(S): In vivo- or in vitro-derived one-cell embryos were cultured in preimplantation-1 medium (P-1). MAIN OUTCOME MEASURE(S): Preimplantation development. RESULT(S): In vivo-derived embryos were cultured in BSA-containing P-1, to which one of the following substances was added: [1] no addition, [2] amino acids (aa), [3] aa+hemoglobin (hb), [4] aa+hb+cysteine (cys), [5] aa+hb and glucose (glu) added at the four-cell, or [6] aa+hb and glu+cys added at the four-cell stage. More (P<0.05) blastocysts developed after aa or aa+hb addition than after no addition, and glu addition to such medium further stimulated the formation (54%). In P-1 with aa+glu, the addition of 1 microg/mL hb was optimal. Additional improvement of blastocyst formation (78%) was achieved by ethylenediaminetetraacetic acid (EDTA), supplementation and bovine serum albumin replacement with polyvinyl alcohol (PVA) did not inhibit the development. P-1 supplemented with aa, hb, glu, EDTA, and PVA also supported the development of in vitro-derived embryos (70%). CONCLUSION(S): A modified P-1 medium was developed, and it supported the development of both in vivo- and in vitro-derived ICR mouse embryos.

Cryopreservation of ICR mouse oocytes: improved post-thawed preimplantation development after vitrification using Taxol, a cytoskeleton stabilizer.

OBJECTIVE: To establish an effective cryopreservation method. DESIGN: In vitro model study. SETTING: Infertility Medical Center, Pochon CHA University. ANIMAL(S): Four-week-old ICR mice superovulated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin. INTERVENTION(S): Vitrified-thawed oocytes were fertilized and subsequently cultured in vitro. MAIN OUTCOME MEASURE(S): Post-thawed development, chromosome/spindle normalities, and blastocyst quality. RESULT(S): More cumulus-enclosed oocytes were fertilized and developed to the 8-cell stage after vitrification and thawing than denuded oocytes. However, cryopreserved oocytes of both types had lower spindle and chromosome normalities than fresh oocytes, which resulted in reduced developmental competence after thawing. The addition of 1 microM of Taxol, a cytoskeleton stabilizer, to vitrification solution greatly promoted the blastocyst formation of vitrified-thawed oocytes, compared with no addition (24.0% vs. 58.6%). No difference in blastocyst quality, which was evaluated by blastomere and inner cell mass cell numbers and inner cell mass cell per trophoblast ratio, was found between fresh oocytes and oocytes vitrified with Taxol. CONCLUSION(S): A vitrification solution consisting of 5.5 M ethylene glycol, 1.0 M sucrose, 10% fetal bovine serum, and 1 microM Taxol greatly improved post-thawed development of vitrified oocytes.

Embryotropic role of hemoglobin and ethylenediaminetetraacetic acid in preimplantation development of ICR mouse 1-cell embryos.

OBJECTIVE: To evaluate embryotropic action of hemoglobin (Hb) and ethylenediaminetetraacetic acid (EDTA) on preimplantation embryo development. DESIGN: In vitro model study using mouse embryos. SETTING: University affiliated hospital, Pochon CHA University. ANIMALS: Four-week-old block strain ICR mice naturally mated after superovulation. INTERVENTION(S): One-cell embryos were cultured in serum-free, modified preimplantation-1 medium, to which 1 microg/ml Hb and/or 0.1 mM EDTA were added. MAIN OUTCOME MEASURE(S): Preimplantation development and blastomere number. RESULT(S): More (P<.05) 1-cell embryos developed to the 4-cell (52% vs. 67%-84%), 8-cell (48% vs. 65%-81%), and blastocyst (40% vs. 61%-79%) stages after the addition of hemoglobin (Hb) and/or EDTA than after no addition. Highest proportion of embryos developed to each stage after the combined addition of Hb+EDTA. EDTA specifically stimulated the development before the 8-cell stage, which was as similar as Hb+EDTA. On the contrary, higher ratio of morula to blastocyst transformation was obtained after the addition of Hb or Hb+EDTA than after no addition (0.76 vs. 0.96-0.98). Significant increases in the cell number of blastocysts (46.5-47.2 vs. 53.2 cells), inner cell mass (ICM) cells (16.7-17.5 vs. 21 cells), and the ratio of ICM cells to trophoblasts (0.3-0.37 to 0.39) were found after the combined addition of Hb+EDTA, compared with no addition or with the addition of EDTA or Hb alone. CONCLUSIONS: Hb and EDTA have stage-specific effects on supporting preimplantation embryo development; Hb promotes both the development before the 8-cell stage and the morula to blastocyst transformation, whereas EDTA mainly promotes the development to the 8-cell stage. The combined exposure of embryos to Hb and EDTA improves not only preimplantation development but also the growth and quality of blastocysts.

Spatial expression of a DNA repair gene, N-methylpurine-DNA glycosylase (MPG) during development in mice.

BACKGROUND: DNA repair is a crucial phenomenon that maintains the chromosome integrity of genome which are continuously damaged by endogenous and exogenous alkylating agents. If the damaged DNA is not repaired, it may lead to mutation, chromosomal aberration, aging and cancer. N-methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair enzyme, removes N-methylpurine and other damaged purines in DNA. MATERIALS AND METHODS: MPG mRNA expression was revealed at various stages of mouse development from day 7.5 p.c. (post coitum) embryo to day 400 mature adult by Northern blot hybridization or RT-PCR. RESULTS: MPG transcripts were abundant in the mouse embryo during pregnancy and in adult testis and ovary. The MPG mRNA level in the testis was low in 1-week-old mice, but the level showed its maximum among the organs tested in 4-week-old young adults. In placenta, the level of MPG mRNA continuously decreased from day 7.5 p.c. to day 17.5 p.c. CONCLUSIONS: The spatial expression of MPG gene is highly regulated. Transcription of MPG is maximum in rapidly dividing and growing tissues during development. These data suggest that an elevated rate of MPG transcription is required for DNA replication.

Pregnancies and deliveries after in vitro maturation culture followed by in vitro fertilization and embryo transfer without stimulation in women with polycystic ovary syndrome.

OBJECTIVE: To improve the efficacy of an IVF-ET program for unstimulated patients with polycystic ovary syndrome (PCOS) with the use of culture for oocyte maturation. DESIGN: Prospective studies with the comparison of different ET procedures from March 1995 through February 1998. SETTING: University-affiliated hospital. PATIENT(S): Ninety-four cycles in 64 consenting patients with PCOS. INTERVENTION(S): Immature oocytes were retrieved from unstimulated patients with PCOS and subsequently cultured and fertilized in vitro. Zygote intrafallopian transfer (ZIFT), uterine ET, or a combined approach of ZIFT + uterine ET was subsequently performed. MAIN OUTCOME MEASURE(S): Laboratory and clinical data. RESULT(S): Among 1, 280 immature oocytes (13.6 +/- 7.5 oocytes per patient) retrieved, 89% (1,139) were morphologically normal, and 62.2% (708/1,139) of the normal oocytes matured in vitro after culture for 48 hours. When intracytoplasmic sperm injection was performed, 68% (481/708) developed to the normal pronuclear stage, and 88.1% of the embryos cocultured with Vero cells (266/302) cleaved. Eighty-five ET cycles were conducted and pregnancy was established in 23 cycles (27.1%), which consisted of 8 after uterine ET and 15 after a combined approach. Seventeen patients delivered 20 normal infants. CONCLUSION(S): The IVF-ET method using no ovarian stimulation followed by in vitro maturation culture can be a feasible assisted reproductive technology for treatment of PCOS with various complications.

Mitochondrial ATPase 6 gene expression in unfertilized oocytes and cleavage-stage embryos.

OBJECTIVE: To compare the level of mitochondrial ATPase 6 gene expression in unfertilized oocytes and cleavage-stage embryos. DESIGN: Reverse transcription polymerase chain reaction was performed in unfertilized oocytes and cleavage-stage embryos derived from tripronucleate embryos to determine ATPase 6 gene expression. SETTING: Department of Obstetrics and Gynecology, Human Genetics Laboratory, Infertility Medical Center of CHA General Hospital, College of Medicine, Pochon CHA University, Seoul, Korea. PATIENT(S): Oocytes were obtained from infertile couples undergoing in vitro fertilization. INTERVENTION(S): Unfertilized oocytes collected at 48 hours after retrieval and cleavage-stage embryos derived from tripronucleate embryos were prepared for evaluation of mitochondrial gene expression. MAIN OUTCOME MEASURE(S): Comparison of ATPase 6 gene expression by using single-cell reverse transcription polymerase chain reaction. RESULT(S): Expression of unfertilized oocytes decreased compared with early cleavage-stage embryos. CONCLUSION(S): Our findings of decreased ATPase 6 expression in unfertilized oocytes suggest that there may be a decrease in the mitochondrial functional capacity of oxidative phosphorylation.

In vitro blastocyst formation of human oocytes obtained from unstimulated and stimulated cycles after vitrification at various maturational stages.

OBJECTIVE: To evaluate the developmental competence and chromosomal normality of oocytes vitrified at various times after maturation culture. DESIGN: In vitro model study. SETTING: A university-affiliated hospital. PATIENT(S): Unstimulated women who underwent cesarean section or oophorectomy and infertile women who underwent a long protocol of GnRH stimulation. INTERVENTION(S): Retrieved oocytes were vitrified at 0 or 48 hours after culture in unstimulated cycles and at 0, 8-15, or 24-28 hours after culture in stimulated cycles. MAIN OUTCOME MEASURE(S): Postthaw morphologic normality, maturation, fertilization, cleavage, blastocyst formation, and chromosome number. RESULT(S): In the 53 oocytes that were obtained from unstimulated cycles, no statistically significant differences were found in rates of morphologic normality (range, 56%-63%) or fertilization (range, 31%-37%) according to the time of vitrification. In the 50 oocytes that were obtained from stimulated cycles, more of those that were vitrified at 24-28 hours were morphologically normal than those that were vitrified at 0 or 8-15 hours. Regardless of these differences, high cleavage rates (83%-100%) were obtained that did not differ significantly among the treatment groups. In both cycles, 20%-43% of cleaved oocytes developed to the blastocyst stage by 6 days after IVF. All the karyotyped blastocysts, three from unstimulated cycles and four from stimulated cycles, had a normal number of chromosomes. CONCLUSION(S): Vitrified and thawed oocytes from unstimulated or stimulated cycles developed to the blastocyst stage, regardless of when vitrification occurred; the number of chromosomes in the blastocysts was normal.

Freezing immature oocytes.

The establishment of a long-term preservation system for mammalian oocytes is important for the development of both biological and medical sciences. A number of efforts have been made to develop this system. In human reproductive medicine, the development of an oocyte cryopreservation system can improve the efficacy of the current assisted reproductive technology (ART) for infertile patients with severe reproductive disorders. In this article, the technical development of cryopreservation programs for human oocytes and its biological background were reviewed. Clinical outcome after the use of this technology was further introduced.

Laparoscopic tubal anastomosis: fertility outcome in 202 cases.

OBJECTIVE: To evaluate the fertility outcome after laparoscopic tubal anastomosis for reversal of sterilization. DESIGN: Retrospective clinical study. SETTING: A private practice affiliated with a university medical school. PATIENT(S): Two hundred two women who desired reversal of tubal sterilization. INTERVENTION(S): Laparoscopic tubal anastomosis. MAIN OUTCOME MEASURE(S): The cumulative pregnancy rate (PR) and factors that influenced the fertility outcome. RESULT(S): The cumulative PR in the 186 patients for whom follow-up data were available was 60.3%, 79.4%, and 83.3% at 6, 12, and 18 months after operation, respectively. Five patients (3.2%) had ectopic pregnancies; one of these patients subsequently conceived normally. There were no statistically significant differences in the PR according to the sterilization method used, the site of the tubal anastomosis, or the length of the fallopian tube after surgery. The intrauterine PR was 87.1% (149/171) with bilateral anastomosis and 60% (9/15) with unilateral anastomosis. The PR decreased with increasing patient age (mean [+/- SD], 35+/-3.6 years) but was still 70.6% (12/17) in patients aged 40-45 years. CONCLUSION(S): Our findings suggest that laparoscopic tubal anastomosis is a highly successful procedure. This less invasive approach could be considered the procedure of choice in patients who desire reversal of tubal sterilization.

Improved human oocyte development after vitrification: a comparison of thawing methods.

OBJECTIVE: To evaluate the developmental competence of vitrified human oocytes thawed using two different methods to establish an effective cryopreservation protocol. DESIGN: In vitro model study. SETTING: University-affiliated hospital. PATIENT(S): Patients who underwent a long protocol of ovarian stimulation with GnRH and gonadotropins. INTERVENTION(S): Vitrified oocytes from the patients were thawed using either a four-step method with 2.5-minute intervals or a four-step method with 5-minute intervals. MAIN OUTCOME MEASURE(S): Morphologic normality, maturation, fertilization, and development of the oocytes to the blastocyst stage. RESULT(S): The two thawing methods did not significantly affect the morphologic normality (84%-100%), maturation (75%-100%), fertilization (38%-71%), polyspermy (more than three pronuclei; 0%-20%), or parthenogenetic activation (only female pronucleus; 0%-8%) of the vitrified oocytes. However, more of the vitrified oocytes developed to the two-cell (71%-100% versus 50%-67%), four-cell (71%-93% versus 0%-50%), eight-cell (46%-71% versus 0%), and blastocyst (23%-36% versus 0%) stages after thawing using the four-step method with 2.5-minute intervals than using the four-step method with 5-minute intervals. CONCLUSION(S): Vitrified human oocytes developed to the blastocyst stage with IVF. A four-step thawing method with 2.5-minute intervals was more effective in supporting preimplantation embryo development than a four-step thawing method with 5-minute intervals.

In vitro maturation and in vitro fertilization of bovine oocytes cultured in a chemically defined, protein-free medium: effects of carbohydrates and amino acids.

This study was conducted to examine the effects of carbohydrates and amino acids on the maturation and fertilization of bovine oocytes. To evaluate the effect of each treatment without any unpredictable interference, oocytes were cultured in a simply defined medium (modified Tyrode's medium; mT) without the addition of hormones and proteins. In Experiment 1, oocyte maturation to the metaphase-II stage was significantly (P<0.0001) enhanced after the addition of glucose (5.6 mM), lactate (10 mM) and/or pyruvate (0.5 mM) to mT (37-74%) than after no addition (0%). In mT supplemented with glucose, the addition of 19 essential and non-essential amino acids (aa; 0, 0.01, 0.1, 1, 5 or 10%) did not further improve in vitro maturation (Experiment 2) or in vitro fertilization (Experiment 3) of oocytes. However, more (P<0.05) pronuclear formation after in vitro-insemination was found in oocytes matured in mT with 1% aa and glucose than in oocytes matured in mT with glucose alone (56% vs. 35%). Penetration of spermatozoa into the ooplasm was initiated at 3 h after insemination and pronuclear formation from 8 h (Experiment 4). When cultured inseminated oocytes were examined up to 192 h post insemination, a significant (P<0.05) increase in the number of 2-cell (18 v. 38%) and 8-cell embryos, (7 v. 20%) and morulae (0 v. 8%) was found after the addition of 1% aa to mT with glucose than after no addition (Experiment 5). A limited number of oocytes matured in mT with aa and glucose developed to the blastocyst stage (6%). These results indicate that exogenous carbohydrates and amino acids are prerequisites for the maturation and fertilization of bovine oocytes in vitro. Glucose alone promotes the nuclear maturation of oocytes, whereas amino acids aid the pronuclear formation of fertilized oocytes.

Microtubule and microfilament organization in maturing human oocytes.

Various stages of immature human oocytes were imaged for microtubule, microfilament and chromatin organization. After germinal vesicle breakdown, a small microtubule aster was observed near the condensed chromatin. The asters appeared to elongate and encompass the condensed chromatin. At metaphase I stage, microtubules were detected in the meiotic spindle. The meiotic spindle in metaphase II was a symmetric, barrel-shaped structure containing anastral broad poles, located peripherally and radially oriented. After germinal vesicle breakdown, treatment with taxol induced numerous cytoplasmic foci of microtubules, mainly in the cortex of the oocyte. Microfilaments were observed as a relatively thick uniform area around the cell cortex and were also found near the germinal vesicle position. After germinal vesicle breakdown, the microfilaments were seen in both the cortex and around the female chromatin. In conclusion, this study suggests that both microtubules and microfilaments are closely associated with the reconstruction and proper positioning of chromatin after germinal vesicle breakdown and during meiotic maturation in human oocytes.

Tissue-specific expression and activation of N-methylpurine-DNA glycosylase in thymic carcinomas of transgenic mice expressing the SV40 large T-antigen gene.

Simian virus 40 T-tumor antigen (SV40 T-ag) can induce a wide variety of tumors in hamsters and neonatal mice. These tumorigenic effects are predominantly due to the activity of early viral gene products, large T-antigen and small t-antigen. We have analyzed the expression of a DNA repair gene, N-methylpurine-DNA glycosylase (MPG), from different tissues of a non-transgenic (control) and SV40 T-ag expressing transgenic mice at the mRNA level. Expression of the transgene in thymus of adult mice was also detected by the presence of SV40 T-ag mRNA. Non-transgenic mice did not express the SV40 T-ag gene in their thymus, while the mRNA for MPG was found in thymus from both of transgenic and non-transgenic mice. The MPG gene was expressed in various tissues and is regulated in a tissue-specific manner. Northern blot analysis revealed that the transgenic mice showed considerably higher expression of MPG in the thymic carcinomas. The level of MPG mRNA in the thymic carcinoma was elevated about 5.7 fold, as compared with those found in the control thymus. MPG expression was significantly increased, either directly or indirectly, by the SV40 T-ag gene product. These findings provide the first in vivo observations that the SV40 T-ag gene induced thymic carcinomas associated with the activation of the DNA repair gene, MPG.