Effects of dopamine and metoclopramide in polycystic ovary syndrome.

A relative deficiency in dopamine has been suggested to explain the inappropriate gonadotropin secretion and postulated increased GnRH secretion characteristic of polycystic ovary syndrome (PCO). Previous studies demonstrated an exaggerated decrement in serum LH after large iv doses of dopamine (DA, 4-5 micrograms/kg X min). Normoprolactinemic patients with PCO and weight- and estrogen-matched normal women received iv infusions of DA in two doses (0.5 and 4 micrograms/kg X min). After DA, each subject also received iv metoclopramide (MCP; 10 mg). Serum LH decreased (P less than 0.05) during DA infusion to a similar degree in PCO [23 +/- 3% (+/- SE)] and normal women (20 +/- 2%). In PCO patients, the decrease in LH was similar with both DA doses. Serum PRL and TSH responses to DA were also similar in PCO and normal women. After MCP treatment, serum LH did not change, but serum PRL increased more in PCO (801 +/- 100%) than in normal women (467 +/- 73%; P less than 0.05), as did serum TSH. These data suggest that the sensitivity of LH to DA in patients with PCO is not increased. Further, increased responses of PRL and TSH to MCP may reflect increased dopaminergic activity or, in the case of PRL, the influence of chronic hyperestrogenism.

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.

Interrupted circular suture: bleeding control during cesarean delivery in placenta previa accreta.

Rapid accumulation of blood from the placental separation site during cesarean delivery for placenta previa obscures the surgical field and quickly leads to deterioration of the patient's vital signs. We used the following technique in eight cases of intractable bleeding among 49 cesareans for placenta previa. Following failure to control the bleeding by suture at the placental separation site via the lower segment cesarean incision, the vessels were ligated using interrupted 2-3-cm sutures at 1-cm intervals in a circle around the bleeding area on the serosal surface of the uterus. The sutures were placed as deeply as possible in order to reach the endometrium. This led to a marked decrease in bleeding and allowed the small vessels to be easily identified and ligated. The amount of blood transfused and the operation time were gradually reduced as we became adept in the use of this procedure. Our experience suggests that this technique could reduce the use of hysterectomy in cesarean for placenta previa.

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.

Approaches for obtaining sperm in patients with male factor infertility.

OBJECTIVE: To describe methods of sperm retrieval for intracytoplasmic sperm injection (ICSI) in patients with male factor infertility and to review the clinical results using sperm from the different sources. DESIGN: The literature on sperm-obtaining methods and ICSI was reviewed. Studies related to this topic were identified through MEDLINE. RESULTS(S): This review describes the evolution of sperm retrieval methods. Sperm can be obtained by microepididymal sperm aspiration (MESA), percutaneous sperm aspiration (PESA), and testicular sperm extraction (TESE), from patients with congenital absence of the vas deferens or acquired vas obstruction. When ICSI is performed with ejaculated, epididymal, or testicular sperm, good fertilization and pregnancy rates are achieved without significant differences among the various sperm sources. The original percutaneous sperm aspiration method has been modified slightly and yields successful results. CONCLUSION(S): Viable pregnancies can be achieved with ICSI by using not only ejaculated sperm, but also epididymal and testicular sperm. Microepididymal sperm aspiration, percutaneous sperm aspiration, modified percutaneous sperm aspiration, and testicular sperm extraction can be considered standard procedures to treat male factor infertility.

Correlation of the bioactivity of luteinizing hormone in follicular fluid with oocyte maturity in the spontaneous cycle.

This study was done to measure the bioactivity of luteinizing hormone (LH) in follicular fluid (FF) and to correlate this and other hormonal values with oocyte maturity in the spontaneous cycle. Twenty follicles (greater than 18 mm) from 18 ovulatory patients were obtained, and FF, according to oocyte maturity, was divided into three groups (I to III) of increasing maturity. FF progesterone correlated with maturity and was highest (P less than 0.01) in group III; estradiol, prolactin, and follicle-stimulating hormone were similar in the three groups. Immunoreactive LH was similar in groups I and II but was highest (30.5 +/- 7 mIU/ml) in group III (P less than 0.02). Bioactive (bio) LH was much higher in group III follicles (1307 +/- 387 mIU/ml); the bio:immunoreactive LH ratio also increased significantly (P less than 0.05). FF progesterone correlated positively with bio LH (r = 0.89, P less than 0.005) and the bio:immunoreactive LH ratio (r = 0.9, P less than 0.001); immunoreactive LH did not correlate. These data suggest that bioactivity of LH is an important correlate of oocyte maturity and that local ovarian factors may modulate these concentrations in FF.

Fertility outcome after laparoscopic microsurgical tubal anastomosis.

OBJECTIVE: To evaluate fertility outcome after laparoscopic microsurgical tubal anastomosis. DESIGN: A retrospective study. SETTING: Infertility Medical Center affiliated with University Medical School. PATIENT(S): Fifty-four patients, who had previously undergone tubal sterilization, seeking reversal. INTERVENTION(S): Laparoscopic microsurgical tubal anastomosis was performed. MAIN OUTCOME MEASURE(S): Pregnancy success was assessed. RESULT(S): The overall pregnancy rate (PR) was 77.5% (38/49) and 29 patients already have delivered healthy offspring. Pregnancy in seven patients is ongoing and one case ended in abortion. There was one case of ectopic pregnancy. The pregnancy success according to the method of previous tubal sterilization was 16 of 24 with the Fallope-ring method, 14 of 15 in cases of cauterization, and 8 of 10 in patients in whom the Pomeroy technique was used. The pregnancy success according to the site of anastomosis was 3 of 4 in cornual-isthmic, 4 of 5 in isthmic-isthmic, 26 of 35 in isthmic-ampulla, 3 of 3 in cornual-ampulla, and 2 of 2 in ampulla-ampulla. The pregnancy success according to the tubal length was 5 of 7 at a length < or = 4 cm, 3 of 5 at 5 cm, 15 of 17 at 6 cm, and 15 of 20 at lengths > or = 7 cm. CONCLUSION(S): Considering the high PR in our minimal follow-up period of 12 months, laparoscopic microsurgical tubal anastomosis could be an alternative procedure to microsurgical laparotomy in patients requesting reversal of tubal sterilization.

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.

Pregnancy after in vitro fertilization of human follicular oocytes collected from nonstimulated cycles, their culture in vitro and their transfer in a donor oocyte program.

This study describes the results with immature human follicular oocytes harvested from unstimulated ovaries, matured in vitro, fertilized, and transferred to an agonadal recipient. Two hundred seventy immature oocytes were aspirated from 23 ovaries removed for various gynecological indications from August 1988 to October 1989. The numbers of follicular oocytes collected from ovaries were compared by patients' ages and the stages of menstrual cycle. Immature oocytes in vitro were incubated with either mature follicular fluid (FF) or fetal cord serum (FCS). The maturation rate in the mature FF group was 55.8%, significantly higher than the 35.9% in the FCS group. In addition, mature FF group was shown to provide a significantly higher fertilization rate than the FCS group (81.0% versus 31.6%). More fertilized eggs developed into normal embryos in the nonstimulated cycle group than in stimulated cycles with routine treatment. Finally, five embryos were transferred to a woman with premature ovarian failure on day 18 of a steroid replacement cycle. She subsequently delivered healthy triplet girls. These results suggest that in vitro maturation of immature oocytes from unstimulated ovaries with mature follicular fluid could be used successfully in a donor oocyte program after in vitro fertilization.

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.

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.

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.

Effects of 1,2-propanediol and freezing-thawing on the in vitro developmental capacity of human immature oocytes.

OBJECTIVE: To investigate effects of 1,2-propanediol and freezing-thawing treatment on the maturation and developmental capacity of the human immature oocytes obtained from unstimulated ovaries. DESIGN: Intact cumulus-enclosed immature oocytes collected from unstimulated ovaries were divided into three groups, such as no treatment as control (group 1), only 1,2-propanediol-treated (group 2), and cryopreserved group (group 3). Oocytes in group 1, group 2, and survived oocytes from cryopreservation in group 3 were cultured for 48 hours. A random selection of matured oocytes was inseminated with normal donor sperm to evaluate the fertilization and developmental capacity. SETTING: Infertility Medical Center at the CHA General Hospital, Seoul, Korea. PATIENT(S): Oocytes were obtained from patients undergoing gynecological surgery. MAIN OUTCOME MEASURE(S): Rates of survival, maturation to metaphase II, fertilization, and cleavage. RESULT(S): Survival rate after freezing-thawing in group 3 was 55.1% (54/98). Oocytes were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal bovine serum, 10 IU/mL pregnant mare serum gonadotropin, and 10 IU/mL hCG. Maturation rates were 76.8% (63/82), 67.1% (47/70), and 59.3% (32/54) in the groups 1, 2, and 3, respectively. Maturation rate in group 3 was significantly lower than that of group 1. Fertilization rates were 90.5% (19/21), 81.0% (17/21), and 42.9% (6/14), and cleavage rates were 94.7% (18/19), 88.2% (15/17), and 16.7% (1/6) in groups 1, 2, and 3, respectively. Fertilization and cleavage rates of survived oocytes in group 3 also were significantly lower than those of groups 1 and 2. CONCLUSION(S): Results suggest that the pretreatment with 1.5 M 1,2-propanediol itself before the freezing has no inhibitory effect on the maturation, fertilization, and cleavage of human immature oocytes in vitro. However, the freezing-thawing procedure used had detrimental effects on the maturation and developmental capacity.

Chromosome and spindle configurations of human oocytes matured in vitro after cryopreservation at the germinal vesicle stage.

OBJECTIVE: To investigate effects of cryoprotectant and cryopreservation on the chromosome and microtubule configuration of human immature oocytes. DESIGN: Intact cumulus-enclosed immature oocytes were collected from unstimulated ovaries and divided into three groups: group 1, no treatment (control); group 2, only 1,2-propanediol treatment, and group 3, cryopreserved oocytes. Oocytes in groups 1 and 2, and oocytes that survived after cryopreservation in group 3 were cultured for 48 hours. SETTING: Infertility Medical Center at the CHA General Hospital, Seoul, Korea. PATIENT(S): Oocytes were obtained from patients undergoing gynecologic surgery. MAIN OUTCOME MEASURE(S): Maturation rate and abnormality in chromosomes by fluorescence in situ hybridization and in the spindle by immunostaining for tubulin. RESULT(S): There was no effect of propanediol-only treatment on the chromosomal (41.4%) and spindle abnormalities (35.3%) in group 2 compared with control oocytes (31.8% and 22.2%, respectively), whereas a statistically significant increase in abnormalities in chromosomes (77.8%) and spindles (70%) was found in group 3. CONCLUSION(S): Human oocytes matured in vitro after cryopreservation at the germinal vesicle stage showed increased incidence of chromosomal and spindle abnormalities. These abnormalities may impair the capacity for further development of the embryos derived from frozen-thawed oocytes.

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 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.

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.

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.