Assessment of global DNA methylation in stem cells with reverse-phase high performance liquid chromatography.

Objective: We tested the possibility of the use of reverse-phase high performance liquid chromatography (RP-HPLC), a sensitive, simple and cost effective technique, for evaluation of global DNA methylation alteration in stem cells. Methods: We detected genomic methylation in four cell lines of mouse embryonic stem cells and in the cell at various developmental stages, including mouse embryonic fibroblast (n=5) and mouse tissues (n=9), using RP-HPLC. The samples were extracted for single nucleosides and investigated for the concentrations of deoxy-cytidine (dC), deoxy-guanosine (dG) and deoxy-methyl-cytidine (5-dmC) using RP-HPLC. The methylation levels were obtained by a ratio of the entire genomic 5m-C to dC. Results: Our results demonstrated that the RP-HPLC technique can be used for analysis of global methylation on the stem cell genome. It can display the discrepancy of methylation levels among the cells at different degrees of cell differentiation. Conclusion: We present the evidence that the RP-HPLC technique can be used for the entire-genomic methylation detection in stem cells and should be further developed as a strategy for monitoring the global DNA methylation of stem cells in advanced research.

Comparison of EffectiveNESS between Recombinant Follicle Stimulating Hormone and Human Menopausal Gonadotrophin Administration in those with Diminished Ovarian Reserve.

The study was carried out to compare the effectiveness of recombinant follicle stimulating hormone (recombinant FSH) and human menopausal gonadotrophin (HMG) in those with diminished ovarian reserve. A total of 106 ovarian stimulation cycles from 88 poor responders were included in this study. Either recombinant FHS or HMG was administered in order to stimulate the ovary for each cycle. The pregnancy rate of the recombinant FSH group (22.5%) was higher than that of the HMG group (9.1%). The cancellation rate of the recombinant FSH group (10.0%) was lower than that of the HMG group (19.7%). In in vitro fertilisation-embryo transfer (IVF-ET) cycles, the fertilisation rate of the recombinant FSH group (62.8%) was higher than that of the HMG group (51.8%). The pregnancy rate and the implantation rate of the recombinant FSH group (23.0 and 9.1%, respectively) were higher than those of the HMG group (13.6 and 5.9%, respectively). Although this did not achieve statistical significance, only the recombinant FSH group achieved pregnancies using gamete intrafallopian transfer (GIFT). In conclusion, recombinant FSH is probably more effective than HMG in improving the IVF and pregnancy rate in poor responders.

Comparative study of embryo culture media containing and without containing glucose as energy substrate.

The aim to increase pregnancy rate by in vitro fertilization (IVF-ET) is to improve the embryo culture system, especailly the culture media. Most of the conventional IVF media has been deve-loped from somatic cell culture which contains glucose as the energy substrate. Many recent studies in mammals, including human, reported that glucose was not the preferred nutrient for the zygote and early cleaving embryo, and may stimulate premature glycolysis, which induce the Crabtree effect; thus depressing respiratory rate and energy production. This study was conducted to evaluate the embryo culture outcome that grows in conventional IVF media containing 5 mM of glucose (Medicult media) as compared to the home-made media without glucose (SJ-media). Ten infertile couples were enrolled in the study for eleven treatment cycles. Seventy eight oocytes were retrieved, of which sixty three were fertilized as 2 PN embryo, that were randomly allocated into two culture groups: 32 embryos in group I( Medicult media), and 31 embryos in group II (SJ-media). Twenty eight hours after the culture, the embryos in both groups were compared. There was no statistical difference between the embryos of the two culture groups (p>0.05), but there was a trend in which embryos in group I had more 3-4 blastomere than group II (68.8%vs 51.5%); embryos in group II; however; had less fragmentation (67.7% vs 50% for fragmentation< 5%) and were of better quality than group I (51.6% vs 40.6% for embryo grade A, and 32.2% vs 25% for grade B). In conclusion, this study has confirmed that human embryos can grow in media devoid of glucose ; at least; as well as glucose containing media. A larger sample size study ; however ; need to be carried out.

Higher pregnancy rate with intrafallopian tubal gamete or zygote transfer (GIFT or ZIFT) than intrauterine embryo transfer (IVF-ET).

From November 1996 to September 1998, we had treated infertile couples who required assisted reproductive conception by allocating to either one of the three treatment methods: IVF-ET for those with bilateral tubal obstruction; ZIFT for those with at least one tubal patency but poor sperm ; and GIFT for those with at least one tubal patency and normal sperm (unexplained infertility). The ovarian stimulation protocol was all the same by using GnRH analogue (Suprefactฎ) for pituitary suppression and daily hMG (Metrodinฎ) injection for ovarian stimulation. The oocyte pick up was due when the leading follicle reach 18 mm. For IVF-ET and ZIFT, the fertilization was obtained by conventional in vitro fertilization or by ICSI depending on the sperm quality. Laparoscopic intrafallopian tubal gamete or zygote transfer was preformed on day 0 ( ovum pick up day) for the GIFT or on day 1 for the ZIFT group. Intrauterine embryo transfer was performed on day 2- 3 for the IVF-ET group. Every treatment cycle was conducted by the investigator group to minimize the variation of technical bias. Of all the 213 treatment cycles, 82 were IVF-ET, 92 were ZIFT, and 39 were GIFT. The average female patient age in each groups was not different. The pregnancy rate achieved in the GIFT and ZIFT groups were significantly higher than the IVF-ET group ( 46.2%, 40.2% and 23.2% respectively , p < 0.05). For the pregnancy outcome, the abortion rate seemed to be highest in the IVF-ET group ( 36.8%) whereas the multiple pregnancy rate seemed to be higher in the fallopian tubal transfer group ( 27% for ZIFT and 38.9% for GIFT), although there were no statistical significance. The benefit of the higher pregnancy rate for the intrafallopian tubal transfer treatment group could be due to the more suitable environment for the early stage embryo and the the more synchronize of the endometrial receptivity and the embryo arrival timing provided by the fallopian tube. In conclusion, until the optimal in vitro embryo culture system can be developed, gamete and zygote intrafallopian tubal transfer should yield higher pregnancy rate than intrauterine embryo transfer.

Cryopreservation of human spermatozoa: The effect of cryostorage time on the post – thaw sperm outcome.

A prospective study was reported comparing the effect of short-term (1 week) and long-term (6 months) cryostorage time on the post-thaw sperm outcome in four different freezing techniques. Eighty five semen samples were included in the study. Each sample was divided and subjected to four cryopreservative techniques: CEG-NCR, CEG-CR, TEST-EYG-NCR, and TEST-EYC-CR. The cryopreservation of each technique was performed in similar way by mixing the semen with the cryopreservative media (CEG or TEST-EYG) in ratio 1:1, then the mixture was aspirated into two straws. The temperature was then reduced (by NCR or CR freezing method) to -80 C, then the straws were transferred into liquid nitrogen. One straw of each technique was thawed after 1 week and the other straw after 6 months of cryostorage time for assessing the post-thaw sperm outcome. As the results, the post-thaw sperm motility and survival rate of the 1 week cryostorage group was slightly higher than the 6 months group in the CEG-NCR and the CER-CR freezing technique, but these did not have clinical significance because the differences were into small. There were no differences of post-thaw sperm outcome between 1 week and 6 months group in the TEST-EYG-NCR and TEST-EYG-CR freezing techniques. We concluded that the duration of cryostorage did not have clinical effect on the post-thaw sperm outcome.

Basal body temperature chart as a guide for donor insemination.

The use of BBT in a single donor insemination and the conception rate according to cycle day was studied. For 107 such cycles, inseminations were performed on day 0 in 21.5% of cycles, on days-1,+1 and –2 in 17.8%, 16.8% and 13.1% respectively. The overall pregnancy rate achieved was 44%. The success rate per cycle was 15%, and the highest success rates were obtained on day 0 (43.7%) and day –1 (31.2%). No pregnancies occurred when insemination was done before day -3 and beyond day +1. In conclusion BBT provides a reasonable guide to the 2-to 3-day period on either side of the nadir in donor insemination.