Human Mitochondria Transfer With Self-granular Cells To Improve Oocyte's Quality And Embryo's Development

Backgroud and Objective:The female fertility reach climax at 25 years old, and it begins to decline very quickly over 35. In assisted reproductive program the pregnancy rates over 37 is just around 20%. Some researches suggested that it was mitochondrial function defection that caused the pregnancy rates declining with female aging and repeated IVF failure for poor embryo quality. The mitochondria in oocyte produce little ATP, and can't maintain the normal chromosomes segregation, biosynthesis, mitosis and other physiological functions of blastomere, which may disturb or cease embryo development. There are three parts in this study. First immunocytochemistry are used to detect the expression of PGC-1 in ovary of young and old rats for exploring mitochondria biogenesis changes. Second polarized light microscope was used to facilitates a non-invasive visualization of the meiotic spindle in the live human oocytes of different female ages in order to explore mitochondria function. And the microstructure of comulus cells was observed by transmission electro microscope. At last self-mitochondria tansfer was used to improve oocytes quality and embryo development from granular cells for women with repeated IVF-treatment failure and advanced reproductive age.Part I Expression of PGC-1 in rat's ovary Material and Methods: The ovary of 2- month-age and 14-month-agerats were cut to frozen tissue sections. And immunocytochemistry were performed with antibody PGC-1 and Goat ABC Staining System to detect the expression difference of PGC-1 in ovary of young and old rats.Result: There were ovarian cortex, medulla and parts of fallopian tube on slides. Primordial follicles and developing stage follicles can be found. There were comullar complex in developing stage follicles; oocytes were dyed to brown, granular cells showed no positive color. The muscle cells in medulla and blood vessels, fallopian tube muscle cells and column cells all showed positive color.There were thinner cortex and more medulla in older rat's ovary as compared with young rat's ovary. The number of primordial follicles and developing stage decreased, and positive color showed no different in all tissue cells.Part II Spindle imagining of human oocyte and embryo developing qualityMaterial and Methods: 31 patients who came to the reproductive medical center, Nanfang Hospital for ICSI treatment from August 2003 to April 2004, were divided into younger group and elder group (17 patients). The younger group include 14 patients from age 24 to 30, with the average age of 27+2.3. The study group had 17 patients from age 37 to 42, with the average age of 38+2.1. All the patients were treated with the long GnRH-a regimens. The meiotic spindles of mature oocytes were visualized before being done ICSI. The spindle imaging rates, fertilization rates and good embryo rates on day 3 were compared between the two groups.Reault: 74.6% mature oocyte spindles (126 in 169 oocytes) could be imaged in younger group and 52.1% in elder group. There was significant difference in spindle image rates between the two groups, P < 0.01. The fertilization rates showed no different, 80.5% and 75.6% in younger and elder group. There was marked difference in good embryo rates (57.6% : 39.1-6-) on day 3 between the two groups ( P < 0.01). But the good embryo rates of oocytes with spindle imaging between the two groups showed no significant difference ( P 0.05), 64.6: 48.9%; neither does the good embryo rates of oocytes without spindle imaging (P > 0.05), 38.2% : 28.6%.Part III The microstructure of human luteinized granular cellsof cumulus complexMaterial and Methods: Human cumulus complex were collected from patients who came to the reproductive center of NanFang Hospital for ICSI treatment. The microstructure of human granular cells in cumulus complex was observed by transmission electro microscope.Result: Three kinds of granular cells can be observed by transmission electro microscope: normal cells, necrosis cells and solidification cells. Intact cell me...