The Effects Of Developmental Microenviroment On Mitochondria In Oocytes And Early Embryos

Part One:The effect of superovulation on mitochondria in oocytes and early embryosObjective:The mitochondria are important organelles, not only because they are the power house of the cell but also because they contribute to Ca+homeostasis. They are the only organelles that can transmit DNA information to their offspring. They play a pivotal role in determine the developmental competence of oocytes and embryos. During oogenesis, mitochondria display stage-specific changes in quantity, function, morphology and distribution. Superovulation is a common assisted reproductive approach that stimulates ovaries into developing multiple oocytes synchronously. The phase in which superovulation acts is exactly the stage during which the oocytes complete their maturation. Many studies have revealed that oocytes collected after superovulation had poor quality. Further investigation is needed to investigate the safety of superovulation. In this study, we compared the in vitro developmental status of oocytes and embryos acquired following superovulation with non-hyperstimulation oocytes. The quantity, function, morphology, distribution and mtDNA heterogeneity of mitochondria from these oocytes or embryos were compared. Based on the results, we describe the effect of superovulation on mitochondria in oocytes and early embryos, and discuss the relationship to their function. Methods:C57BL/6J mice were randomly assigned to a superovulation and a control group. We injected pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG) sequentially to induce superovulation. In the control group, only HCG was injected at the estrus phase. The oocytes and embryos acquired through in vitro fertilization were collected. For these oocytes and embryos, we detected the copy number of mtDNA by real time PCR, mitochondrial ATP by the luciferase method, and mitochondrial membrane potential (△Ψm) by JC-1fluorescein stain. The distribution, ultrastructure and mtDNA heterogeneity of mitochondria in oocytes was analyzed by MitoTracker Red stain, electronic microscopy and denaturing high performance liquid chromatography (DHPLC) respectively. All these mitochondria indices, nuclear maturation, fertilization, cleavage and blastulation rate were compared between the superovulation and the control group.Results:1. Compared with control group, the superovulation group had a higher success rate of oocytes retrieval (P<0.01). The number of oocytes retrievaled and fertilized, as well as cleavage embryos and blastocysts were larger in the superovulation group than the control group (P<0.01, P<0.01, P<0.01, P<0.01), though a higher proportion of immature and low quality oocytes were acquired in the superovulation group.2. The mtDNA copy number of M Ⅱ oocytes and zygotes acquired from the superovulation group was lower than that from the control groups (P<0.01, P<0.05). There was no significant difference in2cell embryos and blastocysts between the two groups.3.The ATP content of M Ⅱ oocytes and zygotes acquired from the superovulation group was lower than that from the control group (P<0.01, P<0.05). There was no significant difference in2cell embryos and blastocysts between the two groups.4.△Ψm of M Ⅱ oocytes acquired from the superovulation group was lower than that from the control group. There was no significant difference in2cell embryos and blastocysts between the two groups.5. Electronic microscopy revealed scarce mitochondria in some M Ⅱ oocytes from the superovulation group. The superovulation group was observed to have a higher proportion of oocytes with mitochondria having a polarized cluster distribution pattern than was found in the control group.6. No obvious increase of D-loop region heterogeneity was found in the superovulation group compared to the control group.7. No significant difference was observed in the oxidative products of ovaries in superovulation group compared to the control group. The total anti-oxidative capacity was found to be higher in the superovulation group compared to the control group.Conclusion:1. Superovulation increases the success rate in oocytes retrieval in this mouse model. Superovulation increases the number of oocytes harvested and the number of oocytes functioning with high competence.2. Superovulation produces more immature oocytes and more oocytes with low mitochondrial counts and functions, these likely will be selected out in the fertilization and cleavage process. There was no significant difference in the number and function of mitochondria of cleavage embryos and blastocysts between the superovulation and control groups.3. Single superovulation does not increase the ovarian oxidative products and the mutation frequency of the mtDNA D-loop region. Part Two:The effect of endometriotic peritoneal fluid on mitochondria in early embryosObjective: Endometriosis(EM) and infertility in women are closely associated.Usually, there are few or no visible anatomic abnormalities in the peritoneal cavity of women with mild endometriosis. Endometriosis is both an immune and an inflammatory disease. There is a high level of inflammatory factors and reactive oxygen/nitrogen species (ROS/RNS) in peritoneal fluid (PF) of the patients with endometriosis. Many studies have suggested that endometriosis PF may inhibit embryonic development though unknown mechanisms. Mitochondria are important factors in the developmental competence of embryos, because they are sensitive to the surrounding environment. Mitochondria are major target organelle for inflammatory factors and ROS/RNS. Apoptosis could be induced if mitochondria were injuried. In this article, we investigate the IVF outcome of patients with minimal or mild endometriosis, examine the nitric oxide (NO) concentration in their peritoneal fluid, and observe the effect of PF on the development potential of mouse embryos, and the effect of PF on embryo mitochondria. We describe the effects on the pelvic microenvironment in patients with minimal or mild endometriosis, and attempt to describe the possible relationship to appropiate treatment strategies in patients with endometriosis.Methods:Clincal information regarding IVF treatment of endometriosis and tubal infertility patients was collected and analyzed. Peritoneal fluid was obtained from these women during laparoscopy. The NO level in peritoneal fluid was measured and the effect of different sodium nitroprusside (SNP, donor of NO) concentration on embryo development was evaluated.2PN embryo were retrievaled and cultured in human tubal fluid medium with or without10%PF. Cleavage and blastulation rates between the two groups the were compared along with the mtDNA copy numbers, ATP,△Ψm and ultrastructure of the embryo mitochondria. Results:1. The minimal and mild endometriosis patients have lower fertilization rate than tubal infertility patients. However, there was no difference between the oocytes harvested, cleavage rate, embryos quality, implantation rate, pregancy rate and abortion rates.2. The NO level in peritoneal fluid is higher in endometriosis patients.10-8M SNP had no effect on cleavage and blastulation rate. However,10-6M and10-6M SNP reduced both the cleavge and blastulation rate.3. The PF-E group had a lower cleavage rate and blastulation rate compared to the PF-NE and the control group.4. The PF-E group had a higher ATP and△Ψm level in2-cell embryos and4-cell embryos than the PF-NE and control groups. No significant difference were found in mtDNA copies among the three groups.Conclusion:1. There is no significant obvious difference tin the clinical outcome of IVF-ET treatments between the mininal or mild endomtriosis patients compared with the tubal infertilty patients.2. Endometriosis PF has a higher NO level than was observed in the control group. NO in high concentration was shown to inhibit early embryo development.3. Endometriosis PF induced functional changes in embryo mitochondria, this may contribute to its adverse effect on early embryos.