The Preliminary Research On The Effect Of Linolenic Acid On Human Oocyte Maturation

Backgroud: In vitro maturation(IVM) technology means the germinal vesicle stage or metaphaseⅠstage oocytes from infertile patients cultured in vitro until to the second meiotic metaphase phase(metaphase Ⅱstage), the matured oocytes can be developed, fertilized and implanted normally through in vitro fertilization and embryo transfer to treat infertility.The successful pregnancy through IVM of human immature oocyte has a history of more than 20 years.With the successful pregnancy increasingly in recent years, IVM has made a great progress in assisted reproductive techniques,which had made more and more pregnancies for the patients whose oocytes mature disability,especially for patients with PCOS.Although human oocytes were able to develop and obtain pregnancy in vitro,there are still several problems, such as the low rates of cleavage,fertilization and pregnancy. The IVM medium had an effect on the regulation of meiosis.In recent,many studies add a variety of nutrients to improve the oocytes’ microenvironment to advance the maturation of oocytes and improve the quality of embroys.So developing a complete culture system that would support human oocyte development.and improve the outcomes of IVM have been a hot research area nowadays.The function of mitochondrial palys an important role in oocyte maturation and subsequent embryo development.Currently,it is unclear whether metabolic capacity or mt DNA replication is essential for supporting oocyte maturation, fertilization, and subsequent embryo development, and a direct comparison between them is lacking. Collectively, these studies indicate that mitochondrial DNA(mt DNA) copy number plays a critical role in oocyte maturation and subsequent embryo development. Based on these analyses,mt DNA copy number in oocyte has been suggested to be an important marker of oocyte quality.But it’s still inconclusive of the effect of mitochondrial DNA on oocyte maturation and subsequent embryo development.Linolenic acid(ALA;18:3 n-3) is all-cis 9,12,15 octatecatrienoic acid,which belongs to n-3 polyunsaturated fatty acids.ALA is an important component of cell membrane,and plays roles through transforming into EPA and DHA.ALA not only increase the composition of PGE2 and c AMP of oocytes but also decrease the peroxidation reaction and decrease the creation of MDA through advancing the vitality of antioxidase such as SOD and GPx,which contribute to the oocyte maturation.Some cases have showed that ALA had an effect on the mammal oocyte maturation in vitro,but it has not been reported the effect on human oocyte maturation in vitro of ALA until now.According to our study before,that were,adding some mature follicular fluid to the IVM medium advanced the rates of maturation,fertilisation and cleavage,which were better than adding gonadotropin. So we add some mature follicular fluid based on the optimal ALA concentration to explore the effect of ALA on human oocyte maturation and subsequent embryo development.Objectives The experiment cultures immuture human oocytes with ALA to explore the effect of ALA on human oocytes maturation and development. At the same time we measure the mt DNA copy number and MDA levels and T-SOD activity in the spent medium after oocyte maturation to explore the potential mechanism contributing to human oocyte development.Materials and methods A total of 689 morphologically normal immature oocytes were recruited from 423 infertile women with whose husbands were sterile for ICSI cycle in Reproductive Medical Research Center of the Third Affiliated Hospital of Zhengzhou University from October 2014 to October 2015 and Gn RHa long stimulate ovulation was adopted.Part I: There were 273 oocytes at MI stage and 262 oocytes at GV stage.All subjects selected randomly were devided into 5 groups according to the same proportion of MI and GV,then they were put into 5 groups of medium with different ALA concentration(0,10Μm,50μM,100μM,200μM).After being cultured 24 hours,we observed the maturation of these oocytes and measured the mt DNA copy number of matured oocytes from group ALA 0μM and group ALA 50μM..Part II:A total of 248 morphologically normal immature oocytes were recruited from 150 infertile women There were 87 oocytes at MI stage and 161 oocytes at GV stage. All subjects selected randomly were devided into 3 groups according to the same proportion of MI and GV,then they were put into 3 groups of medium with different content((1)50μM ALA+50%follicular fluid+50% basal IVM medium;(2)50%follicular fluid+50% basal IVM medium;(3)basal IVM medium),that were group A,B,C.After being cultured 24 hours,we observed the rate of maturation、fertilization、2PN cleavage, available embryos and blastocysts of these oocytes and determined the content of MDA and the activity of T-SOD in the spent medium of three groups at the same time..Statistical methods: SPSS17.0 statistical software was used to make statistical analysis. The statistical result of measurement data was expressed by mean±standard deviation( x ±s), t test was used to compare the two groups independent samples, one-way analysis of variance was used to compare many groups if the data conform to normality and homogeneity of variance;if not,we use K groups independent samples or Kruskal-Wallis test.The Bonferroni method was used to compare between groups.α=0.05 was chosen as inspection level,According to the comparison times adjust the level of significance in multiple comparison α’=0.05/k,differences of P<α’ were considered significant.Results: Part I:(1)Culturing the immature oocytes with a range of concentrion(0μM,10μM,50μM,100μM,200μM) of ALA 24 hours.The total maturation rate of group ALA 50μM(70.1%) significantly higher than that of group ALA 0μM(42.1%) and group ALA 200μM(34.1%),the differences were all statistically significant(P<0.005);(2)The rates of MI oocytes maturation from a range of concentrion(0μM,10μM,50μM,100μM,200μM) of ALA were 61.3%,68.4%,78.9%,74.4%,60.6% after 24 hours culture,respectively.There were no statistically significant between any two groups(P>0.005).(3)Culturing GV stage oocytes with a range of concentrion(0μM,10μM,50μM,100μM,200μM)of ALA 24 hours.The total maturation rate of group ALA 50μM(64.4%) significantly higher than that of group ALA 0μM(28.9%) and group ALA 200μM(18.2%),the differences were all statistically significant(P<0.005);(4)The mt DNA copy number of matured oocytes from group ALA 50μM(3.64×107±1.85×106) were more than group ALA 0μM(3.42×106±1.95×105),the differences were statistically significant(P<0.05).Part II:(1)The rates of oocytes maturation from group 50μM ALA+follicular fluid(group A),group follicular(group B),control(group C) were 75.9%,57.3%,38.0% after 24 hours culture,respectively.The maturation rate of group A in 24 h was higher than group B,which was higher than group C,the differences were statistically significant(P<0.016).(2)As for as the maturation rate of MI in 24 h between the 3 groups,group A was higher than group C and the difference was statistically significant(P<0.05). As for as the maturation rate of GV in 24 h between the 3 groups,group A was higher than group B(46.3%)and group C(29.4%),the difference both were statistically significant(P<0.016).(3)The rates of fertilization from the 3 groups were 78.8%,74.5%,63.3% after ICSI,respectively. There were no statistically significant among the 3 groups(P>0.016);The rates of 2PN cleavage from the 3 groups were 92.3%, 88.6%,57.9%,respectively. The rates of 2PN cleavage in group A and group B were higher than group C,the differences were statistically significant(P<0.016); The rates of available embryos were 50%,45.2%,36.4%,respectively and the rates of blastocyst formation were 22.9%,16.1%,9.1% among the 3 groups. There were no statistically significant among the 3 groups(P > 0.016). But there were some increasing trend between the number of blastocyst formation among the 3 groups;The content of MDA in group A was lower than group B and group C.The differences were both statistically significant(P<0.016).Conclusions: 1.ALA had the effect of promoting human oocyte maturation in vitro,which proper concentration is 50μM,and the effect on GV stage oocytes were more obvious than the effect on MI stage oocytes.2.ALA may advanced the developmental potential of human oocyte through increasing the mt DNA copy number and decreaing the oxidative stress.