The study population comprised 125 patients who underwent their first cycles of in vitro fertilization (IVF)/ intracytoplasmic sperm injection (ICSI) treatment from March 2018 to December 2018 at the Reproductive Medicine Center of the First Affiliated Hospital of Zhejiang University School of Medicine. This study was approved by Research Ethics Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University (Reference Number:2017-734) and written consent was obtained from all the patients. According to the following criteria, the patients were divided into control group( 37 patients ), young occult form of premature ovarian insufficiency (OPOI) group( 39 patients )and advanced age group( 49 patients ).
Inclusion criteria:(1) Control group: 20≤ age ≤ 36 years old, normal menstrual cycle, normal basal hormone level (bFSH < 10 IU/L, bE2 < 50 pg/mL), AMH (>1.1 ng/mL), AFC (≥7);(2) OPOI group: 20 ≤ age ≤ 36 years old, bFSH ≥10 IU/L or normal, AMH ≤ 1.1 ng/mL and AFC <7;(3) advanced age group: age (≥37 years old).
Exclusion criteria: (1) PCOS(Polycystic Ovary Syndrome) patients; (2) endometriosis stage II or above, adenomyosis; (3) endocrine diseases such as hyperprolactinemia, thyroid diseases; (4) immune system diseases; (5) repeated implantation failure; (6) patients with uterine cavity adhesion, endometrial polyps and other factors affecting the history of embryo implantation; (7) surgical history of genital malignant tumors; (8) uterine malformation; (9) contraindication of IVF.
The concentration of serum Sirt1, Sirt2(bSirt1,bSirt2), AMH, FSH, LH, and E2 were measured on the 2nd to 5th day of menstruation. Besides, the concentration of serum Sirt1, Sirt2 were measured again on HCG day and follicular fluid Sirt1 and Sirt2 were determined on OPU(oocyte pick up) day. The serum AMH ,Sirt1, Sirt2 and the follicular fluid Sirt1, Sirt2 were measured by enzyme-linked immunosorbent assay (ELISA). The ELISA test kits were from Shanghai Yuduo Company.
Clinical and laboratory data collection
The number of antral follicles with a diameter of 2-8 mm in bilateral ovaries was recorded by transvaginal ultrasonography in our center on the 2nd to 5th day of menstruation. Other data collected included infertility years, age, factors of infertility, body mass index (BMI) , number of eggs, fertilization rate, rate of high-quality embryos, blastocyst formation rate, cumulative clinical pregnancy rate, continued pregnancy and live birth rate etc.
Ovulation stimulation program
Ovulation promotion program was selected according to the individual ovarian function of patients including long-term luteal regimen, short-term regimen, micro-stimulation and natural cycle regimen. Vaginal ultrasound monitored follicular development. If there were more than two dominant follicles with diameter of 18 mm, combined with serum sex hormone level, Gn was discontinued. One HCG 4000-10000 IU or rhHCG (250ug/branch) was injected at night. Oocyte retrieval was performed 34-36 hours after injection.
Definition of Relevant Indicators
Criteria of high-quality embryos: blastomeres are basically uniform in size, regular in shape, homogeneous in cytoplasm or granular cells, with debris less than 20%. Clinical pregnancy: In addition to biochemical indicators, there are clinically visible evidence of pregnancy, including intrauterine pregnancy sac, ectopic pregnancy, abortion or curettage visible chorionic tissue. Cumulative Pregnancy Rate: That is, after transferring all the embryos in an egg retrieval cycle, the percentage of those pregnant women accounted for the total number of egg retrieval cycles in all patients.
SPSS 19.0 statistical software was used for data analysis. Quantitative data were expressed as a mean±standard deviation. Qualitative data were expressed as counts or percentages. Coparison of quantitative variables between two groups were performed using two-tailed Student's t-test. Comparison of qulitative variables was perfomed using chi-square test. Coparison of intergroup among the OPOI group, advanced age group and control group was perfomed using one-way ANOVA followed by the Tukey's or Bonferroni correction. To correlated the level of Sirt1 Sirt2 with other parameters such as age, AMH, AFC, number of eggs and so on, data were analysed with the Person correlation coefficient. Serum and follicular fluid Sirt1 and Sirt2, serum AMH, FSH, AFC and age were used to construct the Receiver Operator Characteristic Curve (ROC Curve) to evaluate the value in predicting cumulative fertility outcomes. p value < 0.05 was considered statistically significant.