Value of serum and follicular uid Sirt1,Sirt2 protein levels in predicting the outcome of assisted reproduction

Background: To explore whether serum and follicular uid Sirt1, Sirt2 can reect ovarian reserve and predict the outcome of assisted reproduction. Methods: The study population comprised 125 patients , 39 in OPOI (cid:0) occult form of premature ovarian insuciency (cid:0) group, 49 in advanced age group , and 37 in control group. The levels of serum Sirt1, Sirt2 were measured on the 2nd to 5th day of menstruation (bSirt1,bSirt2) and HCG day. Follicular uid Sirt1 (FFSirt1) and Sirt2 (FFSirt2), were determined on OPU (oocyte pick up) day. Results: The level of FFSirt2 in the advanced age group was signicantly lower than those in other two groups. FFSirt 2 and Sirt 2 (HCG day ) were negatively correlated with age (r=-0.35, r=-0.19), but there were no value of them for assessing DOR (diminished ovarian reserve). The level of bSirt2 in (cumulative) pregnant group was signicantly higher (r=0.24, P=0.00). Conclusions: This was the rst study to show that FFSirt2 and Sirt2 (HCG day) might be negatively correlated with age and antral follicle count (AFC). bSirt2 could predict cumulative pregnancy outcome together with anti-Mullerian hormone (AMH), AFC and age.

and oocyte quality. Zhang J (8) found that the expression levels of Sirt1, Sirt3 and Sirt6 were signi cantly decreased in the ovaries of aged mice and mice receiving chemotherapy, and were positively correlated with the number of primary follicles. These results suggested that Sirt1, Sirt3 and Sirt6 are closely related to ovarian reserve.
Sirt2 participates in the mediation of mitosis, oxidative stress, microtubule movement, chromatin agglutination and cell movement (9). Liang Zhang (10) et al. suggested that the level of Sirt2 protein in oocyte of aged mice was signi cantly decreased, and the age-related meiotic defect could be improved after over expression of Sirt2 in oocyte of aged mice. Sirt2 may be related to the formation or assembly of spindle. Subsequently, Danhong Qiu et al (11) con rmed that the speci c deletion of sirt2 could lead to the damage of the kinetochore-microtubule junction of mouse oocyte, thus destroying the maturation process of oocyte and the spindle/chromosome tissue.
The serum Sirt1 concentration had been demonstrated to decreases with age in healthy people (12). This decline was more obvious in patients with AD and MCI, suggesting that Sirt1 may be an early predictor of AD. As the Sirt1 and Sirt2 were proved to be correlated with aging process and age related disease, ovarian reserve was also believed to be correlated with aging process, the present study try to evaluate the clinical value of serum and follicular uid Sirt1 and Sirt2 proteins in predicting the ovarian reserve and assisted reproductive outcomes in women of different ages and ovarian reserve. 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).

Hormone measurements
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 uid Sirt1 and Sirt2 were determined on OPU(oocyte pick up) day. The serum AMH ,Sirt1, Sirt2 and the follicular uid 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.

De nition 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.
Statistical analysis 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 coe cient. Serum and follicular uid 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 signi cant.

Results
Analysis of the differences of laboratories and clinical related indicators among the three groups The control group mainly used the luteal phase long-term regimen, which was statistically different from the other two groups. The OPOI group and the advanced age group mainly used short-term and microstimulation schemes, and there was no statistical difference between the two groups. In the infertility causes, although the proportion of male factor was higher in control group, there was no statistical difference among the three groups. The level of follicular uid Sirt2 (FFsirt2) in the advanced age group was signi cantly lower than that in OPOI group and control group. There were no differences between the value of baseline serum Sirt1\Sirt2 and HCG Day serum Sirt1\Sirt2. The high quality embryo formation rate of OPOI group was signi cantly higher than that of other groups. However, the cumulative pregnancy rate of the control group was 73%, signi cantly higher than that of the advanced age group and OPOI group, while there was no signi cant difference between the advanced age group and OPOI group (Table 1). Analysis of the differences of laboratories and clinical related indicators between pregnancy group and non-pregnancy group According to the cumulative pregnancy outcome, all patients were divided into pregnancy group (56 cases) and non-pregnancy group (69 cases). The level of bsirt2, AMH, age, AFC, egg number, fertilization number, number of high-quality embryos, blastocyst culture number, blastocyst formation number and number of transplanted high-quality embryos in pregnant group were signi cantly higher than those in the non-pregnant group( Table 2).     ROC curve analysis of serum and follicular uid Sirt1 and Sirt2 protein levels in predicting DOR and cumulative pregnancy outcomes The diagnostic criteria of DOR were AMH less than 1.1 ng/ml. Serum and follicular uid Sirt1 and Sirt2 protein levels did not show statistically predictive meaning(P > 0.05) (Fig. 1). Figure 2 showed that the AUC values of bsirt2, AFC, AMH and age were signi cantly different from those under the opportunity reference line (P < 0.05) (Table 4).
Subsequently, ROC curves of basic serum Sirt2 protein, AMH, AFC and age-predicted cumulative pregnancy outcomes were analyzed in three groups (Table 5). Unfortunately, there was no statistical signi cance in the three groups.

Discussion
Currently, most of the indicators predicting fertility outcomes are those predicting ovarian reserve and response, such as AMH, AFC and age. However, their value are not uncertain. Take OPOI patients for example, the decrease of the number of antral follicles is the main problem of OPOI patients, if OPOI patients could obtain eggs, and the egg quality is good enough to form high-quality embryos, the patient could get good clinical pregnancy rate of IVF cycle (13). In our study ,we found that though the AMH and AFC in OPOI group was less than the other groups ,the high quality embryo formation rate was higher than advanced age group and control group, live birth and continued pregnancy rate was also not lower than control group. Besides, patients in the advanced age group might be superior to the OPOI group in ovarian reserve, the BMI and spontaneous abortion rate were higher than OPOI group and control group.
As the age increases, the body's metabolic function declines, and older women are more likely to become overweight and obese than younger women. But the biggest BMI in advanced age group is 27.5 kg/m2, mean is 23.7 ± 3.1 kg/m2 .Overweight and obesity, de ned by World Health Organization (WHO) as a BMI of 25-30 kg/m2 and ≥ 30 kg/m2 (WHO, 2004).There is no obesity patient in our group. The sudy of Antonio MacKenna etc showed that BMI does not in uence the outcome of ART performed in women of Latin America (14). Also some study (15) showed that among women undergoing rst FET with highquality embryo transfer, obesity was associated with worse IVF outcomes, but not overweight. In our study, BMI also did not show statistical differences between the pregnant and non-pregnant groups. And for advanced age patients, the main reason of lower fertility and higher abortion rate might not be low ovarian reserve, but the quality of eggs decreases, such as non-segregation of meiotic chromosomes of eggs, chromosome arrangement and the abnormal composition of spindle matrix appear (16). The cumulative pregnancy rate in control group was higher. This might be related to the higher ovarian reserve in the control group, which can obtain more eggs and transplantable embryos.
Sirt1 and Sirt2 proteins. are important members of the SIRTUIN protein family of deacetylases, and the role of energy receptors plays an irreplaceable role in various elds,such as cell biology, metabolism, fate and so on .Previous studies have shown that under CR (Calorie restriction) conditions, an increase in endogenous sirt1 reduces the expression of p53 (instead of foxo3a), reduces follicular apoptosis or atresia, and maintains ovarian reserve (17).Sirt2 is involved in the mitotic structure, from the central body of the pre-mitotic stage to the central body during the mid-term spindle and cell division, ensuring normal cell division (18) .The study in 2017 (11) further found that sirt2 participates in the regulation of normal oocyte meiosis devices by deacetylating BubR1 and mediates the effects of advanced age on mouse oocyte quality. To explore the relationship between sirt1 and sirt2 and fertility ,we tested serum and follicle uid Sirt1 and Sirt2 protein in our study. Among the three groups, only the level of FFSirt2 in advanced age group was signi cantly lower than other groups. The results suggested that FFsirt2 may be associated with age and AFC, which was con rmed in the subsequent correlation analysis. And there were no difference between baseline serum sirt1/sirt2 and HCG Day serum sirt1/sirt2. This may be related to the highly conserved state of the gene, so it is relatively stable in serum and is not susceptible to ovulation induction drugs.
In the comparison between the cumulative pregnancy group and the cumulative non-pregnant group, the bSirt2 in the pregnancy group was signi cantly higher..This result is consistent with the ROC curve analysis of predicting the cumulative pregnancy value. The cut-off value was 29.95 IU/l. This may suggest that the bSirt2 may be superior to FFsirt2 in predicting overall reproductive outcomes. Pregnancy is related to ovarian reserve, egg quality, uterine environment, and general condition. Serum SIRT2 levels may be more responsive to a patient's whole body state than FFsirt2. The serum and follicular uid SIRT1 did not show any difference between two groups. Because the sample size of this study is small, further research is needed to verify this.
In the correlation analysis, we found the correlation between FFsirt2 and age and AFC, and also found that the serum level of Sirt1(HCG day) was negatively correlated with blastocyst formation but was positively correlated with the number of abortions. The serum levels of Sirt2(HCG Day)was positively correlated with AFC, the number of eggs, the number of matured eggs, the number of fertilization, the number of blastocyst formation and the number of frozen embryos but negatively correlated with age and basal estradiol levels(P < 0.05). Besides bSirt2 was positively correlated with the cumulative number of pregnancies. SIRT1 promotes cell proliferation and also inhibits cell proliferation too (19). Xiong et al (20) showed that down-regulation of SIRT1 expression inhibited apoptosis of mouse granulosa cells. This may also partially explain the results of HCG day serum Sirt1 in our study. Sirt2 not only participates in oocyte meiosis, but also participates in a variety of in ammatory and oxidative pathways.Sirt2 interacts with NF-κB in oxidative stress (21), regulates intracellular ROS levels which is also closely related to the quality of eggs and embryos (22).Those may be the reasons why serum and follicular uid Sirt2 have much more positive results in our study. But much more similar researches are still needed.
In the ROC curve analysis of predicting the ovarian reserve value, all the levels of Sirt1, Sirt2 did not show signi cant predictive value like AFC, which was inconsistent with the results of mice model. This may be due to the lower sensitivity of Sirt1 and Sirt2 in serum and follicular uid. However, due to the lack of relevant research, it is currently unclear.
The main limitations of the study is that the sample size is not large enough, we only use one method of ELISA to detect the serum and follicular uid concentration of Sirt1 and Sirt2. Since there was no such related research before, longitudinal studies with larger sample size, and more accurate detection methods were needed to clarify the correlation between serum and follicular uids Sirt1 and Sirt2 and ovarian reserve, egg quality, embryo quality, and reproductive outcome in the future.

Conclusions
In this study, the correlation between serum and follicular uid Sirt1 and Sirt2 and the assisted reproductive outcomes of patients of different ages and ovarian reserve was studied for the rst time.
The results suggested that FFsirt2 and HCG day serum Sirt2 may be a candidate biomarker to evaluate the ovarian reserve and bSirt2 may be the predictive parameter of the pregnancy outcome of ART. .Since there is no such related research at present, further studies are needed to clarify the correlation between serum and follicular uids Sirt1 and Sirt2 and ovarian reserve, egg quality, embryo quality, and reproductive outcome. We still need to expand the sample size, and use a variety of kits for testing, Page 20/25 evaluation, and comparison, so that the clinical and scienti c value of Sirt1 and Sirt2 are further discovered and con rmed.

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All presentations of reports in the present study have consent for publication.

Availability of data and materials
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Competing interests
The Authors declare that there is no con ict of interest.  ROC curve in predicting ovarian reserve. ROC curve in predicting ovarian reserve. The diagnostic criteria of DOR were AMH less than 1.1ng/ml. All the biomarkers except AFC have no value in predicting DOR.