In this cross-sectional study, the concentrations of RBP4 and hs-CRP in the plasma of DOR patients were explored for the first time. We found lower RBP4 levels and higher hs-CRP levels in DOR subjects and a positive association between RBP4 and AMH. After adjustments were made for age and BMI as covariates, this relationship still existed. hs-CRP was not associated with AMH; therefore, RBP4 seems to be associated with ovarian reserve.
Like vitamin E, vitamin A (retinol),, is an antioxidant[8]. Because retinol is easily decomposed when exposed to light, coupled with the limitations of detection technology, RBP4 is considered to be a good indicator of serum retinol levels[18]. RBP4 has been recognized as an adipokine and as one of the oxidative stress markers involved in the pathogenesis of obesity-related metabolic diseases[20]. However, most of these studies were focused on adipose tissues, in which the effects of RBP4 were believed to be attributed to apo-RBP4, a form of RBP4 that does not bind retinol [21–23]. Some studies have shown that the antioxidative effects of RBP4 may be related to holo-RBP4, which refers to RBP4 combined with retinol[24]. Due to limitations in detection technology, it is impossible to distinguish the above two kinds of RBP4. In our study, we set an even stricter boundary and exclude all people who were obese, and 94.5% of the participants had a BMI of 25 kg/m2 or less in our study. Additionally, those at risk of any metabolic diseases were excluded. Therefore, to a certain extent, we controlled for the factors that led to changes in apo-RBP4 levels so that RBP4 levels could better predict blood retinol levels. This view was verified in another study. Hermsdorff et al[25] found that vitamin A intake and RBP4 levels were positively correlated in Hispanic women who were healthy and not obese (BMI˂30 kg/m2).
Ovarian dysfunction caused by oxidative stress has been reported to be associated with DOR. Long-term low-dose use of antioxidants containing vitamin A can protect the ovary from ROS induced by oxidative stress[5, 6, 9]. According to prior studies, the accumulation of ROS in granulosa cells caused ovary response poor to FSH in older women through downregulation of follicle-stimulating hormone (FSHR) expression and dysregulation of the FSHR signal transduction pathway[26, 27]. The physiological function of granulosa cells and follicular development is inseparable from the communication between FSH and its receptor. Any changes in FSHR, including reduced contact between FSHR and its ligand and reduced signaling after contacting, may result in decreased ovarian reserve[28]. To the best of our knowledge, fewer studies have evaluated the effect of abnormal retinol alone on ovarian reserve. However, these studies indirectly evaluated the correlation between RBP4 and FSHR. Overexpression of RBP4 can upregulate the expressions of FSHR in granulosa cells[29]. This fact was further supported by Rebeca et al[30]. As ovarian reserve decreases, FSHR expression in the ovary decreases. Similar to this study, Zeinab et al[31] also showed a downward trend in FSHR transcript expression in DOR patients. However, in this study, the difference was not significant, and they attributed it mainly to the limited sample size. Contrary to the above findings, Hattori, M.et al[32] found that retinoic acid (RA), the main active form of retinol in vivo, inhibited FSHR expression, thereby preventing immature granulosa cells from further developing into mature cells. However, the contradictory findings on the effects of retinol and its binding protein and derivates on granulosa cells during follicle development may be because the studies involved different species, examined different groups of subjects, were conducted in diverse geographical areas, had variability in sample size and used different experimental methods. These studies collectively suggested that retinol may regulate ovarian reserve. Hence, it is reasonable to speculate that abnormal RBP4 levels may be associated with DOR. According to our study, the serum RBP4 levels of DOR patients were lower than those of NOR patients, and RBP4 levels were positively correlated with AMH. This association has not been studied or proved before. This association may just be a link, rather than a necessary causal relationship. Nevertheless, it provides a reference basis for our future research.
Oxidative stress induces an abnormal immune response, together with an increase in inflammatory markers. Among them, CRP is a classic marker of chronic inflammation[33] and has been shown to be significantly related to DOR. Two cross-sectional studies found increased CRP in young women (under the age of 35 years) with DOR[34, 35]. Our observation that hs-CRP levels are higher in DOR patients is in line with previous studies. However, after we adjusted for covariates, hs-CRP was not associated with AMH. In previous studies that addressed the relationship between hs-CRP and AMH, there was either a negative correlation[34, 35] or no correlation[36], so the relationship between them still needs to be explored. In a large cross-sectional study, scientists analyzed a group of women aged 21–64 years and suggested negatively correlations between AMH levels and BMI or, fasting glucose. However, we did not observe the same relationship between AMH and BMI. It is worth noting that the BMI levels of the people in our study were all below 28, and 94.5% of them had a BMI of 25 kg/m2 or less, this criterion was more stringent than the standards used in previous studies. Our study included only women with normal serum levels of lipids and fasting glucose and women with no family history of cardiovascular diseases because abnormalities in these biochemical parameters and a family history of cardiovascular diseases may impact the plasma levels of CRP and AMH.
To the best of our knowledge, this study is the first to explore the potential connection between the ovarian reserve marker AMH and RBP4, hs-CRP. This research has the following advantages: First, we assessed a group of patients within a normal BMI range and who were under the age of 40 years and had no metabolic risk factors, to eliminate the additional effects of these factors on AMH and biochemical indicators in this experiment. Second, all of our blood samples were taken before IVF started, considering that different doses and kinds of ovulation drugs may have unknown impacts on inflammation and oxidative stress, thus minimizing the potential measurement bias caused by IVF treatment and providing the possibility for an accurate assessment of ovarian function.
Our research has limitations. First, the cross-sectional character of the study that did not able to follow-up of participants at a later stage. second, we did not perform a direct analysis of retinol but did use RBP4 instead. This was because RBP4 is more stable than retinol and because RBP4 detection kits are less expensive and more easily obtained than retinol detection kits. Third, we did not go on to assess this association in the follicular fluid of the patients due to the small sample sizes of the follicular fluid we collected. Currently, we are still enrolling patients who meet the criteria and collecting both blood and follicular fluid for further research. Fourth, the small number and scope of the research population is mainly related to our strict inclusion and exclusion criteria. It is necessary to further expand the research to include a broader patient group. Nevertheless, our results may provide insight into the potential mechanisms that regulate ovarian reserve.