Infertility is a disease and social problem. According to the reports9, about 10–12% of couples are suffering from infertility worldwide. In China, relevant studies have shown9–11 that the prevalence of infertility greatly varies among different regions, with the prevalence among women of childbearing age ranging from 6.7–25%. One of the current methods to solve this problem is to conduct IVF for women of childbearing age with confirmed infertility. Therefore, researchers have been exploring how to obtain a good pregnancy outcome of IVF/ICSI-ET conveniently and effectively.
Current systematic studies suggest that Vitamin D may have some positive effects during normal pregnancy. For example, Vitamin D can promote the development of the nervous17–19, motor8,17,20, and respiratory21 systems of the fetus, respectively, and regulate the immunity22 anti-infection, and allergy. In contrast, lower serum 25(OH)D in pregnant women has been linked to a higher risk of preterm birth (PTB) or intrauterine growth retardation (IUGR) 23.
Our study set 30ng/ml as sufficient 25(OH)D concentrations, as defined by the Italian Association of Clinical Endocrinologists in 201824, which recommended maintaining the 25(OH)D levels above 30 ng/mL (75 nmol/L). Furthermore, Rudick B reported racial differences in the effect of 25(OH)D concentrations on clinical pregnancy rates. However, the 25(OH)D deficiency was associated with a lower pregnancy rate among non-Hispanic whites, but not among Asians, with the authors suggesting that this might be attributed to the lower success rate of IVF-ET in Asia25. The present study is thus the first to investigate the relationship between serum 25(OH)D levels and CPR in IVF/ICSI-ET in Chinese populations. 25(OH)D was significantly positively associated with CPR. This result has been supported by similar studies. For instance, a meta-analysis showed26 that among all women who underwent IVF/ICSI-ET in their analysis, women with ample 25(OH)D were more likely to have a good clinical pregnancy outcome than women with deficient or insufficient 25(OH)D. Furthermore, Jing Zhao et al. reached a similar conclusion that 25(OH)D sufficiency increased live birth rates as an IVF/ICSI-ET outcome27. However, to the best of our knowledge, none of the existing studies have reported the nonlinear positive correlation yet.
The results of the present study were supported by many molecular mechanisms. Vitamin D promotes placental calcium transport, stimulating the expression of prolactin and decidualization, regulating the expression of HOXA 10, a target gene that has been associated with the implantation process28–32. Another experimental study reported that the lack of the VDR gene is associated with gonadal insufficiency, down-regulation of aromatase gene expression, low aromatase activity, hypergonadotropic hypogonadism32,33, and features of estrogen deficiency, such as bone malformations, uterine hypoplasia, impaired folliculogenesis, and infertility32,34.
Furthermore, our study showed that age remains an important factor in IVF/ICSI-ET. In the heat map of Fig. 2A the OR was found to exceed 10 and the color changed 30 times faster for women younger. Changes in the same 25(OH)D levels had a more positive effect on changes in pregnancy outcomes in women under 30 years of old. A similar conclusion related to LBR is shown in Fig. 2B. Therefore, we hypothesize that women below the age of 30 are more sensitive to Vitamin D deficiency.
However, our research results were still controversial. Jason M. Franasiak's study showed that the effect of 25(OH)D on pregnancy outcomes was insignificant35. Abbas Aflatoonian suggested that 25(OH)D deficiency treatment did not relate to a higher pregnancy rate in the cycle of frozen-thawed embryo transfer36. Arne van de Vijver37 and Liu Jiang13 also reached the similar conclusion of no significant correlation between the two. The process of IVF/ICSI-ET is complex, and many factors can affect outcomes. Combined with our findings, it is suggested that this difference could be explained from the perspective of age, since the clinical pregnancy outcomes of women over 30 years old, especially the elderly pregnant women (age ≥ 35 years old), are affected by other more important factors implying the presence of a bias in our model. For example, research by Michaël De Brucker has shown that clinical pregnancy rates in older pregnant women were driven by several complications. The incidence of pregnancy-induced hypertension was only 6–8% in the general pregnant women group, while it increased to 15% in the elderly pregnant women38. In addition, it is obvious that women’s fertility decreases with the increase in age39 with a notable change being the deterioration of the ovarian reserve function with age. Therefore, AFC, as one of the indicators for evaluating the ovarian reserve function40, showed a downward trend with aging41. Moreover, the decline in ovarian function adversely affects the outcome of IVF/ICSI-ET. Therefore, if the patients were not grouped by age, it would have been very likely to obtain different results. Moreover, as shown in our study, the relationship between 25(OH)D and pregnancy outcomes of IVF/ICSI-ET patients is nonlinear. This means that if the patient’s 25(OH)D levels were not well grouped or mainly distributed below 25ng/ml, it would be difficult to observe the correct conclusion.
In summary, our study was the first case study on the Chinese population to propose the CPR and LBR as the research outcome of IVF/ICSI-ET, that explored the trends of pregnancy outcomes under the combined effects of covariates such as age. According to PubMed, we were the first to describe this nonlinear relationship using RCS curves. This was important for the subsequent development of IVF/ICSI-ET and for patients to obtain a good pregnancy outcome. To some extent, it bridged and explained previous controversial views in this field. However, our study still possesses some limitations. First, it only included female patients administered by the Second Affiliated Hospital of Wenzhou Medical University. Second, it had been suggested42 that seasonal variations may affect circulating 25-(OH)D levels, and we did not rule out these possible interferences in this study. In addition, there were many factors affecting the pregnancy outcome. For example, measured in early pregnancy 25(OH)D levels are likely to fail to better represent 25(OH)D levels in late pregnancy, weakening thus the link between 25(OH)D levels and LBR compared to CPR. Further research could promote understanding of the role and influence of 25-(OH)D in IVF/ICSI-ET better and finally contribute to the identification of methods to improve IVF/ICSI-ET pregnancy outcome.