Literature search and characteristics of included studies

Figure 1 illustrates the systematic review’s flow diagram. In the initial search, 948 articles were potentially relevant (236 from PubMed, 296 from Web of Science, and 416 from Scopus). After removing duplicates, 672 studies remained. A preliminary screening of titles and abstracts excluded 663 records that didn’t meet inclusion criteria. The eligibility of the remaining 9 full texts was assessed, and all meeting the criteria were included in the final assessments.

Fig. 1

Table 1 summarizes the main characteristics of the clinical studies included. These four trials were carried out from 2022 to 2023. All trials were conducted in Iran. The sample size of trials ranged from 40 to 53 participants. The intervention dosage ranged from 6 mg/day to 12 mg/day and duration ranged from 6 to 12 weeks. The trials include samples from different causes of infertility including PCOS [15, 22, 23] and Endometriosis [16].

Table 1 Main characteristics of the included clinical studies

Table 2 outlines the main characteristics of the included animal studies. These five studies were conducted between 2021 and 2023. Four studies were conducted in the turkey [17, 24,25,26] and one in Iran [18]. Four studies developed their models of reproductive disorder in Rats [17, 24,25,26] and one in Mice [18]. The intervention dosage ranged from 0.1 mg/kg to 100 mg/kg and the duration ranged from single dose consumption to 14 days. Some studies used oral forms and some intraperitoneal injections. There were also some studies that did not report the exact route or duration of administration. The studies include experimental models of PCOS [17, 18] and ovarian injury [24,25,26].

Table 2 Main characteristics of the included animal studies

Through a hand search of grey literature, a total of nine clinical trials aiming at the evaluation of the effect of AST on reproductive performance, fertility, or ART outcomes were retrieved, of which 4 articles have not yet been published (Supplementary file S3).

Risk of bias assessmentClinical studies

The summary of the risk of bias assessments for clinical studies according to the review authors’ judgements about each methodological bias is shown in Fig. 2. The detailed risk of bias assessments with review author’s comments for each individual clinical study is presented in supplementary file S4.

Fig. 2

Risk of bias in clinical trials according to ROB 2 Toll; (a) Methodological risk of bias summary: review authors’ judgments about each methodological bias item for each included study; (b) Methodological risk of bias graph: review authors’ judgments about each methodological bias item presented as percentages across all included studies; OS: oxidative stress, ART: assisted reproductive technology

All four studies were randomized and reported their sequence generation methods, so they had low risk in this domain, while selection bias was at high risk in two studies [15, 16] because they did not report concealment. All four studies had a low risk of bias for performance, detection, and attrition domains. We obtained the study protocols for all four studies, but we found some reporting bias in two studies [15, 16]. These studies had some inconsistencies between their protocol and their reported outcomes, and some of their statistical analyses seemed to be post hoc.

Animal models

The summary of the risk of bias assessments for animal studies are shown in Table 3. Also, details of the risk of bias assessments with the reviewer’s comments for each individual animal model study are presented in supplementary file S5.

We evaluated the risk of bias for each animal study using the SYRCLE Risk of Bias Assessment Tool. We found that all five studies had high risk of bias in several domains, such as allocation concealment, random housing, blinding, and random outcome assessment. Only one study [26] had high risk of bias in sequence generation, while the others had unclear risk. All studies had low risk of bias in baseline characteristics and most of them (except for [24]) had low risk of bias in selective outcome reporting. One study had high risk of bias in incomplete outcome data [24], while one study [18] had unclear risk. The rest had low risk of bias in this domain.

Table 3 Summary of potential sources of bias in animal model studies according to SYRCLE risk of bias assessment toolMeta-analysis of clinical studiesEffect of AST on ART outcomes

We assessed the effect of AST (n = 72) compared to placebo (n = 71) on four ART outcomes, including the total number of retrieved oocytes, OMR, total number of embryos, and fertilization rate in three studies [15, 16, 22] with a total of 143 infertile participants. The total number of embryos was not reported in Rostami et al. [16]. The results are summarized in Fig. 3. There was no significant difference between the AST and placebo groups in the total number of retrieved oocytes (MD = 0.71, 95% CI: -3.66 to 5.07, p = 0.75). However, there was a high heterogeneity among the three studies included in this analysis (I2 = 69%).

Fig. 3

Meta-analysis of mean difference of the effect of Astaxanthin (intervention) compared to the placebo in different ART outcomes; (a) total number of retrieved oocytes, (b) oocytes maturity rate, (c) total number of embryos, (d) fertilization rate

The OMR was significantly higher in the AST group than in the placebo group (MD = 8.40, 95% CI: 4.57 to 12.23, p < 0.0001), with no heterogeneity among the three studies (I2 = 0%).

The total number of embryos and the fertilization rate were also higher in the AST group than in the placebo group, but the differences were not statistically significant (MD = 1.00, 95% CI: -0.30 to 2.30, p = 0.13 for total number of embryos; MD = 3.41, 95% CI: -0.79 to 7.61, p = 0.11 for fertilization rate). There was no heterogeneity among the two studies included in these analyses (I2 = 0% for both outcomes).

Effect of AST on pregnancy outcomes

We assessed the effect of AST compared to placebo on chemical and clinical pregnancy rates. The rates of miscarriage and live birth were reported in none of the included studies.

There were three studies [15, 16, 22] enrolling 143 infertile women underwent ART that compared pregnancy outcomes between AST (n:72) and placebo group (n:71). The results are shown in Fig. 4 and supplementary file S6. The mean rate of chemical pregnancy was higher in the AST group than in the placebo group (51% vs. 44%), but the difference was not statistically significant (RR = 1.17, 95% CI: 0.83 to 1.64, p = 0.38). In consonance, the risk difference (RD) was 8% in favor of the AST group compared with the placebo group (RD: 0.08, 95% CI: -0.08 to 0.24; P = 0.34). There was no heterogeneity among the three studies included in this analysis (I2 = 0%).

Fig. 4

Meta-analysis of risk difference of the effect of Astaxanthin (intervention) compared to the placebo on the chemical pregnancy and clinical pregnancy; (a) the chemical pregnancy (b) the clinical pregnancy

The clinical pregnancy rate was also higher in the AST group than in the placebo group (43% vs. 38%), but the difference was not statistically significant (RR = 1.24, 95% CI: 0.63 to 2.43, p = 0.53). In consonance, the risk difference (RD) was only 5% in favor of the AST group compared with the placebo group (RD: 0.05, 95% CI: -0.11 to 0.21; P = 0.53). There was no heterogeneity among the three studies included in this analysis (I2 = 0%).

Antioxidative, anti-inflammatory and antiapoptotic effect of AST in clinical studies

In our meta-analysis of clinical studies, redox status markers were consistently reported, allowing for a robust quantitative synthesis. All included clinical studies provided data on these markers, which were subjected to meta-analysis. In contrast, inflammatory cytokines were reported in a single study [16], precluding a meta-analysis for this marker due to insufficient data. Similarly, apoptosis markers were not reported in any of the included clinical studies, highlighting a gap in the current literature and an opportunity for future research.

Effect of AST on redox status markers in FF

We compared the level of redox status markers between AST (n = 72) and placebo group (n = 71) in three studies [15, 16, 22] with a total of 143 infertile participants. The results are summarized in Fig. 5.

Fig. 5

Meta-analysis of mean difference of the effect of Astaxanthin (intervention) compared to the placebo on different Redox status markers in Follicular fluid; (a) Catalase, (b) Malondialdehyde, (c) Superoxide dismutase, (d) Total Antioxidant Capacity

We measured four markers of redox status: MDA, TAC, SOD, and CAT. All three studies reported MDA, TAC, and SOD levels, while CAT level was reported only by Rosatmi et al. [16] and Gharaei et al. [15]. Gharaei et al. and Rostami et al. used the same enzyme-linked immunosorbent assay (ELISA) (ZellBio GmbH, Germany) that indirectly measured SOD activity by the reduction of a water-soluble tetrazolium salt by superoxide anion at 450 nm. Jabarpour et al. [22] used a different ELISA kit (NAVAND, Iran) that directly measured SOD activity by the inhibition of pyrogallol autoxidation by superoxide anion at 420 nm. These two methods had different sensitivity, specificity, and assay ranges for SOD measurement, which may explain the outlier value reported by Jabarpour et al. Therefore, we excluded Jabarpour et al. from our meta-analysis, as their data were not comparable with the other studies.

There was no significant difference between the AST and placebo groups in the pooled mean difference of CAT (MD = 0.24, 95% CI [-0.59, 1.07], p = 0.57) or MDA (MD = -0.06, 95% CI [-0.26, 0.14], p = 0.55) in FF. There was also no significant difference between the groups in the pooled mean difference of SOD (MD = 0.24, 95% CI [-1.81, 2.28], p = 0.82) in FF. However, the pooled mean difference of TAC was significantly higher in the AST group than in the placebo group (MD = 0.04, 95% CI [0.02, 0.06], p = 0.0002) in FF. There was no heterogeneity among the studies for any of the outcomes.

Effect of AST on redox status markers in serum

We compared the level of redox status markers of serum between AST (n = 72) and placebo group (n = 71) in three studies [15, 16, 23] with a total of 143 infertile participants. The results are summarized in Fig. 6.

Fig. 6

Meta-analysis of mean difference of the effect of Astaxanthin (intervention) compared to the placebo on different Redox status markers in serum; (a) Catalase, (b) Malondialdehyde, (c) Superoxide dismutase, (d) Total Antioxidant Capacity

We measured four markers of redox status: MDA, TAC, SOD, and CAT. All three studies reported MDA, TAC, and SOD levels, while CAT level was reported only by Rosatmi et al. [16] and Gharaei et al. [15]. Also, like the redox status markers of FF, the SOD measurement methods were different, so just the Gharaei et al. and Rostami et al. that used the same ELISA kit (ZellBio GmbH, Germany) were included in meta-analysis.

As shown in Fig. 6a, the pooled mean difference of CAT was higher in the serum of AST group than in the placebo group, but not significantly (MD = 1.43, 95% CI: -4.67 to 7.54). As shown in Fig. 6b, the pooled mean difference of MDA in serum was lower in the AST group than in the placebo group, but not significantly (MD = -3.72, 95% CI: -9.70 to 2.26). As shown in Fig. 6c, the pooled mean difference of SOD of serum was higher in the AST group than in the placebo group, but not significantly (MD = 2.08, 95% CI: -2.15 to 6.32). As shown in Fig. 6d, the pooled mean difference of TAC of serum was also not significantly different between the groups (MD = 0.02, 95% CI: -0.03 to 0.07). There was high heterogeneity among the studies for all redox status markers of serum.

Publication bias in clinical studies

Regarding the assessment of publication bias, our results do not include funnel plot analysis or Egger’s regression test. This decision was based on the number of studies included in our meta-analyses, which did not meet the minimum recommended threshold of 10 studies for these tests to be effective. As a result, we have not reported on publication bias, in line with best practice recommendations for systematic reviews with a limited number of studies [27].

Descriptive synthesis of outcomes reported by animal models

The findings of the studies were heterogeneous in terms of the animal models, the doses and durations of AST treatment, the outcomes measured, and the methods used. However, some common themes emerged from the synthesis of the findings. We grouped the findings into four categories: redox status, inflammation, apoptosis, and ovarian tissue histomorphology. We summarized the findings of each individual study in Table 4.

Table 4 Main findings of the included animal studiesRedox status

All studies reported that AST improved the redox status of the ovarian tissue and/or other reproductive organs by reducing OS and increasing antioxidant capacity. The indicators of redox status varied across studies, but they included markers such as ROS, MDA, TAC, total oxidant capacity, oxidative stress index, SOD, GSH, paraoxonase 1 activity, nitric oxide, thiol, and total thiol. Also, a study reported that AST improved the lipid profile by increasing high-density lipoprotein and decreasing cholesterol, low-density lipoprotein, and triglyceride levels.

Inflammation

Four studies reported that AST reduced the inflammation in the reproductive organs by decreasing the expression or levels of inflammatory markers, such as inducible nitric oxide synthase, C-reactive protein), granulocyte colony-stimulating factor, interleukin-1 beta, interleukin-6, tumor necrosis factor-alpha, and NF-kβ. Some studies also reported that AST improved hyperemia (increased blood flow), inflammatory cells, and necrotic changes induced by methotrexate or ischemia/reperfusion injury [24,25,26]. So, it seems that AST can ameliorate ovarian damage caused by inflammatory reactions.

Apoptosis

Three studies reported that AST reduced the apoptosis in the reproductive organs by decreasing the expression or levels of apoptotic markers, such as caspase-3 and annexin V/propidium iodide. One study also reported that AST improved the expression or levels of the anti-apoptotic signaling pathway, AKT 1,2,3 [18].

Ovarian tissue histomorphology

All studies reported that AST improved ovarian tissue histomorphology by increasing the number or quality of follicles, especially the graafian and corpus luteum follicles, and decreasing the number of cystic, atretic, or necrotic follicles. Some studies also reported that AST improved the GCs organization and reduced the loss of oocytes.