Determination of enterotypes according to the P/B ratio
Gut microbiota analysis was performed on the stool samples of all participants (n=407). The frequency plot of the relative abundance of log (P/B) (Figure 1) demonstrated a bimodal distribution of separation between the two groups. These results suggested that the participants could be classified into two groups: enterotype P (P/B ≥0.01) and enterotype B (P/B <0.01).
The baseline characteristics, sperm quality parameters, and sex hormone data of 407 patients enrolled in this study are listed in Table 1. Patients were divided into normal (BMI<24 kg/m2) group and overweight/obese (BMI≥24 kg/m2) group according to their BMI. The baseline characteristics, sperm quality parameters, and sex hormone data of the two groups are shown in Table 2. The levels of rate of forward progressive sperm motility, rate of total sperm motility, and TT were different between the two groups. In the normal BMI group, there were more patients with normal rate of forward progressive sperm motility and total sperm motility, whereas in the overweight/obese group, there were more patients with low rate of forward progressive sperm motility and total sperm motility, and the difference was statistically significant. However, there were no statistically significant differences in total sperm count and sperm concentration between the two groups.
Logistic regression analysis of risk factors for asthenospermia
As shown in Table 3, the incidence of asthenospermia in the overweight/obese group was significantly higher than that in the normal group (P=0.004). Univariate logistic regression showed that decreased rate of forward progressive sperm motility (OR 1.849; 95% confidence interval [CI] 1.218–2.807; P = 0.004) and decreased rate of total sperm motility (OR 2.406; 95% CI 1.346–3.111; P = 0.001) were significantly associated with obesity, as evident from the data of the overweight/obese group. Second, multivariate logistic regression analysis confirmed that obesity (OR 1.793; 95% CI 1.177–2.729; P = 0.006) was a risk factor for the development of asthenospermia after adjusting for FSH (Table 3). FSH was identified as a protective factor for asthenospermia.
Obesity is a risk factor for asthenospermia under enterotype P
As stated, obesity is significantly associated with decreased sperm quality. Considering that enterotypes modulate the risk of disease, we evaluated the effect of BMI on sperm quality in patients with enterotypes P and B.
First, rate of forward progressive sperm motility (Figure 2A), total sperm motility (Figure 2B), sperm concentration (Figure 2C) and total sperm count (Figure 2D) decreased with increasing BMI in patients with enterotype P but not significantly in patients with enterotype B (Figure 2E, F, G and H). Specifically, as shown in Table 4, in men with enterotype P, obesity was a risk factor for decreased rate of forward progressive sperm motility (OR 3.350; 95% CI 1.881–5.966; P<0.001) and decreased rate of total sperm motility (OR 4.298; 95% CI 2.365–7.809; P<0.001). On the contrary, in patients with enterotype B, no significant difference between obesity and sperm motility was found (Supplementary Table 2). Second, in enterotype P, obesity was still an independent risk factor (OR 3.131; 95% CI 1.749–5.607; P<0.001) after adjusting for FSH (Table 4).
Prediction Performance of enterotypes for categorizing asthenospermia
As mentioned above, in the enterotype P group, the increase in BMI was strongly associated with decreased sperm motility. Thus, using ROC curves, we further confirmed the correlation between BMI and asthenospermia in patients with enterotype P. In enterotype P, BMI resulted in higher area under the curve (AUC) of 62.7% and moderate sensitivity (78.8%) and highest specificity (49.5%) for decreased rate of forward progressive sperm motility (Figure 3A). Similarly, the BMI levels also resulted in the higher AUC of 67.5%, with sensitivity (81.1%) and highest specificity (52.3%) for decreased rate of total motility in men with enterotype P (Figure 3B). In contrast, BMI showed poor prediction performance for asthenospermia in men with enterotype B (Figure 3A and B).