Overview of small RNA library sequencing in spermatozoa and sample grouping
To evaluate the role of miRNAs in spermatozoa in the process of embryo development during IVF, miRNA profiles in the spermatozoa of 102 patients who underwent IVF were investigated by small RNA deep sequencing. Quality control (QC) assessment showed that approximately 50% of reads were filtered after QC. Fifty percent of reads after QC could be mapped to the human reference genome (hg19). However, most of the samples had only 5% mappable reads that could be annotated for known miRNAs (Figure 1A). We identified a total of 797 of 2042 known human miRNAs. Hierarchical cluster analysis indicated that the miRNA expression levels were significantly different in 102 samples (Figure 1B). Samples were divided into different groups based on the FR(fertilization rate), EER(effective embryo rate) and HQER(high-quality embryo rate) to detect differentially expressed microRNAs. Based on the FR, we divided the sequencing results into four groups: patients with an FR < 20%, accounting for 9.8% of all patients; patients with an FR between 20%-60%, accounting for 11.76% of all patients; patients with an FR between 60%-80%, accounting for 30.39% of all patients; and patients with an FR > 80%, accounting for 48.04% of all patients (Figure 1C). Based on the EER, we divided the sequencing results into four groups: patients with an EER < 20%, accounting for 25.51% of all patients; patients with an EER between 20%-50%, accounting for 32.65% of all patients; patients with an EER between 50%-80%, accounting for 9.18% of all patients; and patients with an EER > 80%, accounting for 32.65% of all patients (Figure 1D). Based on the HQER, we divided the sequencing results into three groups: patients with an HQER < 10%, accounting for 39.22% of all patients; patients with an HQER between 10%-70%, accounting for 24.51% of all patients; and patients with an HQER > 70%, accounting for 36.27% of all patients (Figure 1E). We demonstrated that most patients have a high FR but not enough embryos. This result may indicate that there is no direct link between fertility and embryo development.
Analysis of differentially expressed miRNAs
A total of 11 miRNAs were identified as differentially expressed, with intergroup differences in the FR (p<0.05): hsa-mir-191-5p, hsa-mir-320a, hsa-mir-320b, hsa-mir-190b, hsa-mir-423-5p, hsa-mir-20a-5p, hsa-mir-548ay-5p, hsa-mir-153, hsa-mir-548d-5p, hsa-mir-1 and hsa-mir-618 (Figure 2A). A total of 10 miRNAs were identified as differentially expressed, with intergroup differences in the EER (p<0.05): hsa-mir-191-5p, hsa-mir-891a, hsa-mir-101-3p, hsa-mir-345-5p, hsa-mir-664a-3p, hsa-mir-19a-3p, hsa-mir-92b-3p, hsa-mir-153, hsa-mir-22-5p and hsa-mir-497-5p (Figure 2B). A total of 13 miRNAs were identified as differentially expressed, with intergroup differences in the HQER (p<0.05): hsa-mir-191-5p, hsa-mir-200b-3p, hsa-mir-891a, hsa-mir-500a-3p, hsa-mir-423-5p, hsa-mir-101-3p, hsa-mir-345-5p, hsa-mir-92b-3p, hsa-mir-140-5p, hsa-mir-548o-3p, hsa-mir-149-5p, hsa-mir-451a and hsa-mir-497-5p (Figure 2C and supplementary table1). By overlapping three sets of differentially expressed miRNAs, we identified only one miRNA, hsa-mir-191-5p, that was differentially expressed in the FR, EER and HQER groups (Figure 2D). This result indicates that hsa-mir-191-5p may be a key factor in both fertility and embryo development.
Comparison of miR-191-5p expression in different FR, EER and HQER groups
We used the independent T test to compare differences in hsa-mir-191-5p expression in the FR, EER and HQER groups. The results showed that the expression of hsa-mir-191-5p in the highest FR group was significantly higher than that in the lowest FR group (p< 0.01) (Figure 3A). The expression of hsa-mir-191-5p was significantly higher in the highest EER group (p<0.05) (Figure 3B), but there was no significant difference between the other EER groups. In the highest HQER group, the expression of miR-191-5p was significantly higher than that of the lowest HQER group (p<0.01) (Figure 3C), but there was no significant difference between the other HQER groups. This finding suggests that hsa-mir-191-5p expression in patients with a high FR, EER and HQER was higher than that in those with a low FR, EER and HQER following IVF. To further clarify the function of hsa-mir-191-5p during fertilization and embryonic development, we conducted receiver operating characteristic (ROC) curve analysis. The results showed that the area under the ROC curve (AUC) for the FR, EER and HQER groups predicted by hsa-mir-191-5p was 0.612, 0.637 and 0.686, respectively, which indicates that miR-191-5p could be used to predict whether the patients belonged to the highest FR, EER and HQER groups, especially in the high HQER group, with an AUC close to 0.7 (Figure 3D, E, F). In addition, the results further indicated that high hsa-mir-191-5p expression could lead to improved embryo quality, although its low expression did not indicate that the IVF results would be poor.
Analysis of correlations between hsa-mir-191-5p and routine sperm parameters
We investigated the correlation between hsa-mir-191-5p and 3 sperm routine parameters: sperm density, sperm morphology and sperm viability. The results showed that the correlation between hsa-mir-191-5p and sperm density as well as sperm viability was not significant (Figure 4A and C, p> 0.05), while the correlation between hsa-mir-191-5p and sperm morphology was significant (Figure 4B, p< 0.01), but the correlation coefficient was only 0.29, indicating a weak correlation between the two. As shown above, hsa-mir-191-5p may be one of the key molecules involved in maintaining normal sperm morphology.
Overview evaluation of the function of sperm-carrying hsa-mir-34c in the early development of human embryos
The function of sperm-carrying mmu-mir-34c in early embryonic development has been discussed in mice, but its function in the early development of human embryos has not been studied. In this study, our results showed no significant difference in hsa-mir-34c between different FR, EER and HQER groups (Figure 5A). We also investigated the correlation between hsa-mir-34c and three routine sperm parameters, namely, sperm density, sperm morphology and sperm viability (Figure 5B). The results showed that hsa-mir-34c had a weak negative linear relationship with sperm morphology (p<0.05) but was not linearly related to sperm density or sperm viability(table 1). This evidence does not indicate that sperm-carrying hsa-mir-34c plays an important role in the early development of human embryos. However, hsa-mir-34c may have a certain function during the normal development of sperm morphology.