Fig. 1. Genomic localization (exon, intron, intergenic, and noncoding RNAs) and function of the 648 required SREs. Most of the required SREs are located in exons of annotated genes (585 of 648; 90.3%), and the remainder are in intronic regions (12 of 648; 1.9%), intergenic regions (9 of 648; 1.4%), or match to noncoding transcriptional elements including small nuclear RNAs, microRNAs, and long noncoding RNAs (42 of 648; 6.5%) with potential regulatory function. About 40% of the genes that contain one or more SREs have a known role in spermatogenesis, sperm physiology (sperm energy production or acrosome reaction), fertilization, and/or early embryogenesis. Additionally, 20% have a known role in cellular process such as transcription regulation, protein transport, ubiquitin-like conjugation pathway, and lipid metabolism. The potential function of the remaining transcripts has yet to be defined.
Fig. 2. Distribution of the 648 required SREs. The percentile rank distribution of the SREs in control (group I) and test set [group II: (i) exclusively use TIC or IUI, (ii) unsuccessful IUI followed by ART, and (iii) directly use ART] is presented. The percentile rank of each element is indicated in a gradient color scale. Red indicates the complete absence of an element (zero percentile rank). In contrast, elements that are present range from yellow for the 60th abundance percentile to green that corresponds to >90th percentile of abundance. (A) (Left) Seven TIC individuals who were used to identify the SREs because they were considered to have successful natural conception and presented with all 648 SREs. (Middle) Thirty-seven samples from group II (25 group II-i, 5 group II-ii, and 7 group II-iii) with the complete set of SREs. (Right) Nineteen samples from group II (4 group II-i, 8 group II-ii, and 7 group II-iii) with at least one missing SRE. The first 33 sperm elements were absent in at least one sample from the test set (right). The remaining SREs (34 to 648) were present in all samples from groups I and II, showing that the vast majority of SREs are uniformly abundant in all samples surveyed. (B) Couples with all SREs using TIC/IUI or ART have a success rate of 72% (16 of 22) during the first two treatment cycles (6 months) after sperm RNA evaluation.
Fig. 3. Analysis of treatment outcome as a function of required SREs. (A). Most of the 37 group II couples with all SREs present underwent TIC/IUI, achieving an LB rate of 73% (22 of 30). The remaining samples reflecting patient preference along with the previously unsuccessful TIC/IUI cases were treated by ART, achieving a 75% (9 of 12) LB rate. (B) RNA analysis from samples with at least one SRE absent. Note that only 3 of the 11 TIC/IUI samples with an absent SRE achieved LB. The success ratio of LB using ART is similar to the ratio observed in samples with all SREs. (C) The percentage of LB using a noninvasive treatment for couples presenting with the complete set of SREs is higher compared to those with at least one SRE absent (two-tailed Fisher’s exact test, P = 0.012).
- Table 1. Characteristics of the study population.
The table details the distribution and characteristics of the study population in relation to fertility treatment used and procedural outcome [LB (live birth) versus NLB (no live birth)]. Group I subjects achieved an LB pregnancy in their first attempt using TIC during the first spermatogenic cycle after semen assessment (90-day cycle) and were considered as a natural conception. Samples from test set II include different subgroups based on treatment: (i) IUI or TIC delayed past the first 90-day cycle, (ii) ART preceded by unsuccessful IUI or TIC, and (iii) ART. The independent set of samples (set III) was composed of two subgroups: (i) samples from an independent fertility clinic, and (ii) patients who never achieved LB and in whom a female factor was subsequently diagnosed. Sample characteristics include male and female ages and semen parameters comprising total million sperm cells per sample, sperm motility (%), total motile sperm per sample (TMC), sperm morphology [% of normal forms (NFs)], and sperm DNA fragmentation [DNA fragmentation index (DFI)]. (A) The type of fertility treatment used did not correlate with any individual or sperm sample parameter evaluated. (B) When all patients were considered as a group, female age showed a negative correlation with LB (two-tailed t test, P = 0.024). (C) Female age was significantly higher in patients who were unsuccessful when treated by ART (two-tailed t test, P = 0.004) but not in patients who were unsuccessful when treated by TIC/IUI.
A Natural conception Test set Independent set P Group I II III Subgroup i ii iii i ii Male age 35 34.6 33.6 34.2 37 36.2 0.683 Female age 32.3 32 31.1 33.6 35.2 32 0.304 Total million sperm 193.4 172.9 235.8 159 349.9 160.2 0.090 Sperm motility (%) 54 52.3 50 50 39.5 52.5 0.441 TMC 113.9 93.9 125.5 83.8 175.5 86.7 0.342 Sperm morphology (% NF) 10.8 10.6 5.7 9.2 4.5 10.2 0.271 DNA fragmentation (DFI) 14.7 16.4 17.3 19.4 — 18.4 0.734 B Final outcome P LB (n = 62) NLB (n = 10) Male age 34.6 35 0.770 Female age 32 34.9 0.024* Total million sperm 192.4 212.4 0.122 Sperm motility (%) 50.4 52.8 0.583 TMC 103 116.7 0.182 Sperm morphology (% NF) 8.8 10.9 0.342 DNA fragmentation (DFI) 17.5 14.6 0.303 C TIC/IUI P ART P LB (n = 35) NLB (n = 17) LB (n = 27) NLB (n = 6) Female age 32.3 31.5 0.377 31.5 37 0.004* *Statistically significant differences.
- Table 2. Required SREs in the test group of samples (group III).
The 648 RNA elements describing fertile sperm were tested in nine samples. (i) All samples that were obtained from an independent fertility clinic and achieved LB presented with all required SREs. LB was achieved spontaneously or by IUI for samples 4 and 5, whereas the remaining three cases (1 to 3) directly used ART. (ii) Samples with known female factor. A single instance (sample 6) shows the absence of two SREs as well as a known female factor. It is possible that with a GC (gestational carrier), the pregnancy was rescued by ART despite the absence of these two SREs.
Treatment Final
outcomeRequired
SREs absent(i) Samples from independent fertility clinic 1 IVF (LB) LB 0 2 IVF (NLB)/ICSI (LB) LB 0 3 IVF (NLB)/ICSI (LB) LB 0 4 IUI (LB) LB 0 5 Natural conception LB 0 (ii) Samples with known female factor 6 ICSI (LB with GC) LB 2 7 ICSI (NLB)/ICSI (LB with GC) LB 0 8 ICSI (NLB)/ICSI (LB with GC) LB 0 9 TIC (NLB)/IUI (NLB) NLB 0
Supplementary Materials
www.sciencetranslationalmedicine.org/cgi/content/full/7/295/295re6/DC1
Materials and Methods
Fig. S1. Distribution and junctions of RNA-seq reads of a selected required SRE, GPX4.
Fig. S2. No correlation between the number of absent SREs and sperm parameters or partner age.
References (32–43)
Additional Files
- Supplementary Material for:
Absence of sperm RNA elements correlates with idiopathic male infertility
Meritxell Jodar, Edward Sendler, Sergey I. Moskovtsev, Clifford L. Librach, Robert Goodrich, Sonja Swanson, Russ Hauser, Michael P. Diamond, Stephen A. Krawetz*
*Corresponding author. E-mail: steve{at}compbio.med.wayne.edu
Published 8 July 2015, Sci. Transl. Med. 7, 295re6 (2015)
DOI: 10.1126/scitranslmed.aab1287This PDF file includes:
- Materials and Methods
- Fig. S1. Distribution and junctions of RNA-seq reads of a selected required SRE, GPX4.
- Fig. S2. No correlation between the number of absent SREs and sperm parameters or partner age.
- References (32–43)
