File S1 - Semen Levels of Spermatid-Specific Thioredoxin-3 Correlate with Pregnancy Rates in ART Couples

Supplemental figures and tables. Figure S1. Western blotting of SPTRX3 in semen of three ART patients and three presumed fertile donors (semen parameters are shown in Table S10). Relative levels of SPTRX3 (top panel) and beta-tubulin (loading control; middle panel) are shown for three presumably fertile sperm bank donors with acceptable clinical semen parameters (lanes 1-3) and three teratozoospermic infertile men (lanes 4-6) suffering from dysplasia of the fibrous sheath. Diagram of SPTRX3/tubulin band density ratios is shown on the bottom. Note the high density of SPTRX3 band in donor #1, who was presumed fertile. Figure S2. Smoking has a modest effect on sperm SPTRX3 levels. Heavy smokers consuming 15-30 cigarettes per day had higher average levels of sperm SPTRX3 than other groups shown. Of note, two thirds (10/15) of these heavy smokers had indication/clinical diagnosis of male infertility. The numeric difference between smokers consuming 2-5 cigarettes per day and smokers consuming 15-30 cigarettes per day, was not statistically significant (p>0.1). Figure S3. Scatter diagram illustrating the relationship between subjective, light microscopic assessment of sperm SPTRX3 content (% spermatozoa with SPTRX3-positive heads; x-axis) and flow cytometry (%M3 SPTRX-value; y-axis). This simple light microscopic analysis was conducted as a potential precursor of a test suitable for clinical andrology laboratories. Samples from 150 randomly chosen donors, processed for flow cytometry, were re-evaluated by epifluorescence microscopy for the percentage of SPTRX3 positive sperm heads, sperm tails, and total % of SPTRX3 positive spermatozoa. Correlations were found between all three categories of light microscopic evaluation and % of SPTRX3-positive spermatozoa as measured by flow cytometry (%M3). The highest correlation coefficient (r = 0.46) was between % SPTRX3-positive sperm heads by light microscopy and %M3 SPTRX3 by flow cytometry (see Table S4B). Table S1. Receiver Operator Characteristic (ROC) analysis. As anticipated, Specificity increases at the expense of Sensitivity as the cutoff values increase. The area under the ROC curve is 0.74, which shows that the %M3 performs well for predicting the male infertility. Table S2. Semen SPTRX3 levels and live births. Pregnancy outcomes were available for 57/60 pregnant couples. One of those 57 pregnancies was an ectopic pregnancy in a case of female-only infertility; it is not included in the above table. Live birth was achieved in 81% (46/57) of those analyzed pregnant couples. One of those three male factor patients had a vasectomy. Table S3. Parameters of zygotic development in 239 couples divided into subgroups based on percentages of SPTRX3-positive spermatozoa (A) or clinical indication (B). SPTRX3 values, but not the clinical indication, were predictive of good zygotic development after IVF or ICSI. Couples with lowest SPTRX3 levels (A, top row, <5% M3) produced the highest percentage of normal, two-pronuclear (2 PN) zygotes out of all fertilized oocytes (A, column 8), and also when calculated based on all oocytes harvested (A, column 9). These couples also produced the most embryos suitable for transfer or cryopreservation (column 13). Note that couples with low or medium levels of SPTRX3 produced more 2PN zygotes per couple on average than couples with >15% SPTRX3 (A, column 10). Idiopathic couples in which men recorded more than 15% SPTRX3-positive spermatozoa had the lowest yields of two-pronuclear zygotes. Numbers shown in red are highest & lowest values for each column. No significant correlations were found between parameters of early embryo development and flow cytometric SPTRX3 levels. Table S4. Relationship between the clinical treatment assignment and various sperm quality parameters. Table S5. A: Pregnancy rates in 150 infertile couples, divided by percentages of SPTRX3-positive spermatozoa. In accordance with flow cytometric SPTRX3-data, couples with lowest levels of SPTRX3 had highest pregnancy rate (column 2). Also similar to flow cytometric results, the percentage of couples treated by ICSI increased progressively with sperm SPTRX3 content (column 6). Spermatozoa were evaluated for the presence of SPTRX3 in the sperm head (B) or tail (C; combined head and tail labeling has not been observed), adding up to third category of total SPTRX3-positive spermatozoa (A). Numbers shown in red are highest & lowest values for each column. With the exception of one SPTRX3 parameter (A-% of all positive spermatozoa), pregnancy rates (column #2) in light microscopic analysis were dose-dependent on SPTRX3 values. B: Correlations between light microscopic SPTRX3-evaluation (STIX assay) and % of SPTRX3-positive spermatozoa as measured by flow cytometry. Bold font indicates highest correlation coefficients for flow cytometric and light microscopic parameters. Table S6. Sperm SPTRX3 levels and oocyte quality parameters in 238 couples divided by female partners’ age, with the threshold of 35 years of age. Female age subgroups (in rows) were further divided based on male partners’ semen content of SPTRX3-positive spermatozoa (Column 4), and based on the treatment (IVF or ICSI, columns 6 & 7). Regardless of female partner’s age, the combined IVF & ICSI pregnancy rates (column 9) as well as the ICSI pregnancy rates (column 10) were numerically higher when the male partner had <10% SPTRX3-positive spermatozoa. Table S7. Analysis of subgroups divided by indication (male or female infertility) and female age. Combined and idiopathic cases were not included in analysis. Table S8. Correlations between flow cytometric SPTRX3 values and clinical semen parameters. R-values by Person’s correlation analysis are shown. Most significant values are printed bold. In general, SPTRX3 values correlate negatively with sperm count, motility, and percentage of morphologically normal spermatozoa in samples, assessed by conventional light microscopic semen evaluation. Ratio of spermatozoa to debris (%TOTAL) correlates positively with sperm count, motility and normal morphology. Bold font indicates highest correlation coefficients. Table S9. Spearman correlations between the most informative flow cytometric SPTRX3 parameters and the conventional semen parameters (the second numbers in the cells are the p-values). Table S10. Conventional semen parameters for semen samples used for Western blotting. Table S11. Discriminant analysis using SAS PROC discrim and stepdisc procedures applied to study the relationship of pregnancy rate with the sperm quality parameters and SPTRX3 levels, with consideration of treatment assignments. Table S12. Structure matrix of the discriminant analysis conducted to study the relationship of the treatment assignment, SPTRX3 and the sperm quality parameters. Table S13. Variables and treatment groups in the discriminant analysis conducted to study the relationship of the treatment assignment, SPTRX3 and the sperm quality parameters. Table S14. Canonical structure matrix of the discriminant analysis using SAS proc discrim procedure, conducted to explore the relationships between the clinically diagnosed male infertility and sperm quality parameters. Table S15. Means of the nine variables shown separately for infertile and fertile male in the discriminant analysis (SAS proc discrim) conducted to explore the relationships between the clinically diagnosed male infertility and sperm quality parameters.
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