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Spermatid specific thioredoxin-3 (SPTRX3 or TXNDC8) is a testis/male germ line specific member of thioredoxin family that accumulates in the superfluous cytoplasm of defective human spermatozoa. We hypothesized that semen levels of SPTRX3 are reflective of treatment outcome in assisted reproductive therapy (ART) couples treated by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Relationship between SPTRX3 and treatment outcome was investigated in 239 couples undergoing ART at an infertility clinic. Sperm content of SPTRX3 was evaluated by flow cytometry and epifluorescence microscopy, and correlated with clinical semen analysis parameters, and data on embryo development and pregnancy establishment. High SPTRX3 levels (>15% SPTRX3-positive spermatozoa) were found in 51% of male infertility patients (n = 72), in 20% of men from couples with unexplained, idiopathic infertility (n = 61) and in 14% of men from couples previously diagnosed with female-only infertility (n = 85). Couples with high SPTRX3 produced fewer two-pronuclear zygotes and had a reduced pregnancy rate (19.2% pregnant with >15% SPTRX3-positive spermatozoa vs. 41.2% pregnant with <5% SPTRX3-positive sperm; one-sided p<0.05). The average pregnancy rate of all 239 couples was 25.1%. Live birth rate was 19.2% and lowest average SPTRX3 levels were found in couples that delivered twins. Men with >15% of SPTRX3-positive spermatozoa, a cutoff value established by ROC analysis, had their chance of fathering children by IVF or ICSI reduced by nearly two-thirds. The percentage of SPTRX3-positive spermatozoa had predictive value for pregnancy after ART. Gradient purification and sperm swim-up failed to remove all SPTRX3-positive spermatozoa from semen prepared for ART. In summary, the elevated semen content of SPTRX3 in men from ART couples coincided with reduced incidence of pregnancy by IVF or ICSI, identifying SPTRX3 as a candidate biomarker reflective of ART outcome.

The purpose of this study was to investigate the relationship between fertility and quantitative measures of boar semen quality, including various patterns of sperm cytoplasmic droplet (CD) retention, as determined by high power differential interference contrast (DIC) microscopy. A total of 116 ejaculates were collected from a nucleus herd of 18 Large White boars over an eight month period. Semen quality parameters were analyzed for each ejaculate by calculating the percentage of normal spermatozoa, spermatozoa possessing a CD in the proximal, distal, or distal midpiece reflex position, total spermatozoa with an attached cytoplasmic droplet, spermatozoa with non-CD related aberrations and total spermatozoa with abnormalities. Of the 116 ejaculates received, 71 ejaculates from 13 boars had corresponding fertility data from single-sire inseminations of multiparous sows. The fertility data included farrowing rate (FR) and total number born (TNB). The monthly FR encompassed one month before and one month after the date of semen collection. Detection of differences for fertility and semen quality parameters was performed by separating the boars into either an above-average or below-average group based on the mean FR (74.01 +/- 1.43%) or TNB (12.34 +/- 0.17) for the study. For FR, the boars in the below-average group had a significantly lower percentage of normal spermatozoa and significantly higher percentage of spermatozoa possessing distal CDs, total attached CDs and total abnormalities compared to the boars in the above-average group. Conversely, for TNB there were no significant differences between the above- and below-average groups for the semen quality parameters. These data suggest that the attached CD may negatively affect FR, but not TNB. The detection of relationships between the boar fertility parameters and the retention of the sperm CD after ejaculation, document the advantage of high power DIC microscopy in conventional semen evaluation.

High content of the platelet activating factor (PAF) and its plasma membrane receptor (PAFr) in semen is thought to benefit fertility in farm animals and humans. We used flow cytometric, biochemical, and immunocytochemical analysis to examine PAFr levels alone (Trial 1, n = 156 bulls) or in a dual assay with sperm defect marker ubiquitin (UBI; Trial 2, n = 88 bulls), in semen samples from 160 yearling bulls undergoing Breeding Soundness Evaluations (BSE). In both trials, we observed increased PAFr levels in semen samples with high content of white blood cells (WBC). Consequently, PAFr levels within such semen samples correlated negatively with several subjective parameters of BSE, including palpation, satisfaction of evaluation, and scrotal circumference. Due to a high WBC content, increased semen sample dilution had to be applied for microscopic evaluation. There was a negative correlation between semen PAFr and conventional sperm morphology, while the increased levels of PAFr correlated positively with sperm UBI content. Immunofluorescence microcopy revealed high expression of PAFr on the surface of leukocytes and morphologically normal spermatozoa, while reduced immunoreactivity was observed in defective spermatozoa immunoreactive to anti-UBI antibodies. A single PAFr band of appropriate mass was observed in Western blots of ejaculated spermatozoa, while testicular and epididymal spermatozoa also displayed several larger bands indicative of posttranslational processing or modification. Collectively, these data suggest that high levels of semen PAFr in young bulls are indicative of semen contamination with WBC. In the future, objective protein marker-based semen analyses in young bulls will likely require additional parameters distinguishing between marker expression in the spermatozoa and in the contaminating WBC. While identification of high sperm PAFr levels may support fertility, this assay alone is not reliable, due to the expression of PAFr in WBC that contaminate semen samples.

Background: Ubiquitin, a house-keeping protein that marks other proteins for proteasomal degradation, tags defective sperm during epididymal passage. To establish ubiquitin as a biomarker of human infertility, the present study examines the relationships between sperm ubiquitin content and clinical semen parameters among men from an infertility clinic population with varied aetiologies., Methods: Anti-ubiquitin immunoreactivity was measured by flow cytometric sperm-ubiquitin tag immunoassay (SUTI) in sperm samples of 28 infertility patients and 15 fertile donors. Semen analyses were performed by computer-assisted semen analysis and World Health Organization morphology., Results: Median values of ubiquitin-induced fluorescence had a strong negative correlation with sperm count (r = -0.63, P = 0.0003) and a positive correlation with % abnormal morphology (r = 0.55, P = 0.01). Infertility patients (n = 28) had significantly higher levels of sperm ubiquitin. Out of 28 patients, six reported possible occupational exposures to solvents, three were current smokers and six were ex-smokers. Within the patient group, men with known male factor infertility, those with self-reported occupational exposure to solvents and current smokers had the highest sperm ubiquitin levels. When men with jobs involving potential occupational exposure to solvents were combined with current smokers, the highest correlations were found between sperm ubiquitin and motility (r = -0.74), count (r = -0.82) and % sperm abnormalities (r = 0.73)., Conclusions: Increased sperm ubiquitin was inversely associated with sperm count, motility and % normal morphology, supporting the use of ubiquitin as a biomarker of human semen quality. SUTI assay confirmed poor semen quality in all men with poor clinical semen parameters, but also was high in some patients with seemingly good clinical semen parameters. Occupational exposure to solvents and smoking may have contributed to high levels of sperm ubiquitin in some of these patients.