Sperm concentration in mail-in semen consistently reflects results obtained on freshly collected semens

Objectives

Semen analysis on samples produced by men in the clinic and assessed on the same day is the standard for assessment of male reproduction function. In clinical and investigational scenarios when sperm motility/morphology examination is not required (e.g., monitoring agents that target spermatogenesis in hypogonadotropic hypogonadism, male contraceptive trials, or environmental effects on sperm production) (1, 2), sperm concentration may be assessed in some countries by at-home test kits. Prior studies using mail-in semen samples with preservatives when analyzed up to 8 days showed no decrease in sperm concentration, but sperm motility and normal morphology were decreased (3, 4). We hypothesized that sperm stored in seminal fluid would allow accurate assessment of sperm concentration in mail-in samples. Our objective was to determine whether sperm concentrations in preservative-free semen samples stored in the laboratory, and in mail-in samples by postal delivery remained consistent with assessment on collection day. All samples were assessed in the same laboratory.

Study Design

We utilized deidentified residual semen samples from IRB approved clinical studies, where participants allowed use of residual semen samples for laboratory-based research. After completing the required semen analysis on collection day using methods recommended by the World Health Organization Semen Manual (5), for the initial experiments, we stored residual samples at 4°C (n=57) or at room temperature (n=10) for 5 days. We compared sperm concentration determined using hemocytometer by three technologists on day of collection (day 0), with sperm concentration assessed every day for five days after ejaculation. For the second set of experiments, after completing semen analysis on collection day in 114 samples from 114 men, we transferred the residual semen sample to a 30 ml self-standing conical tube with no preservatives, packed in USPS Padded Envelope and mailed the samples by priority mail from postboxes at different locations in Los Angeles to our laboratory. On the day of receipt of mail-in samples (ranging from 2 to 14 days after mailing), we determined the sperm concentration.

Results

We showed sperm concentration assessed at day 0 (day of semen collection) exhibited high positive correlation (R²≥ 0.9) with those examined at day 1 (R²=0.99), day 2 (R²=0.97), day 3 (R²=0.95), day 4 (R²=1.0) and day 5 (R²=0.97) stored at 4°C in the laboratory, and samples examined at day 5 stored at room temperature (R²=1.0 ) after ejaculation (Supplemental Fig 1a and b).

The mail-in samples demonstrated high correlation (R²=0.90) in all 114 samples for sperm concentration between day of collection and receipt of mail-in samples (Fig 1) with or without adjustment for days of abstinence (Supplemental Table 1). Sperm concentration assessed on collection day showed high correlation with samples received within 6 days (n=80) of collection (Day <2 (R²=0.99), 3 (R²=0.93), 4 (R²=0.91), 5 (R²=0.97), 6 (R²=0.96), the correlation was lower on Day 7 (R²=0.83), and even lower after >7 days (R²=0.64) (Supplemental Fig. 2). Bland-Altman analysis showed sperm concentrations determined in mail-in samples were within 2SDs of the mean of samples assessed on collection day except for five samples (sperm concentration on day 0 between 49.5 to 170.5 ×10⁶/ml); four of which were received ≥ 6 days after the collection date. Large aggregates in 38 samples affected the correlation with initial analyses (R²=0.84) whereas little or no aggregates in 76 samples yielded high correlation (R²=0.94). Aggregation was independent of receipt day (supplemental Fig 3 and 4).

Figure 1.

Regression analysis showed sperm concentration assessed in mail-in samples and determination on collection day exhibited high positive correlation (R²=0.90) for all 114 samples.

Conclusion

We conclude that sperm concentration in semen samples collected and stored in the laboratory and those collected at home and mailed to the laboratory within 5 or 6 days could serve as an alternative assessment when laboratories are not open, the male cannot attend clinic or unable to produce samples in the clinic. Mail-in samples could replace the in-person sample collection and assessment on the same day at a laboratory, reducing the burden for subfertile patients and participants of contraceptive trials and epidemiological studies when sperm concentration assessment is the key parameter.

Figure 2.

Bland and Altman Plot showed that all samples (n=114) assessed on the same day or after mail-in were within 2SDs of mean, except for five samples with sperm concentrations between 49.5 to 170.5 ×10⁶/ml on day of collection (Day 0).