Laura M Keller

Electroejaculation versus vibratory stimulation in spinal cord injured men: sperm quality and patient preference.

PURPOSE We compared semen quality and patient preference between penile vibratory stimulation and electroejaculation in spinal cord injured men. MATERIALS AND METHODS We treated 11 spinal cord injured men with penile vibratory stimulation and electroejaculation in random order. End points examined were semen analysis, sperm functional assessment, and patient pain scores (1 to 10) and preferred procedure. Differences between the procedures were determined with the paired Student t test. RESULTS There was no difference in antegrade sperm count but penile vibratory stimulation specimens had greater motility (26.0 versus 10.7%), viability (25.2 versus 9.7%) and motile sperm count (185.0 x 10(6) versus 97.0 x 10(6)). The retrograde sperm count was greater (but not significant) in electroejaculation patients. The total (antegrade plus retrograde) and motile sperm counts were not different. There was no difference in immunobead test (all negative), cervical mucus penetration or sperm penetration assay, although the percent hamster egg penetration approached significance (53.7% for penile vibratory stimulation versus 22.1% for electroejaculation, p = 0.06). There was no difference in the peak blood pressures and no complications were noted. Pain scores were significantly greater for electroejaculation compared to penile vibratory stimulation (5.2 versus 1.7, respectively). All patients preferred penile vibratory stimulation. CONCLUSIONS There was a slight advantage in sperm quality and a high patient preference in favor of penile vibratory stimulation. Penile vibratory stimulation should be attempted first to induce ejaculation in spinal cord injured men, with electroejaculation reserved for failures.

Isolation of motile spermatozoa from semen samples using microfluidics

A microfluidic device was designed with two parallel laminar flow channels where non-motile spermatozoa and debris would flow along their initial streamlines and exit one outlet, whereas motile spermatozoa had an opportunity to swim into a parallel stream and exit a separate outlet. Motile sperm samples were prepared with density gradient separation (n = 5). Sperm motility was assessed the following day after exposing aliquots to polydimethylsiloxane (PDMS) used to construct the device. There was no difference in sperm motility when compared with unexposed aliquots (P > 0.05). Unprocessed semen samples (n = 10) were placed in wider channels and sperm motility and strict morphology were assessed from sorted outlets. Sperm motility increased from 44 +/- 4.5% to 98 +/- 0.4% (P < 0.05) and morphology increased from 10 +/- 1.05% to 22 +/- 3.3% (P < 0.05) following processing. Finally, density gradient prepared samples (n = 6) containing 5 x 10(6) motile spermatozoa/ml and 50 x 10(6) round immature germ cells/ml were sorted and assessed in a similar fashion. The ratio of motile spermatozoa to round immature germ cells in the wide inlet (1:10) was significantly improved in the thin outlet (33:1) (P < 0.05). This microfluidic device provides a novel method for isolating motile, morphologically normal spermatozoa from semen samples without centrifugation. This technology may prove useful in isolating motile spermatozoa from oligozoospermic samples, even with high amounts of non-motile gamete and/or non-gamete cell contamination. A movie sequence showing streaming and sorting of spermatozoa may be purchased for viewing on the internet at www.rbmonline.com/Article/847 (free to web subscribers).

Functional characteristics of sperm obtained by electroejaculation

Sperm obtained by electroejaculation in 32 anejaculatory men were examined for functional characteristics. Raw specimens showed high sperm counts but motility averaged only 11%. Average viability was 10% for antegrade and 5% for retrograde fractions. Bovine cervical mucus penetration was normal (30 mm. or more in 30 minutes) in only 24% of the electroejaculation samples but it was normal in all of the donor samples tested. Processed sperm motility averaged 30% with 71% forward progression. At 20 hours patient samples retained 46% of the original motility, while donor controls retained 81%. In the hamster egg penetration assay patient sperm penetrated 14% of the oocytes while donor sperm penetrated 40%. Therefore, we identified 4 characteristics of sperm obtained by electroejaculation: 1) low viability, 2) poor survival after overnight incubation, 3) moderately impaired cervical mucus penetration and 4) moderately poor fertilizing capability as measured by the hamster egg penetration assay. Poor sperm survival and impaired function may explain the low pregnancy rates from insemination with electroejaculated sperm.

Temporal Decreases in Sperm Motility: Which Patients Should Have Motility Checked at Both 1 and 2 Hours After Collection?

A decrease in sperm motility, and thus total motile sperm count (TMSC), over a period of hours might have clinical implications in counseling couples considering intrauterine insemination (IUI), in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI). The objective of this study was to identify patients with decreases in sperm motility from 1 to 2 hours after collection and examine predictive relationships with semen analysis parameters. Between 2001 and 2005, 2313 semen samples were analyzed. Sperm motility was evaluated at both 1 and 2 hours after time of collection. Relevant seminal parameters were compared between patients, with a decrease in 1-hour to 2-hour motility (n = 384) compared with those that showed no change (n = 1929). The same analysis was performed in a subset of patients with a TMSC between 10 and 40 million. In the total patient population, only 16% (384/2313) demonstrated a decrease in 1-hour to 2-hour motility. In patients displaying a decrease in the 1-2-hour motility, sperm concentration (33.5 vs 79 million/mL, P < .0001) and percent normal morphology (7% vs 8%, P < .0001) were significantly lower. Additionally, a significantly higher incidence of 1-2-hour motility decrease was seen in patients with midpiece anomalies (33.3% vs 15.9%, P = .01). Within the subpopulation of 10-40 million TMSC, the only statistically significant difference was in patients with midpiece anomalies (80.0% vs 28.2%, P = .02) who demonstrated a higher incidence of the 1-2-hour motility decrease. Overall, patients with a TMSC between 10 and 40 million showed a significantly higher incidence of 1-2-hour motility decrease compared with the rest of the patient population (29.0% vs 14.6%, P < .0001). Because decreases in 1-2-hour sperm motility affect only a small portion of patients, it is not necessary to check 2-hour motility on all patients. However, because patients with a TMSC between 10 and 40 million were significantly more likely to show a decrease in sperm motility-a decrease that could have possible clinical implications in couples deciding between IUI, IVF, or ICSI--checking 2-hour sperm motility should be considered in this population.

Optimizing human semen cryopreservation by reducing test vial volume and repetitive test vial sampling

OBJECTIVE To investigate optimal test vial (TV) volume, utility and reliability of TVs, intermediate temperature exposure (-88°C to -93°C) before cryostorage, cryostorage in nitrogen vapor (VN2) and liquid nitrogen (LN2), and long-term stability of VN2 cryostorage of human semen. DESIGN Prospective clinical laboratory study. SETTING University assisted reproductive technology (ART) laboratory. PATIENT(S) A total of 594 patients undergoing semen analysis and cryopreservation. INTERVENTION(S) Semen analysis, cryopreservation with different intermediate steps and in different volumes (50-1,000 μL), and long-term storage in LN2 or VN2. MAIN OUTCOME MEASURE(S) Optimal TV volume, prediction of cryosurvival (CS) in ART procedure vials (ARTVs) with pre-freeze semen parameters and TV CS, post-thaw motility after two- or three-step semen cryopreservation and cryostorage in VN2 and LN2. RESULT(S) Test vial volume of 50 μL yielded lower CS than other volumes tested. Cryosurvival of 100 μL was similar to that of larger volumes tested. An intermediate temperature exposure (-88°C to -93°C for 20 minutes) during cryopreservation did not affect post-thaw motility. Cryosurvival of TVs and ARTVs from the same ejaculate were similar. Cryosurvival of the first TV in a series of cryopreserved ejaculates was similar to and correlated with that of TVs from different ejaculates within the same patient. Cryosurvival of the first TV was correlated with subsequent ARTVs. Long-term cryostorage in VN2 did not affect CS. CONCLUSION(S) This study provides experimental evidence for use of a single 100 μL TV per patient to predict CS when freezing multiple ejaculates over a short period of time (<10 days). Additionally, semen cryostorage in VN2 provides a stable and safe environment over time.

Microfluidics for Sperm Selection

Traditional methods for sperm selection such as swim-up and isopycnic (density gradient) centrifugation involve prolonged periods of incubation and/or serial centrifugations. Prolonged incubation of sperm with other cellular components of semen and serial centrifugation expose motile sperm to reactive oxygen species. The exposure affects the sperm chromatin with deleterious effects on embryo developmental potential and pregnancy rates. The use of microfluidics in human assisted reproduction for sperm selection has recently been proposed. Sperm selection with microfluidic technologies is an alternative which circumvents the long periods of incubation associated with standard sperm selection techniques. Fabrication of microfluidic devices with soft lithography to mold polydimethylsiloxane (PDMS) allows easy production of self-powered microfluidic devices. Microfluidic devices for selection of motile sperm are easy to operate and effective in selecting high quality sperm; however, clinical trials to determine their impacts on clinical outcomes are still required. Microfluidic devices can be combined with other modes of sperm selection such as chemotaxis. Microfluidic technologies applied to assisted reproduction is an emerging field and preliminary results are promising. This chapter describes sperm selection with microfluidic devices and how they can be integrated with chemotaxis.

Prospective assessment of follicular growth and the oocyte cohort after ovarian stimulation for fertility preservation in 90 cancer patients versus 180 matched controls

A lower number of metaphase II oocytes eligible for vitrification after controlled stimulation in cancer patients has recently been reported, suggesting that cancer may impair the dynamics and quality of follicular growth. In this prospective, non-interventional study, the pattern of follicular growth and oocyte cohort after ovarian stimulation in cancer patients was analysed. Ninety cancer patients, recruited before starting chemotherapy, were compared with 180 time- and age-matched healthy controls undergoing intracytoplasmic sperm injection. Primary outcome was total number of metaphase II oocytes and metaphase II /total oocytes rate. Basal anti-Müllerian hormone levels (P < 0.05) and antral follicle count (P < 0.0001) were significantly lower in cancer patients. Recombinant FSH total dose was significantly higher in the cancer group (P < 0.0001). No differences were found in duration of stimulation, mean number of mature follicles on day of ovulation induction and total oocyte number after retrieval; the number of metaphase II oocytes retrieved (6.2 ± 4.7 versus 8.8 ± 4.2; P < 0.0001) and number of metaphase II oocytes-total oocytes ratio were significantly lower in cancer patients (56% versus 78%, P < 0.0001). Fewer metaphase II oocytes were eligible for vitrification and lower maturation rate in the cancer group.