A retrospective analysis was performed on a cohort of 19 men with SCI who underwent sperm cryopreservation in the CECOS (Centre d’Etudes de la Conservation des OEufs et du Sperme: Egg, Sperm Embryo banking) of Dijon’s Hospital from 1995 to 2011. Spermatozoa were retrieved using either penile vibration stimulation (PVS, n=9), surgical sperm extraction (SSR, n=9) or after sexual intercourse (SI, n=1). Two groups were defined, the antegrade ejaculation group (PVS and SI, n=10), the SSR group (testicular sperm extraction: TESE and microsurgical epididymal sperm aspiration: MESA). From both groups, a total of 8 couples seeking infertility treatment underwent ICSI procedure (Intra Cytoplasmic Sperm Injection). Female partners in both groups underwent a basic infertility evaluation by a gynaecologist.
The 11 patients who did not undergo ART (6 and 5 in the antegrade and surgical groups respectively) were contacted by telephone. Nine of these patients were single at the time of the accident and 2 were married with children. Two patients out of 11 were lost to follow-up. The patients were asked the following questions.
For the singles at the time of the accident: are you still single? If not, are you planning on having a baby? Does having frozen sperm make you feel more secure about your fertility and your future parenthood?
For the patients married at the time of the accident: are you still married? Did you have another baby or are you planning on having another baby?
Sperm retrieval methods
For one of the patients, sperm was retrieved and frozen after sexual intercourse using non-spermicidal condoms (Male factorPak, Inhealth, Dublin, Ireland). Sperm was collected and allowed to liquefy for 30 min at room temperature (RT). Then, sperm parameters were determined and the freezing performed (see procedures below).
Penile vibratory stimulation
PVS has been recommended as the first line of treatment for anejaculation in men with SCI. Therefore, PVS (Ferticare®, Multicept, Albertslund, Denmark) was performed on all patients except for the patient who could ejaculate via sexual intercourse. PVS was performed by placing the vibrating device on the dorsum or frenulum on the glans penis until antegrade ejaculation occurred with a maximum of 10 min. The amplitude and the frequency used were 2.5 mm and 100 Hz respectively .
For 9 patients, semen samples after PVS were collected. Four out of the 9 patients underwent either 2 or 3 semen retrievals.
Surgical sperm retrieval
In case of PVS failure, no retrograde ejaculation was looked for and TESE and MESA were offered as an alternative (n=9). The decision on performing MESA was taken by the urologist the day the surgery was performed and based upon the aspect of the epididymis (dilated or not). If MESA was performed, it was always combined with a testis biopsy in order to extract sperm (TESE) but also to look for Intratubular Germ Cell Neoplasia (histopathological analysis of the excised tissue). If MESA was not performed, only a testicular biopsy was undertaken.
For MESA, spermatozoa were aspirated from the caput epididymis and the fluid was examined under a light microscope to check for the presence of motile spermatozoa. Two patients underwent MESA and TESE and the remaining only TESE.
For TESE, two testicular specimens approximately 0.5 cm long and 5 mm thick were excised from two different locations on the same testis and minced using sterile needles in a dish containing 2 ml of culture medium (Ferticult flushing, Fertipro, Belgium). The minced tissue was incubated at 37°C under 5% CO2 for 30 min before checking for motile spermatozoa under an inverted microscope and spermatozoa were frozen.
Immediately after liquefaction (30 min at 37°C), semen parameters were determined in accordance with the guidelines of the World Health Organization . Briefly, concentration, progressive motility, total motility, viability and morphology were assessed. The morphology was determined using David’s classification, the French method,  and the viability was measured in accordance with WHO guidelines  using the eosin-nigrosin test  and as described previously by Bechoua et al. .
In the antegrade group, the parameters analyzed after thawing were progressive motility (PM: a+b), total motility (TM: a+b+c) and viability.
For the patient who underwent SSR, we determined only if motile spermatozoa were present after thawing (a, b or c). Therefore, because of a low sperm count, the number of motile spermatozoa was determined on several 10 μl drops of the sperm suspension covered with mineral oil.
Sperm cryopreservation was performed as an option for fertility preservation. Sperm characteristics were evaluated before freezing and after thawing. Semen samples obtained either by antegrade ejaculation (PVS and SI) or by SSR were cryopreserved into freezing straws (Cryo Bio Systems, France) in liquid nitrogen using a commercial freezing medium (SpermFreeze, Fertipro, Belgium) according to the manufacturer’s instructions.
Sperm preparation for ICSI
After thawing, semen obtained by antegrade ejaculation was washed with a culture medium (Ferticult flushing, Fertipro, Belgium) and centrifuged at 600 g for 5 min at RT. The sperm pellet was re-suspended into 0.5 ml of culture medium (Ferticult flushing, Fertipro, Belgium) and loaded onto 2 puresperm density gradients (0.5 ml of 95% and 47.5%; Puresperm 100, Nidacon, Sweden) and centrifuged at 300 g for 20 min. After centrifugation, the sperm pellet was washed once with a centrifugation at 600 g for 5 min at RT with 1 ml of culture medium and used for ICSI. For testicular spermatozoa, only a wash with a centrifugation at 600 g for 5 min at RT with 1 ml of culture medium (Ferticult flushing, Fertipro, Belgium) was done.
ICSI was offered to 8 couples (4 patients from the antegrade ejaculation group and 4 patients from the SSR group). The female partners of the 8 SCI patients underwent ovarian stimulation. Briefly, pituitary suppression was performed with a daily injection of 0.1 mg of decapeptyl (IPSEN PHARMA, Paris, France) and ovarian stimulation was controlled with recombinant FSH. Serum estradiol levels and vaginal ultrasound measurements of follicles were used to evaluate the ovarian stimulation. hCG injection was scheduled when at least 3 follicles > 17 mm were visible. Oocyte retrieval was performed under general anesthesia, 34 to 36 hours after hCG administration. Assisted fertilization by ICSI was performed 1–3 h following oocyte collection on morphologically mature oocytes. Depending on the female age, the number of cycles, the number and quality of embryos obtained, 1, 2 or 3 embryos were transferred at either day 2 or day 3 after the oocyte retrieval. Progesterone was administered daily vaginally from the day of oocyte retrieval until the time of a negative pregnancy test or until 8 weeks of gestation. Embryo cryopreservation and frozen/thawed embryo transfer were performed as described previously .
Data from the literature
We gathered the data from the studies published in the past 10 years and focused on ICSI outcomes using fresh and/or frozen/thawed sperm obtained after PVS and SSR. A multi-criteria literature search was carried out. Filters: past 10 years, languages (French and English). Key words used for the searches: spinal cord injury – quadriplegic – paraplegic – man – men – male – males – pregnancy –pregnancies - live birth - live births – deliveries – fertility – infertility - intracytoplasmic sperm injection - in vitro fertilization – IVF – ICSI - penile vibratory stimulation - surgical sperm retrieval.
A Wilcoxon matched-pairs signed-ranks test was performed comparing PM (progressive motility), TM (total motility) and viability before freezing and after thawing on 8 patients for whom complete data were available (antegrade group).