This retrospective study included 159 ICSI cycles from 126 couples, with 98 cycles treated with fresh testicular sperm and 61 cycles treated with frozen-thawed testicular samples. Before assisted reproduction, 126 males were evaluated by clinical history, physical examination, and hormonal assay. Female partners were also assessed by full history and infertility investigation, and informed consent was taken from all patients.
The etiology of azoospermia was classified as obstructive azoospermia OA (91 cycles) and non-obstructive azoospermia NOA (68 cycles).
In all cases, surgical sperm was retrieved by the surgeon, and either testicular sperm aspiration under local anesthesia or testicular sperm extraction under general anesthesia was done for them.
Testicular specimens placed in petri dish with HEPES-buffered culture medium were processed by mechanical shredding [22], and the suspension was explored under an inverted microscope at × 400 magnification, when spermatozoa found in specimens was used either freshly for ICSI or when done for diagnostic specimens cryopreserved for next cycle.
Ovarian stimulation and oocyte retrieval
In all patients, short agonist protocols were used to stimulate follicular development. The short protocol includes pituitary desensitization with gonadotropin-releasing hormone agonist (Decapeptyl 0.1 mg, Ferring GmbH, Germany) at day 2 of the cycle and ovarian stimulation with follicle-stimulating hormone (fostimon, IBSA, Lugano 3, Suisse; Gonal-f, Laboroteries Serono S.A, Switzerland; Puregon, Schering-Plough, NV Organon, Oss, Netherlands) or combined LH and FSH (Merional, IBSA, Lugano 3, Suisse; Menegon, Ferring GmbH, Germany) starting at day 3 of the cycle. Measuring serum E2 and performing transvaginal ultrasound monitored the follicular development. Ovulation and final maturation of the ova were induced with human chorionic gonadotropin (hCG) (Choriomon, IBSA, Lugano 3, Suisse) as a single dose of 10,000 IU, when the leading follicle reached 18 mm in average diameter in addition to the presence of at least two other follicles of more than 16 mm in size and E2 > 500 pg/ml, then the oocytes were retrieved. Oocytes were aspirated 34–36 h after hCG administration. Oocyte retrieval was performed by transvaginal ultrasound-guided puncture using 16-gage, 35-cm double lumen aspiration needle (William A. Cook, Australia Pty Ltd.) with a negative pressure of 20 mmHg.
Oocytes denudation and evaluation
Removal of the surrounding cumulus cells was accomplished by a combined enzymatic and mechanical treatment carried out under a stereoscopic dissecting microscope. Oocytes were denudated from cumulus oophorus by exposure to 80 IU/ml hyaluronidase enzyme in HEPES-buffered medium (Hyase, FertiPro N.V., Belgium) followed by mechanical removal of the corona radiate with the use of plastic pipette stripper tips (EZ strip, Research Instruments Ltd, UK) with decreasing inner diameters of 290 and 135 μm.
Oocytes are assessed for their maturation and for their morphology under an inverted microscope (intgera Ti, R.I., Olympus, IX51/IX70, Tokyo, Japan) at × 400 magnification. Metaphase II oocytes were separated from the immature oocytes (metaphase I oocytes and germinal vesicle) just before sperm injection (3–4 h after retrieval).
Sperm preparation
In the case of azoospermia, sperms are retrieved by the urologist, under local anesthesia, either by percutaneous epididymal sperm aspiration (PESA) or testicular sperm aspiration (TESA).
The technical procedure for PESA involved the insertion of a needle attached to a syringe through the scrotal skin into the epididymis. Originally, the use of a larger butterfly needle was described. Currently, most experts use a fine needle (26 gage) attached to a tuberculin syringe containing sperm washing medium. The epididymis is stabilized between the index finger, thumb, and forefinger. After creating negative pressure by pulling the syringe plunger, the tip of the needle is gently and slowly moved in and out of the epididymis until fluid is aspirated. If motile sperm are not obtained, PESA may be repeated at a different site (from the cauda to caput epididymis) until an adequate number of motile sperm is retrieved.
In TESA, the testis is divided into three poles: upper, middle, and lower poles. A needle was inserted through the scrotal skin into the anteromedial or anterolateral portion of the upper pole at an oblique angle toward the medium and lower poles. These aspirations are usually carried out using either fine (testicular fine-needle aspiration (TEFNA)) or a scalp butterfly cannula gauge 23 attached to a syringe. The testicular parenchyma is aspirated by creating negative pressure, and the tip of the needle is moved within the testis to disrupt the seminiferous tubules; the specimen was sent to the laboratory for microscopic examination. TESA can be carried out in the contralateral testis if no sperms are obtained during the first attempt.
The obtained tissues from the epididymis or the testis were placed in a special media for handling gametes. By using a scalpel or surgical blade, the tissue was sliced or dissected mechanically, and wet slides prepared were examined under phase contrast microscope (Olympus BX41 Tokyo, Japan) at × 200 magnification. It was then placed in a flushing media for washing. After that, if sperm was found then it is dissected to remove tissues and blood cells, and then the content was placed into an Eppendorf tube and kept in an incubator for 30 min under 37 °C then centrifuged for 5–10 min. The supernatant was removed. After adding 1 ml of flushing media, it was then kept in incubator for another 30 min and centrifuged for 5–10 min. The supernatant was discarded, and the remaining was used as fresh sample for ICSI when done for diagnostic specimens cryopreserved for the next cycle.
Cryopreservation of samples
After the semen is allowed to be liquefied in the room temperature, it was then mixed with freezing media 1:0.7 ml from SpermFreezeTM (FertiPro) in drops with gentle swirling, then we leave the mixture at room temperature for 10 min for equilibration. The mixture was put in cryovial then fixed in the freezing straw; leave the straw fixed in a liquid nitrogen vapor for 15 min then transfer quickly into the liquid nitrogen and store at 196 °C.
Thawing
We removed as many cryovials as required from the liquid nitrogen and placed the cryovial in tap water for 5 min. We placed the cryovial mixture into the centrifuge tube diluted with sperm wash media (FertiCult TM Flushing medium) of 3 ml per 0.5 sperm mixture mixed thoroughly then centrifuged it for 15 min at 300–350g. We resuspended the pellet in sperm wash media for 15 min then used for ICSI.
Intracytoplasmic sperm injection
Intracytoplasmic sperm injection was performed in MII oocytes according to the technique described by [18]. Oocytes were transferred to the ICSI dish prepared with drops of IVF media (ferticult-IVF, FertiPro N.V., Belgium) covered by mineral oil.
Assessment of fertilization, embryo cleavage, and embryo transfer
Fertilization assessments were performed 17 ± 1 h post-injection. Normally, fertilized oocytes should be spherical and have two polar bodies and two PNs. PNs should be juxtaposed, approximately the same size, and centrally positioned in the cytoplasm with two distinctly clear, visible membranes [23].
Embryo quality was evaluated under an inverted microscope. The following parameters were recorded: (1) the number of blastomeres, (2) the fragmentation percentage, (3) variation in blastomere symmetry, and (4) defects in the zona pellucida and the cytoplasm.
High-quality (grade A) embryos were defined as those having all of the following characteristics: either 4–6 cells on day 2 or 8–10 cells on day 3 of development, less than 15% fragmentation, symmetric blastomeres, colorless cytoplasm with moderate granulation with no inclusions, absence of perivitelline space granularity, and absence of zona pellucida dysmorphism. Embryos lacking any of the above characteristics were considered as low quality [24]. For each couple, 1–4 embryos were transferred, depending on the embryo quality and the female’s age. Embryo transfer was canceled if no embryos were available. Embryo transfer was performed on day 2 or day 3 using a Gynetics catheter (Gynetics Medical Products N.V, Lommel, Belgium). Transfers were performed with transabdominal ultrasound guidance.
Statistical analysis
All the statistical analysis procedures are performed using SAS 9.4 statistical software (SAS institute Inc. Cary, NC, USA© 2014). The differences for continuous variables were examined using Wilcoxon-Mann-Whitney test for samples without assumed normal distribution. The chi-square test was used for categorical variables and proportions, and for categorical variables with cell counts less than five, Fisher’s exact test was used. p values less than 0.05 are considered statistically significant.
