Patients
A retrospective study was carried out from June 2014 to June 2018. The inclusion criteria was those patients using motile or immotile testicular spermatozoa in order to perform ICSI treatment. The exclusion criteria were as follows: (1) no transplantable embryos available, including abnormal fertilization, no cleavage embryos or embryo degeneration on day 3 and (2) those embryo transfers performed at the day 1 and 2 zygotic stages. The patients were divided into a motile testicular spermatozoa ICSI group (control) and a group who had laser-assisted selection of viable but immotile testicular spermatozoa (laser group) according to whether motile spermatozoa were observed after in vitro cultures of 2~4 hours.
Ovarian stimulation
Ovarian stimulation was performed using a routine protocol developed by our clinic. Briefly, all female patients were down-regulated by use of leuprolide acetate (Lupron; TAP Pharmaceuticals, Lake Forest, Illinois). Ovarian stimulation was achieved with the use of recombinant follicle stimulating hormone (Gonal-F or Puregon; Merck Serono, Italy). When two or more follicles reached 18 mm in mean diameter, 5,000–10,000 IU human chorionic gonadotropin (hCG) (Serono, Switzerland; or Livzon, China) was administered. Oocytes were collected by follicular aspiration with the use of vaginal ultrasonography 36 hours after hCG administration.
Testicular sperm aspiration
The patients were placed in the supine position and disinfected following routine procedures. Anesthesia was performed by using 2% lidocaine to block the spermatic cord. A 50-mL syringe containing 0.5mL of fertilization Quinn’s 1020 medium (Sage, Trumbull, CT, USA) and a 16-gauge needle were used for aspiration of the seminiferous tubules. The tubules were independently minced using two sterile needles in a culture dish containing 2mL of Quinn’s 1020 medium. Then, the processed samples were observed under high magnification (×200 magnification). If no motile sperm were found either immediately or after 2~4 hours of culture in a 6% carbon dioxide incubator maintained at 37 ℃, the sperm were considered to be immotile.
Laser selection of immotile spermatozoa
Using a protocol based on the method of Aktan et al [10], the tips of immotile sperm were targeted with a laser beam of approximately 200μJ with an irradiation time of about 2 ms (RI Saturn 5TM Laser System, UK). Those spermatozoa which presented with curling of the tails after the laser shot, were regarded as viable, while others which did not respond in this way were considered to be non-viable. The viable but immotile sperm selected were subsequently used for ICSI injections.
ICSI
All ICSI procedures were performed 39–40 hours after hCG administration. After injection with hCG, oocytes were transferred to culture dishes and kept in fertilization medium (Quinn Advantage medium, ART-1020) supplemented with 10% Quinn Advantage serum protein substitute (SPS, ART-3010; Sage) until pro-nucleation was observed.
Fertilization checking, embryo culture and transfer
Fertilization was confirmed after observation of two pro-nuclei and two distinct polar bodies at 16–18h after ICSI. Quinn’s medium was used for culture of embryos. Zygotes displaying two pro-nuclei were transferred to the cleavage culture media (Quinn Advantage medium, ART-1026) supplemented with 10% SPS for further culture. The day of ICSI manipulation was considered as day 0. All of the day 3 embryos from patients with good prognoses or the surplus day 3 embryos after transfer or vitrification were delayed in culture until days 5 or 6. Blastocyst culture media (Quinn Advantage medium, ART-1029) supplemented with 10% SPS was used for this. Fresh embryo transfers were performed on day 3 at the cleavage stage or on day 5 (the blastocyst stage). Frozen-thawed embryo transfers were performed after endometrium transformation on day 3 for cleavage stage embryos or on day 5 for blastocyst transfers, respectively. The serum levels of hCG were measured on day 14 after transfers. Clinical pregnancies were confirmed by transvaginal ultrasonography imaging of the presence of one or more gestational sacs within the uterine cavity after 28 days of transfer.
Embryo vitrification, thawing and transplantation
Vitrification and thawing were performed according to the methods established in our clinic and described previously [16]. The common modality for frozen-thawed embryo transfers was the natural or hormone replacement cycles after endometrial preparation.
Follow up and evaluation indexes
The main outcome measures consisted to be the cumulative live birth rate as well as the obstetric and neonatal outcomes. The cumulative live birth rate was calculated by dividing the number of live births over a period for each egg collection cycle (including both fresh and frozen embryo transfer cycles and when the number of live births was greater than or equal to 2 in one ovum acquisition cycle was considered to be one) by the total number of ovum acquisition cycles. The data from patients undergoing frozen-thawed cycles were included from up to December 2019.
Statistical Analysis
Statistical analysis was performed with the use of the Student t test and chi-squared analysis. P < 0.05 was considered to be statistically significant.