Patients
A retrospective study was conducted in patients who underwent their IVF cycles in Reproductive and Genetic Hospital of the First Affiliated Hospital of University of Science and Technology of China (Hefei, Anhui, China), from January to December 2016. A total of 1223 IVF couples were included in this comparison: 536 couples prepared sperm with DSU technique, 687 couples with DGC-SU technique. The inclusion criteria for the female partner were: (i) age <40 years; (ii) body mass index (BMI) <30 kg/m2; (iii) basal level of follicle-stimulating hormone (FSH) <10 IU/L; and (iv) undergoing their first cycle of IVF. The female partners with diagnosis of uterine or karyotype abnormality were excluded. The study was approved by Ethics Committees on Human Research of the First Affiliated Hospital of University of Science and Technology of China. There is no conflict of interest in the present study.
Preparation of semen samples
Semen samples were obtained by masturbation after at least a 2-day sexual abstinence. After liquefaction at room temperature for 20 minutes, raw semen samples were examined for concentration and motility according to the 2010 WHO guidelines. Spermatozoa were categorized by the following different motility grades: 1) progressively motile, 2) non-progressively motile, and 3) immotile sperm. In order to reduce the artificial differences in sperm characteristics, the routine semen analyses were performed by the same well-trained technician using Computer-aided sperm analysis (CASA). A 500μl semen sample was drawn to evaluate the basal level of sperm motility, viability, DNA fragmentation, high DNA stainable (HDS), acrosomal reaction (AS), mitochondrial membrane potential (MMP), and the remaining sample was split into two aliquots for sperm selection. One aliquot was separated with the DSU technique, and another aliquot was separated with the DGC-SU technique. Similarly, DSU-separated sperm and DGC-SU-separated sperm were examined for motility, viability, DNA fragmentation, HDS, AS, MMP and microbiological contamination.
Density-gradient centrifugation followed by swim-up (DGC-SU)
1ml of semen sample was layered on the top of the discontinuous density gradients (40 and 80%, ORIGIO, Denmark) and centrifuged for 20 min at 350g. Thereafter, sperm pellet at the bottom of tube was collected. At least 3 ml of the culture sperm medium (IVF Sperm Medium, COOK, Limerick) was added to washing sperm pellet and the tube was centrifuged for 6 min at 250g. The supernatant was discarded and 2 ml of the same culture sperm medium was layered gently above the pellet. The tube was inclined at an angle of 45 degrees and incubated for 40 min at 37°C with 6% CO2, and then 1 ml of the middle-clouded layer medium was collected into a new tube for the following experiments [22]. The whole process of DGC-SU takes around 75 minutes (Fig. 1).
Direct swim-up without centrifugation (DSU)
2 ml of the culture sperm medium was prepared into a round-bottomed tube, and then 1ml of semen sample was added to the bottom of the tube. The tube was inclined at an angle of 45 degrees and incubated for 40 min at 37°C with 6% CO2, and then 1 ml of the middle-clouded layer medium was collected into a new tube for the following experiments. The whole process of DSU only takes about 45 minutes (Fig. 1).
Measurement of sperm DNA damage
The level of sperm DNA damage was assessed by DNA fragmentation index (DFI) as described previously [23–24]. Briefly, about one million spermatozoa were treated with hydrochloric acid on ice for 30 sec and then incubated for about 1 min at room temperature with acridine orange (AO, Cellpro Biotech, Zhejiang, China) solution. The spermatozoa were then analyzed by flow cytometry (Accuri C6 cytometer, BD Biosciences, San Jose, CA). More than 5,000 spermatozoa were collected per sample in the gate of sperm population. Data was analyzed using the flow cytometer software (Cellpro Biotech). AO intercalated with double-stranded DNA emits green fluorescence and AO associated with single-stranded DNA emits red fluorescence. The DFI value was calculated by the ratio of red to total (red plus green) fluorescence intensity. Additionally, the fraction of HDS sperm cells was also detected in this experiment. HDS represents immature spermatozoa in semen with incomplete chromatin condensation, which exhibit green fluorescence intensity higher than the upper border of the main cluster of the sperm population.
Evaluation of the acrosomal reaction
Acrosomal reaction (AR) was evaluated by FCM according to manufacturer’s instructions (Cellpro Biotech). In Brief, sperm samples were first diluted and swam up in culture medium for 15 min. After 2-minute fixation in 75% ice ethyl alcohol, calcium ion carrier A23187 (Sigma-Aldrich, St Louis, MO) was added to the sperm suspension to make a final concentration of 10 μmol/L A23187. A23187 stock solution (5mmol/ L) in dimethyl sulfoxide (DMSO, Sigma-Aldrich) was frozen at –20°C. Before use, this was thawed, dissolved in human tubal fluid (HTF) medium. As a control, diluted DMSO (Sigma-Aldrich) was added to the same sperm suspension. Both test and control tubes were incubated for 15 min at 37°C with 5 μg/ml of FITC-conjugated pisum sativum agglutinin (FITC-PSA, Cellpro Biotech) working solution in the dark. Next, spermatozoa were analyzed by FCM. At least 5,000 events were collected per sample in the gate of sperm population. Acquired data was analyzed by flow cytometer software. When more than half the head of a spermatozoon was fluoresced brightly and uniformly with FITC-PSA, the acrosome was considered to be intact. Spermatozoa without fluorescence or with a fluorescing band limited to the equatorial segment were considered to be acrosome-reacted. The ionophore-induced AR was calculated by the acrosome-reacted ratio of test group subtract that of control group.
Measurement of MMP
The mitochondrial membrane potential (MMP) was examined according to the manufacturer’s instructions (Cellpro Biotech). Briefly, one million spermatozoa were incubated with 2 μM of the lipophilic cationic dye 5,5′,6,6′-tetrachloro–1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine iodide (JC–1) working solution for 15 min at 37°C in the dark. Afterwards, the sperm cells were analyzed by FCM. More than 5,000 spermatozoa were collected per sample in the gate of sperm population. JC–1 exists in the cell in the form of polymers and monomers. When the MMP is high, JC–1 spontaneously forms polymers with red fluorescence. When the MMP is low, JC–1 is mainly in the form of monomers and exhibit green fluorescence. The value of MMP was calculated by the ratio of red to total fluorescence using the flow cytometer software.
Microbiological examination
Native semen samples were collected in sterile plastic containers by masturbation in the IVF laboratory. After liquefaction, the semen sample was split into two aliquots for sperm selection. One aliquot was separated with the DSU technique, and another aliquot was separated with the DGC-SU technique. All reserved specimens, including native semen, the upper layer medium after DGC-SU and DSU treatments and the culture medium of simulating fertilization test were collected in sterile plastic containers and examined for aerobic and facultative bacteria within 30 minutes of collection. 10μl sample was inoculated on blood and chocolate agar plates respectively, followed by incubation for 48 hours in 5% CO2 at 37°C. Next, the presence, pattern and number of colonizing bacteria were checked. Identification of bacteria was performed in that case either by means of the VitekTM system (bioMerieux, St. Louis, MO) or a conventional method, as required. All operations of microbiological examinations were carried out in the biosafety cabinets.
IVF protocols
IVF was carried out following routine protocols [25–26]. Briefly, ovarian stimulation of multiple follicles was achieved by purified (FSH, LiZhu pharma, ZhuHai, China) or recombinant (Gonal-f, Merk Serono SA, Geneva, Switzerland) gonadotropins. At 36 h after administration of 10, 000 IU human chorionic gonadotropin (hCG, LiZhu pharma), cumulus-corona oocyte complexes (COCS) were retrieved by vaginal ultrasound-guided aspiration.
Native semen samples were collected in sterile plastic containers by masturbation in the IVF laboratory. All patients were asked to wash their hands and genital areas with soap and water before sperm collection. After liquefaction, semen samples were randomly divided into two groups: one group selected sperm for IVF with DSU technique, and another group selected sperm for IVF with DGC-SU technique. Sperm selected by DSU or DGC-SU technique were used for subsequent insemination of the oocytes at a final concentration of 200, 000 sperm/ml. After co-incubation with spermatozoa for 4 h, the oocytes were completely denuded by pipetting and checked for the presence of the second polar body. The early rescue ICSI were performed in patients who had more than 2/3 oocytes failed to extrude the second polar body after 6 h of insemination [27]. 16 to 18 h after insemination, oocytes were assessed for 2 pro-nuclei presence. 72 h after oocyte retrieval, embryos were classified according to their morphology. Up to two day 3 embryos with good quality were transferred into uterus.
Pregnancy outcomes
A biochemical pregnancy was diagnosed only by the detection of beta hCG in serum or urine 14 days after embryo transfer. Ultrasound was performed at 6 weeks after embryo transfer to confirm clinical pregnancy. A clinical pregnancy was diagnosed by ultrasonographic visualization of one or more gestational sacs or definitive clinical signs of pregnancy. In addition to intra-uterine pregnancy, it includes a clinically documented ectopic pregnancy. Spontaneous abortion was defined as a pregnancy loss of an intra-uterine pregnancy prior to the 22th week of gestation [28]. The implantation rate was calculated as the number of gestational sacs divided by the total number of transferred embryos.
Statistical Analysis
The statistical analysis was performed by using the SPSS, version 20 software. One-way ANOVA was performed to compare the differences among the three groups and the data shown was described as mean ± standard deviation (S.D). Differences regarding rates were evaluated by the Chi-Squared test and the data shown was described as percent (%). p-values <0.05 were considered statistically significant.