2.1 Patients
The research object of this study was 60 infertile couples who undergoing assisted reproduction techniques (ART) treatments including intrauterine insemination (IUI), in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI) treatment in human assisted reproductive medical technology of southwest medical university from May 2019 to May 2020. Inclusion criteria: after regular sexual life for more than one year, there was no natural conceived on the condition of no contraception. The female showed normal ovarian function. Neither of the couple show any inherited disorders, sexual dysfunction and anatomical deformity, mumps history. There were no obvious abnormalities can be found in physical examination and ancillary testing. Male patients preservde pre-treatment sperm samples for routine semen analysis and sperm nuclear DNA integrity detection. For female participants, they were arranged ovums extraction under the ultrasound detection to conduct IVF assisted reproductive technology treatment. Based on DFI, the participants were divided into group A (DFI≤10%), group B (10%<DFI<30%) and group C (DFI≥30%).
2.2 Semen routine
After keeping abstinence for 2-7 days, samples were left in the one-time sperm collectors through masturbation and stored in the environment of 20-37°C with the specimen number, name of patients, semen collection time and days without ejaculation record. It kept notes of total specimens and liquefied duration. According to the Human Semen Examination and Processing Laboratory Manual (version V) published by Word Health Organization in 2010[7], it applied computer-assisted sperm, microbial dynamic (static) state image analysis system (THTF, CASAS-QH-III) to analysis and record the following data: sperm concentration, sperm activate rate, sperm non- progressive motility rate, progressive motility. rate and immotility rate.
2.3 SCD
This study improved the SCD method proposed by scholars such as Meseguer[8]. In this study, it first used phosphate buffer (PBS pH 7.2) to wash semen samples adequately. After centrifugation the supernatant was discarded. The concentration of the specimen was adjusted to 4-10×106/ml. Make 1% 100ml low-melting-point agarose solution, took 70 ul to 1.5ml centrifuge tube to be melted in 65-90℃ water bath and be kept in 47℃ for three minutes. Added treated semen samples 30 ul and do mechanical blending. Dropped 20 ul into the slide with 0.65% prepared standard agarose gel, closed the 18×18cm cover glass to be placed in 4℃ refrigerator for 35 minutes. If the agarose was completely cured, carefully peeled off the surface cover glass. Put into the prepared diluted hydrochloric acid (0.08mmol/L)with avoiding light in order to degeneration and immerse in the sperm cracking liquid (0.4mol/L DTT consists of Tris, DTT, SDS and EDTA). Conducted natural dehydration in 70%, 90%, 100% ethanol and withdraw them into object slide stand to dry naturally for Wright Stain. After completion of dyed, the samples were placed under Olympus microscope (10×40) to observe the account. It was worth stressing that more emphasis was placed on the preparation of reagent when using on the basis of previous test, especially sperm cracking liquid configuration.
Because normal sperm DNA could be attached to the loose chromatin structure and form a dizzy ring, while damaged sperm DNA cannot or can only form a small ring [9]. Through microscope observation, sperm head diameter is d1 and unilateral halo thickness is d2 (see Fig 1). When evaluating d2≤1/3d1, it showed that the existence of sperm DNA fragmentation (small halo ring); if the head was deeply stained, it was considered as no halo ring. As can be seen from Fig 2, big halo ring (d2≥2/3d1) and middle halo ring (1/3d1<d2<2/3d1) were complete DNA sperm.
2.4 Conventional stimulation protocol Fertilization
On day 2 of menstrual cycle, the female participants began to accept intramuscular injectiongonadotrophin releasing hormone agonists (Diphereline, Ipsen Pharma Biotech) 0.1mg/day for 14 consecutive days. B ultrasound and estrogen were used to monitor the condition of follicular development at any time. After reaching the standard of falling tone, gave with recombinant human follicle stimulating hormone injections (Recombinant Human Follitropin for Injection, Swiss Serono pharmaceutical). When the 18 nm above follicles were more than 2, combined with patient blood P and E2 level, they were given intramuscularly injection of human chorionic gonadotropin (HCG, Swiss Serono Company) 6000U. 36 hours after intramuscularly injection, punctured ovum extraction under the guide of B ultrasound. Added Protein insemination culture (G-IVF PLUS, vitrolife, Sweden) and placed in 37℃, 6% CO2 incubator to cultivate sperm egg interaction for five hours.
2.5 Embryological observation and high-quality embryo number selection
Normal fertilization refered to 2 pronuclears (PN) and Polar body2 (Pb2) could be found under a microscope. Abnormal fertilization refered to monopronuclear (1PN) was less than or equal to 1 or multipronuclear (3PN) was equal or greater than 3. According to Peter’s cleavage stage [10] embryo scoring system, embryo quality could be assessed through segmentation sphere volume, form, fragments and cytoplasma granula.
Those I and II level embryos that develoed into 6-10 segmentation spheres on the third day were defined as high quality embryos, and the rest were considered as low-score embryos. High quality embryos were selected to be transplanted.
2.6 Data collection
The study collected the data of clinical sperm concentration, sperm activate rate, sperm progressive motility rate, non-progressive motility rate and immotility rate (see Figure 3). SCD method was used to calculate the sperm number with DNA fragmentation in 400 sperm smears under microscope. Sperm DNA fragmentation index(DFI)=(small halo ring sperm+ no halo ring sperm)/ observed 400 sperm×100%[11]. Figure 4 shows retrieved oocytes, fertilized oocytes, cleavage number and high quality embryo number after the treatment of IVF assisted reproductive technology. Fertility rate=fertilized ocum number/mature ocum number×100%, cleavage rate=cleavage number/fertilized ocum number×100%, high-quality embryo rate=high quality embryo number/total number of embryos×100%.
2.7 Statistical analysis
SPSS17.1 statistical software was applied to deal with all the collected statistics analysis. All the data adopted mean standard deviation and measurement data using to test analysis. One-way analysis of variance was used on the basis of DFI classification. It explored the relationship between DFI and routine semen parameters, fertility rate, cleavage rate and high-quality embryo rate. Correlation analysis used Pearson correlation analysis and the correlation coefficient refers to P<0.05 presents the difference was statistically significant.