Correlation of the sperm DNA fragmentation index with semen parameters and its impact on fresh embryo transfer outcomes—a retrospective study

Study design

From November 18, 2020, to August 29, 2024, 2,317 patients who underwent in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) and fresh embryo transfer (fresh IVF/ICSI-ET) at the Jiangxi Maternal and Child Health Hospital, Reproductive Medicine Center, were retrospectively identified. The Reproductive Medicine Ethics Committee of Jiangxi Maternal and Child Health Hospital approved this study (SZYY-202407). The study was conducted in accordance with the Declaration of Helsinki. All participants provided written informed consent. During the research, we strictly followed the relevant guidelines and regulations of the local research institute. Participants were informed that they could withdraw from the experiment without giving a reason.

The inclusion criteria were as follows: (1) female infertility due to tubal factors and (2) fresh IVF/ICSI-ET with 1–2 transplanted embryos. (3) Oocyte retrieval time: 2020.11.18–2024.08.29. The exclusion criteria were as follows: (1) the female partner has polycystic ovary syndrome (PCOS), endometriosis, congenital uterine malformation or organic uterine lesions, etc.; (2) the male partner has azoospermia, retrograde ejaculation, and ejaculation disorders; (3) cycles with donor sperm and oocytes, and preimplantation genetic testing (PGT) cycle; and (4) missing data. DFI is classified as normal (DFI < 30%) or high (DFI ≥ 30%). A total of 1,110 infertile couples with fresh IVF/ICSI-ET were included in this study, including 81 cases with high DFI levels and 1,029 cases with normal DFI levels. The study flowchart was presented in Fig. 1.

Considering that the sample size ratio between the normal DFI group and the high DFI group exceeded the range of 1:4, a 1:1 propensity score matching (PSM) method was used to match the female age, male body mass index (BMI), female BMI, female infertility duration, female anti-Mullerian hormone (AMH), fertilization method, controlled ovarian stimulation (COS) protocol, and number of embryos transferred from the grouped patients. Finally, 81 patients were included in the high DFI group, and 81 patients were included in the normal DFI group.

Semen routine analysis

Semen processing and testing were performed according to the WHO Laboratory Manual for the Examination and Processing of Human Semen (6th edition) (World Health Organization, 2021). To ensure the accuracy of test results, internal quality control (IQC) measures were implemented throughout the testing process to minimize inter-technician variability. Additionally, the laboratory regularly participated in external quality assessment (EQA) programs (e.g., the Sichuan-Chongqing Regional Quality Control Program in China).

The man abstained from sexual activity for 2–7 days and obtained semen through masturbation. The duration of abstinence was recorded. The weighing method was used to calculate semen volume (Ying Heng, GuangZhou). Each semen sample was directed into a sterile plastic cup and liquefied in an incubator at 37 °C (JingHong, ShangHai). After complete liquefaction of semen, 10 ul of semen sample was taken and dropped into the detection well of the sperm counting plate (SAS, BeiJing). A computer-aided semen analyzer (Suijia, Beijing, SSA) was used to detect sperm concentration, forward movement, and vitality. The SSA automatic detection system captured the movement trajectory of sperm through microscopic camera technology, and further analyzed the kinematic parameters and vitality. Kinematic parameters were divided into motion speed parameters, motion mode parameters, and spatial displacement degree parameters. Sperm motility was classified into forward movement, non forward movement, and inactivity based on their movement status. Obtain ≥5 visual fields and ≥200 sperm from each semen sample for observation and counting of the concentration, total number, and percentage of viable sperm at all levels. The test was performed using a constant temperature console (TC-01H, China). Fresh liquefied semen smears were air dried and analyzed for sperm morphology via Diff-Quik stained method (AnHui, Ankebio, Moscow, Russia). Only sperm with normal head (neck) and tail (middle and main segments) were considered morphologically normal, and all sperm in critical states were classified as abnormally shaped. For each semen sample, at least 200 sperm samples were analyzed using a double-blind method.

Sperm DFI detection

The sperm chromatin structure assay (SCSA) was a reliable and most commonly used assay for the determination of DFI. The flow cytometry-based SCSA method was used to determine the DFI values. A sperm nuclear integrity staining kit (Zhejiang Xingbo Biotechnology, Zhejiang, China) was used for testing, following the instructions in the kit manual. According to the instructions of the reagent kit, the sperm concentration was adjusted to 1 × 10⁶–2 × 10⁶/mL, and 100 μL was used to test DFI. The chromatin in the damaged sperm nucleus forms a single chain after acid treatment, which binds with acridine orange and emits red or yellow fluorescence. The chromatin in normal sperm nuclei can maintain its intact double stranded structure after acid treatment and emit green fluorescence when combined with acridine orange. Fluorescence was detected by flow cytometry (Beckman Coulter, Brea, CA, USA) after the samples were stained with acridine orange. DFI (%) = green fluorescent sperm count/(green fluorescent sperm count + red fluorescent sperm count) × 100%.

Sperm optimization for IVF/ICSI

The male partner abstained from sexual activity for 2–7 days and collected semen through masturbation on the day of oocyte retrieval. After the semen was completely liquefied, the sperm was selected by discontinuous density gradient centrifugation combined with the swimming-up method, as follows. First, sperm was prepared using a processing solution, including density gradient solution and swimming‑up solution. The density gradient liquid was prepared using Vitrolife Ivrine Isolate (Vitrolife, Gothenburg, Sweden), with 0.5 ml of lower layer liquid and 0.5 ml of upper layer liquid loaded into a 15 ml Falcon centrifuge tube (Corning Incorporated, Corning, NY, USA) with a pointed bottom. The swimming‑up solution used was Vitrolife G-IVF plus (Vitrolife, Gothenburg, Sweden), 2 ml was divided into 2 Falcon 2003 test tubes and equilibrated overnight in a 37 °C, 6% CO₂ incubator for future use. Then, (1) discontinuous density gradient centrifugation. Slowly add 2.0 ml of semen to the top layer of density gradient solution. After centrifugation at 300–400×g for 15 min, the supernatant was aspirated and discarded, leaving behind approximately 0.5 ml of sperm at the bottom. Add 1 ml of swimming‑up solution to the sperm and mix thoroughly. Centrifuge again at 300–400×g for 5 min, then discard the supernatant and leave the bottom sperm at approximately 0.5 ml. (2) Sperm swimming‑up. Add 0.5 ml of swimming‑up solution to the sperm. The Falcon 1006 centrifuge tube was tilted at 45 degree angle in a 37 °C incubator with 5% CO₂ and saturated humidity. The process of sperm swimming-up lasted for 15 min, and then the upper sperm suspension was aspirated into another clean Falcon 1006 centrifuge tube for later use.

Controlled ovarian stimulation and oocyte collection

In accordance with the patient’s physical condition, controlled ovarian stimulation (COS) is performed via the ultralong GnRH agonist protocol, the long GnRH agonist protocol, the GnRH antagonist protocol or other protocols. Transvaginal ultrasound and serum hormone level testing (such as estradiol (E₂), luteinizing hormone (LH), follicle stimulating hormone (FSH), and progesterone (P)) are used to monitor follicle growth during the menstrual cycle. When the diameter of the dominant follicle is ≥19 mm or at least 3 follicles have a diameter ≥17.5 mm, a subcutaneous injection of 250 µg of human chorionic gonadotropin (HCG) should be given that evening, and oocyte aspiration should be performed after 36–40 h. Oocytes were cultured at 37 °C in a 5% CO₂ incubator for future use.

Fresh IVF/ICSI-ET

The fertilization method was as follows: (1) IVF fertilization: The oocytes were cultured for 3‒4 h before fertilization, with 50,000-100,000 sperm added to each oocyte. After 4–6 h, the cumulus cells were removed, and 16–18 h after fertilization the second polar body (2 pb) was observed. (2) ICSI fertilization: ICSI is performed 2–3 h after oocyte retrieval. Embryo culture and scoring were conducted according to standard operating procedures. The embryo scoring criteria are mainly based on morphological indicators. On the third day of 2PN fertilization, the embryos underwent cleavage, with 7–10 blastomeres of uniform size and a fragmentation rate of 0–20%, which were classified as high-quality cleavage embryos. The evaluation of blastocysts is based on three aspects: the degree of expansion of the blastocyst cavity, the number of inner cell mass, and the number of trophoblast cells. Fresh D3/D4/D5/D6 embryos can be transferred, and the number of embryos transferred varies from one to two according to the doctor’s advice and the patient’s requirements. Fourteen days after embryo transfer, a positive HCG blood test (HCG ≥ 50 mIU/ml) indicates a biochemical pregnancy. Five weeks after transplantation, B-ultrasound examination revealed a gestational sac and primitive pulsation in the uterine cavity, which confirmed clinical pregnancy.

Outcome measures

MII oocyte rate = (number of MII oocytes/total number of retrieved oocytes) × 100%;

IVF 2PN fertilization rate = (2PN number/number of retrieved oocytes) × 100%;

ICSI 2PN fertilization rate = (2PN number/MII oocyte number) × 100%;

D3 high-quality embryos = sourced from 2PN fertilization, with 7–10 symmetrical blastomere cells, rated as grade I or II; D3 high-quality embryo rate = (number of D3 high-quality embryos/number of 2PN cleavages) × 100%;

Biochemical pregnancy rate = (number of biochemical pregnancy cycles/number of fresh transplant cycles) × 100%;

Clinical pregnancy rate = (number of clinical pregnancy cycles/number of fresh transplant cycles) × 100%;

Miscarriage rate = (number of miscarriage cycles/number of fresh clinical pregnancy cycles) × 100%;

Delivery rate = (number of delivery cycles/number of fresh transplant cycles) × 100%.

Data statistics and analysis

R 3.3.4 was used for statistical analysis of the data. The Shapiro‒Wilk test was used to test the normality of the measurement data. Normally distributed data are presented as the means ± standard deviations (X ± S), and intergroup comparisons were conducted via t tests. Quantitative data that conform to a skewed distribution or approximate normal distribution are represented by medians (M (Q1, Q3)), and intergroup comparisons were performed via the Mann Whitney U test. Count data are expressed as percentages (%) and were compared between groups via the chi square test or Fisher’s test. The bilateral test method was used, and p < 0.05 was considered statistically significant.

Comparison of semen parameters among different sperm DFI groups

After PSM, this study included 162 male patients with a median age of 33 years. The median sperm DFI was 29.79%. The median semen volume was 2.90 ml. The median sperm concentration was 48.98 × 10⁶/ml. The median sperm motility was 40.15%. The forward movement of the sperm was 29.34%. The percentage of normal morphology sperm was 4.46%. According to the DFI value of sperm, there were 81 patients (50%) with a DFI ≥ 30% and 81 patients (50%) with a DFI < 30%. A comparison of the two groups revealed that sperm forward movement (p < 0.001), sperm motility (p < 0.001), and the percentage of sperm with normal morphology (p = 0.001) were significantly greater in the normal DFI group than in the high DFI group. However, there were no significant differences in male age (p = 0.574), abstinence days (p = 0.244), semen volume (p = 0.523), or sperm concentration (p = 0.056) between the two groups (Table 1).

Correlation analysis between the sperm DFI and semen parameters

The correlations between the sperm DFI and routine semen parameters, as well as the associations between routine semen parameters and the DFI, are worthy of further exploration. Spearman correlation analysis revealed a negative correlation between the sperm DFI and sperm motility (r = −0.44, p = 3.32e−08), forward movement of sperm (r = −0.46, p = 3.25e−09), and normal morphology of sperm (r = −0.25, p = 0.000), of course, these linear correlations were not strong. However, there was no correlation between the sperm DFI and male age (r = 0.08, p = 0.31), semen volume (r = −0.15, p = 0.05), sperm concentration (r = −0.16, p = 0.32), or BMI (r = 0.02, p = 0.98) (Table 2 and Fig. 2).

Comparison of pregnancy outcomes between the different sperm DFI group

After conducting PSM, there were no significant differences in terms of infertility duration (p = 1.000), female age (p = 0.615), female BMI (p = 0.671), female basal FSH (p = 0.879), female basal E₂ (p = 0.967), female basal LH (p = 0.683), female AMH (p = 0.875), male BMI (p = 0.693), COS protocol (p = 1.000), number of oocytes retrieved (p = 0.861), fertilization (p = 0.962), or number of embryos transferred (p = 0.747) between the two groups (Table 3). As shown in Tables 4 and 5, when the clinical outcomes of the two groups were compared, there were no significant differences in the 2PN fertilization rate (64.98% vs. 67.18%, p = 0.362), D3 high-quality embryo rate (28.34% vs. 23.91%, p = 0.107), biochemical pregnancy rate (71.60% vs. 71.60%, p = 1), clinical pregnancy rate (65.00% vs. 65.00%, p = 1), delivery rate (50.72% vs. 48.44%, p = 0.928), miscarriage rate (7.25% vs. 6.25%, p = 1), or singleton birth weight (3,350 g vs. 3,200 g, p = 0.599) between the two groups.