Ethical Approval
All protocols used in this study involving human subjects were approved by the Ethics Committee of Sichuan Jinxin Xinan Women’s and Children’s Hospital (approval no.2023-001). It was confirmed to the World Medical Association Declaration of Helsinki: ethical principles for medical research involving human subjects [18]. Written informed consent was obtained from all study subjects for the use of their specimen for research.
Subjects
Ten female patients with an average age of 30.7±4.11(26-34) years and eight male patients with an average age of 32.13±4.45 (26-40) years were diagnosed with COVID-19 and their spouses were negative to COVID-19 by SARS-CoV-2 RNA polymerase chain reaction (PCR) assay. All the recruited patients were asymptomatic or mildly symptomatic without severe fever. They performed assisted reproduction in Sichuan Jinxin Xinan Women’s and Children’s Hospital in December, 2020. Sixteen control couples including 16 female healthy donors with an average age of 30.75±3.00 (24-36) years and 16 male healthy donors with an average age of 31.56±3.24 (26-37) years were enrolled.
Ovarian stimulation
Ovarian stimulation was performed as described previously(19). Briefly, a dose of recombinant FSH (Gonal-f 1050, Merck Europe B.V. Amsterdam, the Netherlands) range from 150 to 225 IU was administrated from the second day of the menstrual cycle. When follicles reached a size of 14mm diameter on average, 0.25mg of GnRH antagonist (Orgalutran 0.25mg, Merck Sharp & Dohme, Haarlem, the Netherlands) was added. The patient’s cycle was monitored according to the individual policy of the clinic. Recombinant hCG (Ovitrellew, Serono) was administered as soon as three or more follicles reached a mean diameter ≥18mm, and oocyte retrieval was performed 36hs later. IVF was used as appropriate for each couple.
Semen preparation and evaluation
The semen samples were obtained from the subjects in the test and control groups by masturbation after three days of sexual abstinence, and then incubated at 37 °C in a humidified incubator with 5% CO2 in air for 30 min to allow liquefaction. Standard semen parameter analysis was performed according to WHO laboratory manual for the examination and processing of human semen [20]. The motile spermatozoa were prepared by a density gradients centrifugation (90–45%) with SpermGrad to separate the seminal plasma and other cellular components [20].
Embryo culture evaluation
Fertilization was confirmed 16-20h after insemination by the presence of two pronuclei and extrusion of the second polar body. Embryos quality and development were assessed on Days 3 and D5/6. Cleavage embryos quality grades were assigned by cell number, fragmentation pattern (defined as the embryonic volume occupied by the enucleated cytoplasm), symmetry and multinucleation. The blastocysts quality grades were assessed by cell number, and embryo expansion, with grades (A, B, or C) given to both the trophectoderm and inner-cell mass of the embryo as described by Gardner and Schoolcraft [21].
Sperm chromatin structure assay (SCSA)
SCSA assay described in our previous study [22] was performed to detect spermatozoa with a high degree of DNA fragmentation. Briefly, 400 μL of acid detergent solution was admixed with 200 μL of the adjusted sperm sample in a Falcon tube. After 30 sec, spermatozoa were stained by adding 1.2 mL of acridine orange (AO)-staining solution containing 6 μg of AO per mL of buffer. Approximately 10,000 events per sample were analyzed. Sperm chromatin damage was quantified by Flow Cytometry measurements (FCM) of the metachromatic shift from green to red fluorescence and displayed as red versus green fluorescence intensity cytogram patterns. The extent of DNA denaturation is expressed as the sperm DNA fragmentation index (DFI).
Detection of SARS-CoV-2 RNA
Real-time Fluorescent Quantitative Polymerase Chain Reaction (RT-qPCR) was employed to quantify SARS-CoV-2 RNA in semen and follicular fluid. SARS-CoV-2 RNA was extracted using the SARS-CoV-2 RNA extracted Kit and PCR amplification was performed using SARS-CoV-2 RNA Detection Kit (Daan Gene, Guangzhou, China) according to the manufacturer’s instructions. Briefly, the genomic RNA (10 µL) was added to the reaction solution (20 µL) provided in the Kit. The amplification was carried out in an instrument SLAN-96S (Hongshi, Shanghai, China). Thermal cycling condition was as follows: after a pre-denaturation at 50 ℃ for 2 min and then 95 ℃ for 2min, 10 cycles were performed, and each cycle included a denaturation (95℃, 5 sec) and an annealing/extension (60 ℃, 35 sec). After pre-amplication, 32 cycles were performed, and each cycle included a denaturation (95℃, 5 sec) and an annealing/extension (60 ℃, 35 sec), a fluorescence signals collection step. CT values of SARS-CoV-2 N and ORF1ab gene were calculated using Rnase P as endogenous reference genes.
Statistical analysis
SPSS 26.0 software was used to analyze the data. Data are presented as the mean values ±standard deviations (SDs). The independent t-test, One-way analysis of variance (ANOVA) and the least significant difference (LSD) post hoc test were performed to evaluate the impact of COVID-19 infection. A P-value of less than 0.05 was considered statistically significant.