Patients aged 20–45 years undergoing their first IVF-ET treatment at the Reproductive Medicine Center of Sichuan Provincial Women’s and Children’s Hospital from January 2021 to December 2022 were prospectively enrolled. The OS protocol was the gonadotrophin-releasing hormone (GnRH) antagonist or agonist protocol. The exclusion criteria were: history of SARS-CoV-2 infection; oocyte and/or sperm donation; pre-implantation genetic testing (PGT); oocyte frozen; adenomyosis, submucosal, or intramural uterine fibroids; uterine abnormalities; and inaccurate information on vaccination. The patients were divided into the vaccinated group (CoronaVac, an inactivated COVID-19 vaccine produced by Sinovac Biotech Ltd., Beijing, China) and the unvaccinated group based on the inoculation status before OS.

The vaccinated group was sub-grouped based on the dose of inoculation (single dose, double dose, and triple dose) or the interval between the first inoculation and OS (<3 months, 3–6 months, and >6 months). In China, the full course of vaccination consists of three doses: the first dose, the second dose (given 1 month after the first dose), and the third dose (a booster dose, given 6 months after the second dose). Prior to the OS, information on the vaccination (i.e., date of vaccination, type, and manufacturer) was recorded by a nurse via Tianfutong (an application program of the Sichuan health database) installed on mobile phones. For all patients, nucleic acid is routinely detected to exclude SARS-CoV-2 infection before OS. The flowchart is shown in Figure 1 . This prospective cohort study was approved by the Medical Ethics Committee of Sichuan Provincial Women’s and Children’s Hospital. All procedures in this study complied with the ethical standards of the relevant national and institutional committees on human experimentation and the Helsinki Declaration 1975 (2013 revision). The trial was registered in the Chinese Clinical Trial Registry (ChiCTR2200055721; https://www.chictr.org.cn/,16 January 2022).

The body mass index (BMI), the antral follicle count (AFC), and the serum levels of the follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E₂), progesterone (P), total testosterone (TT), prolactin, and AMH were measured as described previously (30).

Patients with decreased ovarian reserve (DOR) or polycystic ovary syndrome (PCOS) were mainly treated with the GnRH antagonist protocol, while others were generally treated with the long-acting GnRH agonist protocol.

With the GnRH antagonist protocol, 125–300 IU/day of recombinant FSH (rFSH) (Gonal-F, Merck-Serono KGaA, Darmstadt, Germany; Jinsai Heng, Jinsai Pharmaceuticals, China; Puregon^®, Merck Sharp & Dohme, Rahway, NJ, USA) was injected daily from day 2 to day 3 of the menstrual cycle until the trigger day. The rFSH dose was determined based on the AFC, AMH, BMI, and age of the patient and was adjusted according to follicle development and the serum level of E₂. When the diameter of the leading follicle reached 12 mm or the serum level of LH ≥10 mIU/ml, 0.25 mg GnRH antagonist (Ganirelix, Ocalon, FL, USA) was subcutaneously injected daily until the trigger day. When the diameter of at least one or two follicles has reached 18 mm, 250 μg of recombinant human chorionic gonadotrophin (rHCG; Merck-Sheranova, Darmstadt, Germany) or 0.2 mg of triptorelin (Jinsai Pharmaceuticals, Changchun, China) was injected as the trigger, with the latter used only for all frozen embryo cycles. Transvaginal oocyte retrieval was performed 36.5 h later.

With the GnRH agonist protocol, 3.75 mg of leuprorelin acetate (Beiyi; Shanghai Livzon Pharmaceutical Co., Ltd., Shanghai, China) was injected subcutaneously from day 2 to day 3 of the menstrual cycle. After 28–38 days, 125–300 IU/day rFSH was injected daily when the diameter of most follicles was 5 mm and with serum levels of E₂ <50 pg/ml and LH and FSH <5 mIU/ml. The standard of the rFSH initiation dose and adjusted dose and the timing of the trigger and ovulation were consistent with the GnRH antagonist protocol.

Oocyte retrieval was cancelled for any of the following conditions: follicular growth failure (10 days after OS, diameter of the leading follicle <10 mm), premature ovulation before oocyte retrieval, and personal reasons. Ovarian hyperstimulation syndrome (OHSS) was diagnosed and graded according to Navot et al. (31).

In vitro fertilization (IVF) was carried out 39 h after the trigger for all retrieved oocytes, while intracytoplasmic sperm injection (ICSI) was carried out 3–4 h after the retrieval of mature oocytes. A mature oocyte was defined as being at the metaphase II (MII) stage with the first polar body visible in the cytoplasm. A normal fertilized oocyte was confirmed as containing two pronuclei (2PN). The embryo was cultured to cleavage and blastocyst stage in sequential G1-plus/G2-plus medium (Vitrolife, Gothenburg, Sweden) at 37°C in a culture environment containing 6.0% CO₂ and 5% O₂. Day 3 cleavage embryo was scored based on the number of blastomeres and the degree of fragmentation, with high-quality embryo categorized as grade I or II (32). On day 5 or day 6, morphological scoring was carried out based on the Gardner and Schoolcraft’s system (32). Blastocysts were considered as usable with a grade over 4CC and as high quality for those over 4BB.

Most of the patients were transferred with two cleavage embryos with the highest morphological scores. One embryo was transferred for those with a scarred uterus or with a body height of <1.5 m, and one blastocyst was transferred when eligible (with the number of high-quality cleavage embryos ≥3); otherwise, one cleavage embryo was transferred. Luteal phase support was started on the day after oocyte retrieval by daily injection of 60 mg progesterone oil (Zhejiang Xianju Pharmaceutical Co., Ltd., Taizhou, China) or 90 mg vaginal progesterone (Crinone 8% gel; Merck, Darmstadt, Germany), and 30 mg dydrogesterone (Duphaston; Abbott Healthcare Products B.V., Weesp, the Netherlands) was taken daily.

Embryo implantation, clinical pregnancy, and early miscarriage were defined as previously described (30). Ectopic pregnancy was defined as implantation at any site out of the uterine cavity. Late miscarriage was defined as loss of pregnancy between the 12th and 28th weeks of gestation. Ongoing pregnancy was defined as the detection of fetal heartbeat at the 12th week of gestation. Live birth was defined as delivery of a live fetus after 28 weeks of gestation.

Fresh embryo transfer was cancelled for any of the following conditions: failed oocyte retrieval, no transplantable embryo formation, serum level of P >1.5 ng/ml on the trigger day, diagnosis of OHSS, prevention of OHSS (with number of oocytes retrieved >18 or serum level of E₂ >5,000 pg/ml on the trigger day), and personal reasons. All of the embryos were frozen, and frozen–thawed embryo was transferred at least 1 month later. Only the outcomes of fresh embryo transfer were analyzed.

The primary outcome was the LBR. The secondary outcomes were the rates of MII, 2PN, cleavage embryo, high-quality cleavage embryo, blastocyst, high-quality blastocyst, implantation, clinical pregnancy, early miscarriage, and ongoing pregnancy.

The above outcomes were calculated as follows: MII rate = number of MII oocytes/number of retrieved oocytes; 2PN rate = number of 2PN oocytes/number of oocytes for fertilization; cleavage embryo rate = number of D3 cleavage embryos/number of cleaved embryos on day 2; high-quality cleavage embryo rate = number of high-quality D3 cleavage embryos/number of cleaved embryos on day 2; blastocyst rate = number of blastocysts/number of D3 cleavage embryos cultured for blastocyst; high-quality blastocyst rate = number of high-quality blastocysts/number of D3 cleavage embryos cultured for blastocyst; IR = number of gestational sacs/number of transferred embryos; CPR = number of clinical pregnancy cycles/number of fresh embryo transfer cycles; ongoing pregnancy rate = number of ongoing pregnancy cycles/number of fresh embryo transfer cycles; early miscarriage rate = number of early miscarriage cycles/number of clinical pregnancy cycles; and LBR = number of live birth cycles/number of fresh embryo transfer cycles.

SPSS v26.0 software (IBM, Armonk, NY, USA) was used for statistical analysis. Continuous variables are expressed as the mean ± standard deviation and were analyzed using one-way ANOVA. Categorical variables are expressed as frequency and were compared using chi-square or Fisher’s exact test. Two-tailed p < 0.05 was considered as statistically significant, as we had no prior evidence indicating the effect of vaccination on the outcomes of IVF-ET.

A flowchart of the recruitment is shown in Figure 1 . Infertile women undergoing their first OS cycle with an antagonist or agonist protocol (n = 1714) were included as participants. Women with missing information on vaccination (n = 37), women >45 or <20 years old (n = 36), those who refused to participate (n = 13), and those with disease-affected embryo transfer (n = 22) and PGT cycles (n = 348) were excluded. Finally, 1,258 patients were enrolled. They were divided into the vaccinated group (n = 713) and the unvaccinated group (n = 545) according to their vaccination status before OS.

The age, duration, type and etiology of infertility, BMI, AFC, and serum levels of AMH and basal sex hormones were not significantly different between the vaccinated and unvaccinated groups (p > 0.05) ( Table 1 ).

The proportion of OS protocol; the types of trigger and fertilization; the dose and duration of rFSH; the serum levels of E₂ and P on the trigger day; the numbers of retrieved oocytes, MII, 2PN, cleavage embryos, and high-quality cleavage embryos; the rates of MII, 2PN, cleavage embryo, high-quality cleavage embryo, blastocyst, and high-quality blastocyst; the incidence of mild, moderate, and severe OHSS; and the rates of cancelled oocyte retrieval were not significantly different between the vaccinated and unvaccinated groups (p > 0.05) ( Table 2 ).

Respectively 221 and 223 patients from the vaccinated and unvaccinated groups had cancelled fresh embryo transfer. As a result, 475 and 312 patients from the vaccinated and unvaccinated groups, respectively, underwent fresh embryo transfer. The thickness of the endometrium; the number and age of embryo transferred; and the IR, CPR, multiple pregnancy rate, early miscarriage rate, ongoing pregnancy rate, LBR, gestational age at delivery, and birth weight of infants were not significantly different between the vaccinated and unvaccinated groups (p > 0.05) ( Table 3 ). We further calculated the power to illustrate false negative errors. For the power calculation, we set the target difference for categorical variables as 20%. The results showed that the powers for LBR, CPR, and ongoing pregnancy rate were 0.69, 0.77, and 0.71, respectively, which suggest that the false negative errors for our results were acceptable.

In the vaccinated group, nine patients had ectopic pregnancies and nine patients had late miscarriages, compared with five and three patients, respectively, from the unvaccinated group. There were 282 (137 boys and 145 girls) and 171 (87 boys and 84 girls) live-born infants in the vaccinated and unvaccinated groups, respectively. No birth defects were found in the study. The mean gestational age at delivery was 38 weeks (28–41 weeks), and the mean birth weight was 2,847 g (500–4,430 g).

In both the dose and interval subgroups, no significant differences were found in the IR, CPR, LBR, rates of early miscarriage and ongoing pregnancy, gestational age at delivery, and birth weight of infants (p > 0.05) ( Table 4 ).