The association between pregnancy outcomes of frozen thawed embryo transfers and previous SARS-CoV-2 infections.

This study was approved by the Ethics Committee of Xiangya Hospital, Central South University. This committee determined that the study was exempt from the requirement for informed consent, due to its retrospective design. This study was performed in accordance with the Declaration of Helsinki. This was a multicenter, retrospective case-control study. As the control of COVID-19 has been relaxed since December 7, 2022 in China, the present study concluded couples who had undergone FET, between February 2022 and December 2022, at six reproductive centers in four provinces across China. The SARS-CoV-2-positive cohort included couples in which either partner was infected with SARS-CoV-2 during previous COS and underwent FET, with embryos obtained during a SARS-CoV-2 infection. The SARS-CoV-2-negative cohort included couples in which neither partner had a past history of SARS-CoV-2 infection, who underwent FET before the COVID-19 control policy had been relaxed. The inclusion criteria for the SARS-CoV-2-positive cohort were as follows: (1) SARS-CoV-2 infection during controlled ovarian stimulation (COS); (2) freezing of all embryos after previous COS; (3) recovery from COVID-19; and (4) on FET treatment. The inclusion criteria for the SARS-CoV-2 -negative cohort were as follows: (1) FET treatment; and (2) no history of SARS-CoV-2 infection. Furthermore, couples who met the following criteria were excluded from our study: (1) undergoing oocyte or sperm donation cycles; (2) embryos obtained from oocyte or sperm freezing cycles; (3) no transferable embryos after warming; (4) obvious factors affecting embryo implantation, such as intrauterine adhesions, severe hydrosalpinx, adenomyosis, Autoimmune system diseases, Uncorrected endocrine disease; (5) PGT cycles; and (6) lost to follow-up. As the control of COVID-19 has relaxed since December 2022, an increasing number of infertile couples undergoing ART had become infected with SARS-CoV-2. Therefore, the data of patients in the SARS-CoV-2-negative cohort were obtained from February 2022 to November 2022. To clarify the association between FET, with embryos obtained during SARS-CoV-2 infection and the outcomes of FET by sex, SARS-CoV-2-positive patients were further subdivided into three sub-cohorts: the female SARS-CoV-2-positive sub-cohort (in which only the female partner was infected with SARS-CoV-2 during COS), the male SARS-CoV-2-positive sub-cohort (in which only the male partner was infected with SARS-CoV-2), and the co-infected sub-cohort (in which both partners were infected with SARS-CoV-2). To eliminate possible confounding factors resulting from the different reproductive centers, the same proportion of sample size of the study cohort, as opposed to that of the control cohort was ensured for each reproductive center. Endometrial preparation was performed, as per our previous study 10 . The natural cycles (NC) protocol involved the development of the dominant follicle; and the endometrium was monitored from day 10, by regular vaginal ultrasonography. When the diameter of the dominant follicle was > 14 mm, the serum luteinizing hormone (LH), estradiol (E2), and progesterone (P4) concentrations were measured. Endometrial transformation with P4 was initiated on the day of ovulation or 2 days after the serum LH peak (LH ≥ 45-65IU/L). The artificial cycle (AC) protocol involved the administration of E2 from the third day of the menstrual cycle, which was continued for at least 12 days. Endometrial thickness was monitored using transvaginal ultrasonography every 3 to 5 days. When the endometrial thickness was > 7 mm, serum E2 > 100pg/mL, and P4 < 1ng/mL, additional P4 was initiated for endometrial transformation. Letrozole for ovarian induction treatment was initiated on the third day of menstruation for 5 consecutive days. From the 10th day of the menstrual cycle, transvaginal ultrasonography was performed to monitor the growth of the follicles and endometrium. Human menopausal gonadotrophin (hMG) was used to promote follicular development, when necessary. When the dominant follicle size was > 18 mm, 6000-10000IU human chorionic gonadotropin (hCG) was injected to promote further maturation of the follicles and ovulation. P4 was added for endometrial transformation, starting on the day of ovulation or 2 days after the hCG injection. No more than 2 embryos were thawed and transferred under abdominal ultrasonographic guidance. Cleavage-stage embryos or blastocysts were thawed and transferred on the third and fifth days after endometrial transformation, respectively. Progestin for luteal phase support was continued either until 2 months after embryo transfer in cases of confirmed intrauterine pregnancy, or until pregnancy was excluded. Biochemical pregnancy was determined using the hCG test, 12 days after embryo transfer. A gestational sac visualized by vaginal ultrasonography, 28–35 days after the transfer was defined as a clinical pregnancy. Among them, the presence of two gestational sacs in the uterine cavity was defined as twin pregnancy. Intrauterine pregnancy beyond 12 weeks of gestation was defined as an ongoing pregnancy, and a pregnancy loss before 12 weeks of gestation was defined as an early miscarriage. After 28 weeks of gestation, the delivery of a viable baby was called a live birth. The primary outcome was the ongoing pregnancy rate. The secondary outcomes were biochemical and clinical pregnancy, implantation, and early miscarriage rates. The outcomes were calculated as follows: Clinical pregnancy rate (CPR) = number of cycles with a clinical pregnancy/number of FET cycles; biomedical pregnancy rate (BPR) = number of cycles with positive hCG 12–15 days after embryo transfer/number of FET cycles; implantation rate (IR) = number of gestational sacs/total number of embryos transferred; early miscarriage rate (EMR) = number of cycles with an early miscarriage/number of cycles with clinical pregnancy; ongoing pregnancy rate (OPR) = number of cycles with ongoing pregnancy/number of FET cycles; Twin pregnancy rate (TPR) = number of cycles with twin pregnancy/number of cycles with clinical pregnancy; live birth rate (LBR) = number of cycles with live birth/number of FET cycles. Embryo grading was based on the Istanbul consensus workshop parameters 11 . All data were analyzed using SPSS software (version 22.0; IBM Corp.). Normality was tested using the Shapiro–Wilk test. Continuous variables were expressed as means (SDs) for normally distributed data ( P  > 0.05) and as medians (IQRs) for non-normally distributed data. The Mann–Whitney U or Kruskal–Wallis tests were used for statistical analysis. Categorical data were described as a number (percentage) and analyzed by the χ 2 or Fisher exact tests. Logistic regression analysis was used to adjust for confounding factors of outcomes with statistically significant differences. Statistical significance was set at a 2-sided P  < 0.05.

A total of 440 infertile couples who underwent FET were included in this study, with 110 and 330 cycles in SARS-CoV-2-positive and SARS-CoV-2-negative cohorts, respectively. The baseline patient characteristics are shown in Table  1 . There were no statistically significant differences in the female age, body mass index, infertility type, infertility factor, and previous COS protocol, between the SARS-CoV-2-positive and SARS-CoV-2 -negative cohorts. This was with the exception of the infertility duration, which was longer in the SARS-CoV-2 -negative cohort than that in the SARS-CoV-2 -positive cohort (median [IQR], 3.13 [1.81–5.00] years; 2.0 [1.00–4.00] years, respectively; P  < 0.001). Table 1 Baseline characteristics of SARS-CoV-2–Positive and SARS-CoV-2–Negative Cohorts a . Characteristic SARS-CoV-2 negative ( n  = 330) SARS-CoV-2 positive ( n  = 110) Median difference (95% CI) OR (95% CI) P Female age, median (IQR), y 32 (29.0 to 35.0) 32 (28.0 to 35.3) 0.0(−1.0 to 1.0) NA 0.613 BMI, median (IQR) 22.0 (20.3 to 24.3) 21.9 (20.3 to 24.2) 0.0(−0.6 to 0.7) NA 0.936 Infertility duration, median (IQR), y 3.1 (1.8 to 5.0) 2.0 (1.0 to 4.0) 1.0(0.5 to 1.33) NA <0.001 Infertility type NA 0.9(0.6 to 1.3) 0.544 Primary 172 (52.1) 61 (55.5) Secondary 158 (47.9) 49 (44.5) Infertility factor b NA NA 0.003 Tubal factor 177(53.6) 60(54.6) Ovarian factor 34(10.3) c 29(26.36) d Uterine factor 24(7.3) 10(9.1) Male factor 140(42.4) 43(39.1) Unexplained 43(13.0) 9(8.2) COS protocols NA NA 0.519 GnRH agonist 82 (24.8) 26 (23.6) GnRH antagonist 186 (56.4) 68 (61.8) Others e 62 (18.8) 16 (14.5) Abbreviations: BMI, body mass index (calculated as weight in kilograms divided by height in meters squared); COS, controlled ovarian stimulation; GnRH, gonadotropin releasing hormone. a Data are presented as number (percentage) of study couples unless otherwise indicated. b Ovarian factor, including diminished ovarian reserve, polycystic ovary syndrome and anovulation; Uterine factor, including endometriosis, intrauterine adhesion and adenomyosis. c VS d, P  < 0.05. e Others, including mild stimulation and luteal-phase stimulation protocols. Baseline characteristics of SARS-CoV-2–Positive and SARS-CoV-2–Negative Cohorts a . Abbreviations: BMI, body mass index (calculated as weight in kilograms divided by height in meters squared); COS, controlled ovarian stimulation; GnRH, gonadotropin releasing hormone. a Data are presented as number (percentage) of study couples unless otherwise indicated. b Ovarian factor, including diminished ovarian reserve, polycystic ovary syndrome and anovulation; Uterine factor, including endometriosis, intrauterine adhesion and adenomyosis. c VS d, P  < 0.05. e Others, including mild stimulation and luteal-phase stimulation protocols. As shown in Table  2 , there were no statistically significantly differences in the number of treatment cycles; endometrial preparation protocol, thickness, and pattern; as well as serum LH, E2, and P4 levels on the day of endometrial transformation, between the SARS-CoV-2 -positive and SARS-CoV-2 -negative cohorts. Moreover, statistically significant differences in the number, type, source, and quality of embryos transferred were not present between these two cohorts. The interval time between embryo freezing and transfer was longer in the SARS-CoV-2 -positive cohort than that in the SARS-CoV-2-negative cohort (median [IQR], 86.5 [58.35–137.36] days; 67.0 [52.00–104.50] days, respectively; P  < 0.001). Generally, baseline characteristics were comparable between the two cohorts. Table 2 Fertility evaluation of Frozen–Thawed embryo transfers between SARS-CoV-2–Positive and SARS-CoV-2–Negative Cohorts. Characteristic SARS-CoV-2 negative ( n  = 330) SARS-CoV-2 positive ( n  = 110) Median difference (95% CI) OR (95% CI) P No. of treatment cycles, median (IQR), No. 2 (2 to 3) 2 (2 to 3) 0.0(0.0 to 0.0) NA 0.563 Protocol for endometrial preparation NA NA 0.839 NC 79 (23.9) 27 (24.5) HRT 245 (74.2) 80 (72.7) OI 6 (1.8) 3 (2.7) Endometrial thickness (mm) 9.8 (8.7 to 11.0) 9.4 (8.38 to 11.0) 0.0(−0.2 to 0.5) NA 0.548 Endometrial pattern NA NA 0.141 A 139 (42.1) 40 (36.4) B 152 (46.1) 49 (44.5) C 39 (11.8) 21 (19.1) Hormone levels on endometrial transformation day Estradiol, pg/mL 210.3 (135.7 to 395.7) 192.1 (135.6 to 373.5) 2.6(−33.1 to 40.2) NA 0.822 Progesterone, ng/mL 0.4 (0.2 to 0.8) 0.32 (0.2 to 0.8) 0.0(−0.1 to 0.1) NA 0.725 Luteinizing hormone, IU/L 10.3 (1.8 to 28.0) 11.6 (4.3 to 18.9) 0.2(−3.3 to 4.1) NA 0.810 Source of embryo transferred (%) NA NA 1.000 IVF 238 (72.1) 80 (72.7) ICSI 91 (27.6) 30 (27.3) IVF + ICSI 1 (0.3) 0 (0.0) Type of embryo transferred (%) NA 0.9(0.5 to 1.6) Cleavage 103 (31.2) 38 (34.5) 0.516 Blastocyst 227 (68.8) 72 (65.5) No. of embryo transferred 439 151 NA 1.2(0.8 to 1.9) 0.416 1 (%) 221 (67.0) 69 (62.7) 2 (%) 109 (33.0) 41 (37.3) Quality of embryo transferred (%) NA NA 0.739 Top quality 191(57.9) 59(53.6) Non-top quality 112(33.9) 41(37.3) Top and non-top quality 27(8.2) 10(9.1) Interval time between embryo freezing and transfer (day) 67.0(52.0to104.5) 86.5(58.4 to 137.4) −18.0(−27.0 to −7.0) NA <0.001 a Abbreviations: CI, confidence interval; OR, odds ratio; NA, not applicable; IQR, interquartile range; NC, natural cycle; HRT, hormone replacement treatment; OI, ovulation induction; IVF, in vitro fertilization; ICSI, intracytoplasmic sperm injection. Fertility evaluation of Frozen–Thawed embryo transfers between SARS-CoV-2–Positive and SARS-CoV-2–Negative Cohorts. a Abbreviations: CI, confidence interval; OR, odds ratio; NA, not applicable; IQR, interquartile range; NC, natural cycle; HRT, hormone replacement treatment; OI, ovulation induction; IVF, in vitro fertilization; ICSI, intracytoplasmic sperm injection. The pregnancy outcomes of FET are shown in Table  3 . The implantation (31.1%; 47.2%, respectively; P  = 0.001), clinical pregnancy (42.7%; 54.8%, respectively; P  = 0.028), ongoing pregnancy (35.5%; 48.2%, respectively; P  = 0.020), and live birth (29.1%; 47.3%, respectively; P  = 0.001) rates in the SARS-CoV-2 -positive cohort were statistically significantly lower than that in the SARS-CoV-2 -negative cohort. No statistically significant differences were observed in the early miscarriage rate and twin pregnancy rate, birth weights between the two cohorts. Table 3 Pregnancy outcomes of Frozen–Thawed embryo transfers between SARS-CoV-2–Positive and SARS-CoV-2–Negative Cohorts. Characteristic SARS-CoV-2 negative ( n  = 330) SARS-CoV-2 positive ( n  = 110) OR (95% CI) P Biochemical pregnancy rate (%) 219(66.4) 58(52.7) 0.57(0.37 to 0.88) 0.01 Implantation rate (%) 207 (47.2) 47 (31.1) 0.51(0.34 to 0.75) 0.001 Early miscarriage rate (%) 22 (12.2) 4 (8.5) 0.67(0.22 to 2.06) 0.658 Clinical pregnancy rate (%) 181 (54.8) 47 (42.7) 0.61(0.40 to 0.95) 0.028 Twin pregnancy rate (%) 26 (14.4) 3 (6.4) 0.41 (0.12 to 1.41) 0.223 Ongoing pregnancy rate (%) 159 (48.2) 39 (35.5) 0.59(0.38 to 0.92) 0.020 Live birth rate (%) 156 (47.3) 32 (29.1) 0.46 (0.29 to 0.73) 0.001 Birth weight (g) 3300 (2795 to 3500) 3150 (2675 to 3450) NA 0.875 a Abbreviations: OR, odds ratio; CI, confidence interval. Pregnancy outcomes of Frozen–Thawed embryo transfers between SARS-CoV-2–Positive and SARS-CoV-2–Negative Cohorts. a Abbreviations: OR, odds ratio; CI, confidence interval. Table S1 in the supplement shows the association between infection, based on sex and FET treatment outcomes; and the study population was stratified into female partner, male partner, and both partners infected during COS. No statistical differences were found in the baseline characteristics and fertility evaluation of FET among the sub-cohorts, except for the infertility duration and time interval between the embryo freezing and transfer. Compared with the SARS-CoV-2 -negative cohort, the clinical pregnancy and ongoing pregnancy rates of the three positive sub-cohorts showed a decreasing trend, without a statistically significant difference. The implantation and live birth rates in the female and male SARS-CoV-2 positive-sub-cohorts showed a downward trend, and these rates were statistically significantly lower in the SARS-CoV-2 -positive-sub-cohort where both partners were infected. Multivariate logistic regression analysis revealed that FET, with embryos obtained during SARS-CoV-2 infection was negatively associated with clinical pregnancy outcomes, including implantation and clinical and ongoing pregnancy rates (Table  4 ). Table 4 Logistics regression analysis for the pregnancy outcomes. Clinical pregnancy Ongoing pregnancy rate Implantation rate Variables B P Exp (B) 95% CI of B B P Exp (B) 95% CI of B B P Exp (B) 95% CI of B Lower Upper Lower Upper Lower Upper (Constant) −0.39 0.73 0.68 −0.41 0.71 0.66 −0.18 0.87 0.83 Age 0.09 < 0.01 1.09 1.04 1.14 0.12 < 0.01 1.12 1.07 1.18 0.08 < 0.01 1.08 1.03 1.13 Primary infertility $ 0.48 0.03 1.61 1.05 2.48 0.20 0.36 1.23 0.80 1.89 0.53 0.016 1.69 1.10 2.60 Endometrial thickness (mm) −0.09 0.11 0.91 0.81 1.02 −0.14 0.02 0.87 0.78 0.98 −0.09 0.12 0.91 0.82 1.02 Type of embryo transferred (Cleavage) & −1.11 < 0.01 0.33 0.19 0.58 −0.83 0.01 0.44 0.25 0.78 −1.10 < 0.01 0.33 0.19 0.59 Quality of embryo transferred (Non) # −0.70 0.11 0.50 0.21 1.16 −0.48 0.29 0.62 0.26 1.49 −0.63 0.15 0.53 0.23 1.25 Quality of embryo transferred (Top and non) # 0.15 0.74 1.16 0.48 2.84 −0.07 0.89 0.93 0.37 2.35 0.21 0.65 1.23 0.50 3.01 No. of embryo transferred(1) @ − 0.065 0.02 0.52 0.30 0.90 −0.53 0.06 0.59 0.34 1.02 −0.58 0.04 0.56 0.33 0.97 Group (Positive) ! − 0.530 0.026 0.59 0.37 0.94 −0.91 < 0.01 0.40 0.25 0.66 −0.65 0.01 0.52 0.33 0.84 $ Secondary infertility was set as the reference category. & Blastocyst transferred was set as the reference category. # Top quality of embryo transferred was set as the reference category. @ 2 embryo transferred was set as the reference category. ! SARS-CoV-2 negative group was set as reference category. Logistics regression analysis for the pregnancy outcomes. Primary infertility $ Endometrial thickness (mm) Type of embryo transferred (Cleavage) & Quality of embryo transferred (Top and non) # Group (Positive) ! $ Secondary infertility was set as the reference category. & Blastocyst transferred was set as the reference category. # Top quality of embryo transferred was set as the reference category. @ 2 embryo transferred was set as the reference category. ! SARS-CoV-2 negative group was set as reference category.

This case-control study demonstrated that FET using embryos obtained during SARS-CoV-2 infection was negatively associated with clinical pregnancy. The negative impact of COVID-19 on human reproductive health should be emphasized, and greater efforts are needed to improve clinical interventions and public health policies. Prospectively, continued follow-ups and observations of neonatal outcomes are essential in affected populations.

To the best of our knowledge, this study is the second of its kind, with a larger sample size that investigated the association between the transfer of frozen-thawed embryos, obtained during SARS-CoV-2 infection; and the increased risk of adverse pregnancy outcomes, in a cohort of Chinese couples undergoing FET. The results of this multicenter, retrospective case-control study demonstrated that transfer of frozen-thawed embryos obtained during SARS-CoV-2 infection was negatively associated with clinical pregnancy outcomes. In December 2022, infertile couples who underwent ART and completed the COS procedure and oocyte retrieval, and the number of people infected with SARS-CoV-2 increased sharply, according to the COVID-19 epidemic control policy in China. Regarding the paucity of data on the possible peri-implantation transmission of SARS-CoV-2 to the conceptus, we adhered to a strict freeze-all strategy to avoid conception in SARS-CoV-2 positive patients. Months later, female patients recovered from the COVID-19 illness and came to for FET. The results of our study generally showed that the implantation rate, clinical pregnancy rate, ongoing pregnancy rate, and live birth rates were statistically significantly lower in the SARS-CoV-2 positive cohort, in which the transferred embryos were obtained during SARS-CoV-2 infection compared to that in the SARS-CoV-2 negative cohort. Conversely, Boudry et al. found that the clinical outcomes in eight patients after FET were favorable 9 . Recently, a retrospective study showed that women who tested positive for SARS-CoV-2 during FET cycles have similar clinical pregnancy, ongoing pregnancy rates with women without SARS-CoV-2 infection 12 . However, a statistically significant reduction in pregnancy rates has been observed in patients with a past history of SARS-CoV-2 infection than that in patients without this past history 4 , 5 . Even after recovery from the COVID-19, 3 to 9 months after SARS-CoV-2 infection, IgG antibodies against SARS-CoV-2 in the follicular fluid have been found to be inversely associated with female reproductive outcomes 13 . This observation has confirmed the possible long-term effects of the SARS-CoV-2 infection on embryo implantation and the subsequent course of pregnancy. Nonetheless, the mechanism by which SARS-CoV-2 affects reproductive outcomes has not yet been elucidated. One possible explanation for this involves the endometrial factors. Angiotensin-converting enzyme (ACE)−2 receptors have been found in the endometrium. Moreover, it has been established that the endometrium can potentially be exposed to the SARS-CoV-2 virus, which may negatively interfere with embryo implantation 14 . Additionally, the hypercoagulable state induced by the COVID-19 “cytokine storm” could result in a toxic endometrial environment and microthrombus formation, resulting in hypoperfusion. It remains known whether endometrial toxicity resolves over time. However, an analysis of cell entry factors for SARS-CoV-2, by single-cell RNA-sequencing, in the pre-conceptional human endometrium reveals a low risk of infection 15 . A recently study found that COVID-19 infection after oocyte retrieval does not exhibit a damaging effect on endometrial receptivity for embryo implantation during FET 3 . Another possible explanation involves the presence of embryonic factors. Published small conditional RNA sequencing datasets have revealed that ACE-2 receptors are expressed in early embryos, before the eight-cell stage; and in the trophectoderm (TE) of the late blastocyst 16 . Other studies 17 – 19 , 20 have confirmed that ACE-2 receptors and transmembrane serine protease (TMPRSS)−2 proteins are co-expressed in the TE cells of peri-implantation embryos. Moreover, the co-expression of ACE-2 receptors and TMPRSS-2 proteins in both the ectodermal and trophectodermal cells of human metaphase II oocytes, zygotes, and blastocysts marks a theoretical opportunity of invasion by the SARS-CoV-2 virus. Human embryos possess all the machinery required for SARS-CoV-2 binding, internalization, and replication. Furthermore, one study explored 17 ACE-2 receptor and TMPRSS-2 protein expression patterns in peri-implantation embryos and the maternal–fetal interface, using previously published single-cell transcriptome data. The findings thereof reveal the strong co-expression of ACE-2 receptors and TMPRSS-2 proteins by day 6 TE cells in peri-implantation embryos; as well as by syncytiotrophoblast and extravillous trophoblast cells, at 8 and 24 weeks of gestation, respectively, at the maternal-fetal interface. This is indicative of the susceptibility to SARS-CoV-2 infection. A second ACE-2-receptor independent mechanism has been described recently, involving CD147, also known as Basigin as the cellular receptor; and cathepsin L as the protease 21 , 22 . Additionally, the presence of the CD147 protein has been demonstrated on human oocytes and preimplantation embryos 23 . Our previous multicenter cohort study found that couples infected by SARS-CoV-2 during COS had statistically significantly lower top-quality embryo and blastocyst, blastocyst formation, and available blastocyst rates than that of couples who were not infected 7 . Even recover from COVID-19, women with a previous COVID-19 infection have a reduced proportion of top-quality embryos 24 . Another study found that previous SARS-CoV-2 maternal infections may compromise embryo morphokinetics. This study revealed a higher pregnancy rate and lower miscarriage rate in female patients with a negative SARS-CoV-2 immunoglobulin test than that in female patients with a positive SARS-CoV-2 immunoglobulin test, in the last 6 months 6 . Our study further analyzed the association between FET and the clinical outcomes thereof, in which either the female or male partner, or both, were infected with SARS-CoV-2, during COS. The female and male SARS-CoV-2-positive sub-cohorts showed lower clinical and ongoing pregnancy rates than that of the SARS-CoV-2-negative cohort, without statistically significant differences. These results indicate that a previous SARS-CoV-2 infection during COS in either partner is negatively associated with clinical pregnancy outcomes after FET. However, these results should be cautiously interpreted, due to the small sample sizes of the sub-cohorts. We also found that FET with embryos obtained during SARS-CoV-2 infection was no associated with early miscarriage. Moreover, consistent with the findings of our study, a national cohort study examining administrative claims from 78,283 pregnancies found that SARS-CoV-2 infection during pregnancy was not associated with the risk of a spontaneous abortion (SAB) 25 . A meta-analysis of 27 studies regarding the COVID-19 disease revealed a miscarriage rate of 15.3% and 23.1%, using fixed and random effects models, respectively 26 , which is in the normal population range. Joseph et al. 27 have reported on the effects of COVID-19 on pregnancy and its implications for female reproductive health. Nevertheless, an association between SARS-CoV-2 infection during the early weeks of gestation and situs inversus have been found 28 . Generally, uncertainty exists regarding an association between COVID-19 and an early pregnancy loss or a pattern of birth defects. Firstly, we included samples from six reproductive centers in four provinces across China, with fixed proportions in the study and control cohorts. Secondly, the associations between previous maternal and paternal infections and clinical outcomes were compared separately. Finally, this study had a relatively long follow-up period; in addition to the short-term pregnancy outcomes, such as clinical pregnancy and embryo implantation rates, we followed up the ongoing pregnancies and live birth. This study had some limitations. First, this was a retrospective study with inherent biases in data collection that were not uniformly generated in the study cohort. Second, we did not follow up live birth outcomes after FET. Nonetheless, this association should be further evaluated by extending the follow-up period. Third, we performed a sub-cohort analysis based on sex; however, the sample sizes of the sub-cohorts were relatively small.

Over the past 3 years, the SARS-CoV-2 infection that has resulted in the COVID-19 worldwide pandemic has had a huge impact on human health. According to the official website of the World Health Organization, as of October 12, 2023, a total of 7.71 million people have been diagnosed with COVID-19; and 6.9 million people have died. Definitively, SARS-CoV-2 infection has adversely affected multiple organs and systems of the body, including the human reproduction system 1 . Whether SARS-CoV-2 infection affects the outcomes of assisted reproductive technology (ART) therapy is a growing public concern for infertile couples and reproductive physicians. Due to the limitation of samples and controls, most existing studies on this topic are speculative and have a low level on the hierarchy of evidence. Moreover, these studies focused on the association between previous SARS-CoV-2 infection in infertile couples and the ART outcomes 2 , 3 , 4 , 5 , 6 . Few studies have reported ART outcomes in infertile couples with acute SARS-CoV-2 infection, during ART treatment. Our previous study has demonstrated an association between SARS-CoV-2 infection and impaired oocyte and blastocyst quality 7 . Another study has shown a diminished oocyte utilization rate of two - pronuclear (2PN) zygotes and good-quality embryos in women infected with SARS-CoV-2, within 7 days before oocyte retrieval 8 . Thus far, now, only one study reported the outcomes after frozen-thawed embryo transfer (FET), with embryos obtained during SARS-CoV-2 infection. Moreover, the findings of the study revealed that the fertilization rate, embryo development, and clinical outcomes after embryo transfer are favorable 9 . However, the results there of should be regarded cautiously, due to the small sample size of eight patients. Consequently, thus far, the FET outcomes, with embryos obtained during SARS-CoV-2 infections are yet to be elucidated. Therefore, this study aimed to explore whether the transfer of frozen-thawed embryos, obtained during a SARS-CoV-2 infection is associated with an increased risk of adverse pregnancy outcomes, in Chinese couples undergoing FET.

Below is the link to the electronic supplementary material. Supplementary Material 1 Supplementary Material 1