Endometrial injection of embryo culture supernatant adversely affects assisted reproductive technology outcomes in frozen thawed embryo transfer cycles.

During the study period, 1187 hormone replacement therapy cycles were conducted for FETs involving embryos that reached the blastocyst stage on day five. After exclusion based on the study protocol, 996 cycles were considered eligible. Among these, the SEET group had 118 cycles, whereas the BT group had 878 cycles (Table  1 ). Maternal age and serum estradiol 2 (E2) levels on the day of embryo transfer was predominantly higher in the SEET group than in the BT group (both p  < 0.05), whereas the body mass index did not significantly differ between the groups. There were no significant differences between the groups in serum anti-Müllerian hormone levels, endometrial thickness on the day of embryo transfer, serum progesterone 4 (P4) levels on the day of embryo transfer, GQB rate, and assisted hatching rate. Table 1 Baseline characteristics of the study participants. Characteristics SEET group (118 cycles) BT group (878 cycles) p -value Maternal age 38 (29–48) 37 (22–48) < 0.05 Previous embryo transfer cycles 1 (0–7) 0 (0–9) < 0.001 BMI (kg/m 2 ) 23.3 (15.4–28.9) 21.8 (15.4–38.6) N.S. AMH (ng/mL) 2.4 (0.08–11) 2.8 (0.02–18.5) N.S. Basal FSH (mIU/mL) 7.0 (1.4–15.1) 6.9 (0–31.2) N.S. Basal LH (mIU/mL) 4.7 (0.8–10.9) 4.7 (0–14.8) N.S. Basal E2 (pg/mL) 33.1 (0–95.9) 32.4 (0–99.6) < 0.05 Endometrial thickness on the day of the embryo transfer was determined (mm) 10.4 (6.2–16.1) 9.9 (6.4–19.8) N.S. Serum E2 value on the embryo transfer day (pg/mL) 255 (64.6–1730) 230 (26.7–1745) N.S. Serum P4 value on the embryo transfer day (ng/mL) 12.4 (2.2–30.9) 12.1 (1.2–26.3) N.S. GQB (%[n]) 89.0 (105) 84.9 (745) N.S. Assisted hatching (%[n]) 94.1 (111) 87.6 (769) < 0.05 Infertility causes (total number of causes) 152 1053 N.S.  Male factor 53 (34.9%) 358 (34.0%) N.S.  Tubal factor 12 (7.9%) 55 (5.2%) N.S.  Cervical factor 3 (2.0%) 8 (0.8%) N.S.  Endometriosis 9 (5.9%) 54 (5.1%) N.S.  Polycystic ovarian syndrome 3 (2.0%) 45 (4.3%) N.S.  Luteal phase deficiency 0 (0%) 7 (0.7%) N.S.  Hyperprolactinemia 0 (0%) 9 (0.9%) N.S.  Thyroid dysfunction 5 (3.3%) 26 (2.5%) N.S.  Anti-sperm-antibodies 1 (0.7%) 11 (1.0%) N.S.  Uterine fibroid 6 (3.9%) 34 (3.2%) N.S.  Adenomyosis 0 (0%) 3 (0.3%) N.S.  Asherman syndrome 0 (0%) 2 (0.2%) N.S.  Uterine malformation 1 (0.7%) 14 (1.3%) N.S.  Hydrosalpinx 0 (0%) 2 (0.2%) N.S.  Diminished ovarian reserve 9 (5.9%) 97 (9.2%) N.S.  Hypothalamic-pituitary insufficiency 0 (0%) 3 (0.3%) N.S.  Unexplained infertility 50 (32.9%) 315 (29.9%) N.S.  Oncofertility 0 (0%) 10 (0.9%) N.S. AMH, anti-Müllerian hormone; BMI, body mass index; BT, blastocyst transfer; E2, estradiol; FSH, follicle-stimulating hormone; GQB, good-quality blastocyst; LH, luteinizing hormone; P4, progesterone; SEET, stimulation of endometrial embryo transfer. The data were presented as median (interquartile range). Baseline characteristics of the study participants. AMH, anti-Müllerian hormone; BMI, body mass index; BT, blastocyst transfer; E2, estradiol; FSH, follicle-stimulating hormone; GQB, good-quality blastocyst; LH, luteinizing hormone; P4, progesterone; SEET, stimulation of endometrial embryo transfer. The data were presented as median (interquartile range). The implantation, clinical pregnancy, ongoing pregnancy, and live birth rates were lower in the SEET group than in the BT group, with significantly lower implantation ( p  < 0.05), ongoing pregnancy ( p  < 0.05), and live birth rates ( p  < 0.05), and a significantly higher miscarriage rate ( p  < 0.05) in the SEET group compared to the BT group (Table  2 ). Serum levels of E2, P4, and human chorionic gonadotropin (hCG) 9 days after embryo transfer did not show significant differences between the groups. Table 2 Outcomes of assisted reproductive technology for the study participants. ART outcomes SEET group (118 cycles) BT group (878 cycles) p -value Implantation rate (GQB/non-GQB) 40.2 (44.1/7.1) 51.1(55.7/25.4) < 0.05 Clinical pregnancy rate (GQB/non-GQB) 35.6 (39.0/7.1) 38.3 (42.5/14.8) 0.55 Ongoing pregnancy rate (GQB/non-GQB) 18.2 (20.3/0.0) 26.8 (30.5/8.5) < 0.05 Live birth rate (GQB/non-GQB) 18.2 (20.3/0.0) 26.2 (29.4/8.5) < 0.05 Miscarriage rate (GQB/non-GQB) 48.9 (47.8/100) 31.5 (30.7/42.9) < 0.05 Serum E2 level 9 days after ET (pg/mL) 274 (26–1725) 259 (10.2–1726) 0.84 Serum P4 level 9 days after ET (ng/mL) 11.1 (0.35–25.3) 11.4 (0.06–44.5) 0.68 Serum hCG level 9 days after ET in implantation cycles (mIU/mL [n]) 104 (11–474 [27]) 128 (5–515 [228]) 0.84 ART, assisted reproductive technology; BT, blastocyst transfer; ET, embryo transfer; E2, estradiol; hCG, human chorionic gonadotropin; P4, progesterone; SEET, stimulation of endometrial embryo transfer. The data were presented as median (interquartile range). Outcomes of assisted reproductive technology for the study participants. ART, assisted reproductive technology; BT, blastocyst transfer; ET, embryo transfer; E2, estradiol; hCG, human chorionic gonadotropin; P4, progesterone; SEET, stimulation of endometrial embryo transfer. The data were presented as median (interquartile range). The age-adjusted OR (95% confidence intervals [CIs]) for clinical pregnancy was 1.02 (0.69–1.52) in the SEET group compared with the BT group (Fig.  1 ). This age-adjusted OR remained consistent when stratified by maternal age and the number of BT failures. Fig. 1 Age-adjusted odds ratio for clinical pregnancy after endometrial injection of the embryo culture supernatant. Odds ratio was calculated for maternal age categories of ≥ 37 years and < 37 years, and the number of blastocyst transfer failures of < 2 and ≥ 2. BT, blastocyst transfer. Age-adjusted odds ratio for clinical pregnancy after endometrial injection of the embryo culture supernatant. Odds ratio was calculated for maternal age categories of ≥ 37 years and < 37 years, and the number of blastocyst transfer failures of < 2 and ≥ 2. BT, blastocyst transfer. The age-adjusted OR for miscarriage in the SEET group compared with the BT group was 1.97 (95% CI 1.04–3.72) (Fig.  2 ). This age-adjusted OR remained consistent when stratified by the number of BT failures. However, when stratified by maternal age, a notable difference emerged. For participants aged < 37 years, the age-adjusted OR for miscarriage was 0.92 (95% CI 0.33–2.58), whereas, for those aged ≥ 37 years, it was 3.43 (95% CI 1.40–8.44) in the SEET group compared with the BT group. Fig. 2 Age-adjusted odds ratio for miscarriage after endometrial injection of the embryo culture supernatant. Odds ratio was calculated for maternal age categories of ≥ 37 years and ˂37 years and number of blastocyst transfer failures of < 2 and ≥ 2. BT, blastocyst transfer. Age-adjusted odds ratio for miscarriage after endometrial injection of the embryo culture supernatant. Odds ratio was calculated for maternal age categories of ≥ 37 years and ˂37 years and number of blastocyst transfer failures of < 2 and ≥ 2. BT, blastocyst transfer. Logistic regression analysis showed that maternal age had an adjusted odds ratio (aOR) (95% CI) of 0.885 (0.853–0.918) for clinical pregnancy, and GQBs had an aOR (95% CI) of 4.24 (2.45–7.36), both of which were statistically significant (Table  3 ). In contrast, the aOR (95% CI) for clinical pregnancy associated with endometrial injection of embryo culture supernatant was 1.00 (0.634–1.58), indicating no significant difference. These trends remained consistent when the analysis was stratified by age, including those aged ≥ 37 and < 37 years. Table 3 Evaluation of endometrial injection of embryo culture supernatant as a predictor of clinical pregnancy. Predictors Adjusted odds ratio (95% CI) All ≥ 37 years of age < 37 years of age Maternal ages 0.89 (0.85–0.92)*** 0.87 (0.79–0.96)** 0.92 (0.85–0.99) * BMI 1.00 (0.96–1.04) 1.01 (0.96–1.06) 0.99 (0.92–1.05) AMH 1.03 (0.99–1.08) 1.04 (0.96–1.06) 1.02 (0.92–1.09) Basal FSH 0.98 (0.97–1.03) 1.00 (0.98–1.10) 0.98 (0.90–1.07) Endometrial thickness on the day the embryo transfer date was determined 1.05 (0.97–1.13) 1.00 (0.90–1.11) 1.12 (0.99–1.07) GQB 4.24 (2.45–7.36)*** 6.54 (2.73–15.60)*** 3.53 (1.66–7.51)** Endometrial injection of embryo culture supernatant 1.00 (0.63–1.58) 1.08 (0.61–1.92) 0.75 (0.37–1.55) Serum P4 value on embryo transfer day 1.02 (1.00-1.04) 1.01 (1.00-1.02) 1.02 (0.99–1.05) AMH, anti-Müllerian hormone; BMI, body mass index; CI, confidence intervals; FSH, follicle-stimulating hormone; GQB, good-quality blastocyst; P4, progesterone. * p  < 0.05, ** p  < 0.01, *** p  < 0.001. Evaluation of endometrial injection of embryo culture supernatant as a predictor of clinical pregnancy. AMH, anti-Müllerian hormone; BMI, body mass index; CI, confidence intervals; FSH, follicle-stimulating hormone; GQB, good-quality blastocyst; P4, progesterone. * p  < 0.05, ** p  < 0.01, *** p  < 0.001. For miscarriage, maternal age had an aOR (95% CI) of 1.08 (1.01–1.14), which was statistically significant, and endometrial injection of the embryo culture supernatant had an aOR (95% CI) of 2.08 (1.02–4.24), also statistically significant (Table  4 ). For participants aged ≥ 37 years, the trends remained consistent, and the aOR (95% CI) for miscarriage associated with endometrial injection of the embryo culture supernatant was notably higher at 4.63 (1.61–13.1). However, these findings were not statistically significant for individuals aged < 37 years. Table 4 Evaluation of endometrial injection of embryo culture supernatant as a predictor of miscarriage. Predictors Adjusted odds ratio (95% CI) All ≥ 37 years of age < 37 years of age Maternal ages 1.08 (1.01–1.14)* 1.26(1.06–1.50)* 1.07 (0.95–1.20) BMI 1.00 (0.94–1.06) 1.00 (0.92–1.09) 1.02 (0.92–1.12) AMH 0.98 (0.92–1.05) 1.01 (0.93–1.09) 0.95 (0.85–1.06) Basal FSH 0.99 (0.910–1.08) 0.99(0.88–1.12) 0.99 (0.86–1.12) Endometrial thickness on the day the embryo transfer date was determined 1.02 (0.90–1.15) 0.90 (0.74–1.09) 1.13 (0.96–1.34) GQB 0.68 (0.25–1.88) 1.70 (0.27–10.70) 0.547 (0.15–1.96) Endometrial injection of embryo culture supernatant 2.08 (1.02–4.24)* 4.63 (1.61–13.30)** 0.732 (0.22–2.47) Serum P4 value on embryo transfer day 1.01 (0.99–1.02) 1.01 (0.99–1.02) 1.01 (0.98–1.03) AMH, anti-Müllerian hormone; BMI, body mass index; CI, confidence intervals; FSH, follicle-stimulating hormone; GQB, good-quality blastocyst; P4, progesterone. * p  < 0.05, ** p  < 0.01, *** p  < 0.001. Evaluation of endometrial injection of embryo culture supernatant as a predictor of miscarriage. AMH, anti-Müllerian hormone; BMI, body mass index; CI, confidence intervals; FSH, follicle-stimulating hormone; GQB, good-quality blastocyst; P4, progesterone. * p  < 0.05, ** p  < 0.01, *** p  < 0.001.

This single-center, retrospective, observational cohort study was conducted at the Reproduction Center of Toho University Omori Medical Center, Tokyo, Japan, from January 2017 to March 2022. We included participants undergoing hormone replacement therapy cycles, with embryo transfers occurring on day 5 post-fertilization at the blastocyst stage. To mitigate any potential bias, we excluded participants with cycles involving multiple embryo transfers, transfers based on endometrial receptivity analysis (ERA ® , Igenomix, Valencia, Spain) results, transfers combined with platelet-rich plasma therapy, and transfers involving embryos that underwent preimplantation genetic testing for aneuploidy. We categorized participants into the SEET group, where participants underwent endometrial injection of the embryo culture supernatant during the embryo transfer cycle, and the blastocyst transfer (BT) group, where participants did not. The decision to perform endometrial injection of the embryo culture supernatant was based on information provided by the physician and the patient’s preference, particularly in cases where pregnancy had not been achieved in one or more previous embryo transfer cycles. The study conformed to the US Federal Policy for the Protection of Human Subjects. This study was approved by the Ethics Committee of Toho University Omori Medical Center (approval no. M22240 ) and conducted in accordance with the ethical standards outlined in the 1964 Declaration of Helsinki and its subsequent amendments or comparable ethical standards. The requirement for written informed consent was waived as information regarding the study was provided on the hospital website in an opt-out format, allowing potential research participants to decline participation. Embryo vitrification was performed using a vitrification medium (VT507) (Kitazato Corporation, Shizuoka, Japan). Embryos were equilibrated in a solution containing ethylene glycol, dimethyl sulfoxide, hydroxypropyl cellulose, and gentamicin before being vitrified in a solution containing higher concentrations of ethylene glycol, dimethyl sulfoxide, and sucrose. Subsequently, the samples were placed in liquid nitrogen. When participants requested endometrial injection of the embryo culture supernatant, we collected supernatants in serum tubes and stored them in liquid nitrogen (-196 °C). Embryo thawing was performed using a thawing kit (VT508) (Kitazato Corporation). After warming the embryos, a recovery culture was performed for 2–7 h, depending on blastocele expansion before embryo transfer. Assisted hatching was performed for patients who requested it. Starting from day 3 of menstruation, participants received a transdermal estradiol patch (Estrana tape, Hisamitsu Pharmaceutical Co., Inc., Saga, Japan). Subsequently, one of four transvaginal progesterone administrations (Lutinus vaginal tablets, Ferring Pharmaceuticals, Saint-Prex, Switzerland; luteum vaginal suppositories, ASKA Pharmaceutical Co., Ltd., Tokyo, Japan; OneCrinone vaginal gel, Merck Biopharma, Darmstadt, Germany; or UTROGESTAN Vaginal Capsules 200 mg, Fuji Pharma, Tokyo, Japan) commenced once the endometrial thickness reached at least 8 mm. Embryo transfer occurred five days after the administration of natural transvaginal progesterone. In the SEET group, 50 µL of the thawed embryo culture supernatant was transcervically injected into the uterine cavity two or three days before FET, using an embryo transfer catheter placed just beyond the internal os, with uterus stability confirmed using transabdominal ultrasonography. We compared implantation, clinical pregnancy, ongoing pregnancy, live birth, and miscarriage rates between the SEET and BT groups. The implantation rate, clinical pregnancy rate, ongoing pregnancy rate, and live birth rate were calculated using the total number of cycles as the denominator, while the miscarriage rate was calculated using the number of cycles with confirmed clinical pregnancies as the denominator. Implantation was defined as a serum hCG level of ≥ 5 mIU/mL measured 9 days after embryo transfer. Clinical pregnancy was defined as the presence of a gestational sac confirmed via transvaginal ultrasound between 4 and 6 weeks of gestation. Ongoing pregnancy was defined as a pregnancy confirmed to be continuing at 12 weeks of gestation. Live birth was defined as a delivery occurring at or beyond 22 weeks of gestation. Miscarriage was defined as a pregnancy loss occurring after a clinical pregnancy was diagnosed but before 22 weeks of gestation. To investigate the effects of endometrial injection of embryo culture supernatants on implantation and miscarriage, we calculated age-adjusted odds ratios (ORs) for clinical pregnancy and miscarriage in the SEET group relative to the BT group, adjusting for maternal age as a confounding factor. We also calculated age-adjusted ORs for implantation and miscarriage within maternal age categories of ≥ 37 years and < 37 years and in cases with less than two and two or more BT failures, respectively. We defined good-quality blastocysts (GQBs) as those with a minimum grade of 3BB, based on the blastocyst morphological scoring system introduced by Gardner and Schoolcraft 14 . Specifically, GQBs had blastocele expansion scores of 3, 4, or 5 and trophectoderm and inner cell mass scores of A.A., AB, B.A., or B.B. All other blastocysts were classified as non-GQBs. We used multivariate logistic regression analysis to evaluate the effect of endometrial injection of the embryo culture supernatant on clinical pregnancy and miscarriage, considering covariates, such as maternal age, body mass index, serum anti-Müllerian hormone level, basal follicle-stimulating hormone level, endometrial thickness on the day of embryo transfer, GQB status, and serum P4 level on the day of embryo transfer, noting that these factors may influence ART outcomes. All statistical analyses were performed using SPSS version 28 (IBM Corp., Armonk, NY, USA). For normally distributed data, we used the t-test to compare the means between groups. The Mann–Whitney U test was used to assess nonparametric data. Proportions were compared using the chi-square and Fisher’s exact probability tests, and we presented ORs with 95% CIs. We calculated age-adjusted ORs using the Mantel–Haenszel test. Statistical significance was set at p  < 0.05. Logistic regression analysis was employed to evaluate the relationship between potential predictors and ART outcomes. Covariates were selected based on clinical relevance and statistical considerations. ORs with 95% CIs were calculated to quantify the strength of associations, with adjustments made for confounding factors where appropriate.

This study suggested that endometrial injection of the embryo culture supernatant may adversely affect ART outcomes in FET cycles. This intervention significantly increased the miscarriage rate in women, particularly those aged ≥ 37 years, resulting in a heightened risk of miscarriage. The SEET group had significantly lower rates of implantation, ongoing pregnancies, and live births than the BT group. Moreover, the SEET group exhibited an age-adjusted OR (95% CI) as high as 1.97 (1.04–3.72) for miscarriage, in contrast to the age-adjusted OR for clinical pregnancy, which was 1.02 (0.69–1.52). Furthermore, when considering factors that might influence ART outcomes through logistic regression analysis, the aOR for miscarriage in the SEET group was 2.08 (1.02–4.24). This result contradicts the results reported by Goto et al. 4 , who proposed that blastocyst transplantation with endometrial injection of the embryo culture supernatant results in a higher implantation rate than blastocyst transplantation without endometrial injection. In addition, Goto et al. 4 suggested that the crosstalk between the embryo and the endometrium begins earlier in blastocyst transplantation with endometrial injection of the embryo culture supernatant, leading to a higher implantation rate attributed to the significantly higher serum β-hCG level observed in the SEET group on day 30 after embryo transfer, in contrast to the BT group. Our study compared serum hCG levels on day nine after embryo transfer in cycles with implantation between the SEET and BT groups. However, we found no significant differences between the groups or evidence of crosstalk between the embryo and the endometrium. A 2020 Cochrane review 9 and several other studies 10 – 13 found no evidence supporting the benefits of endometrial injection of embryo culture supernatant in ART 9 – 13 . Kamath et al. 11 reported a significantly lower implantation rate in a group that underwent endometrial injection of the embryo culture supernatant than in a group that did not undergo this procedure (27% vs. 44%, p  = 0.018), and they attributed the lower implantation rate to the catheter insertion into the uterus and the injection of the supernatant fluid 11 , both of which may negatively impact implantation. Additionally, while not statistically significant, the miscarriage rate was higher in the group that underwent the procedure (43% vs. 6%, p  = 0.103), suggesting a potential negative impact of this intervention on pregnancy outcomes. The results of these studies provided no evidence that endometrial injection of the embryo culture supernatant has a positive effect on implantation or pregnancy. After accounting for various factors that might influence ART outcomes through logistic regression analysis, the adjusted odds ratio (aOR) for miscarriage in the SEET group compared with the BT group was 4.63 (1.61–13.3) overall, 0.732 (0.22–2.47) for maternal age < 37 years, and 4.63 (1.61–13.3) for maternal age ≥ 37 years (Table  4 ). The 2020 Cochrane Review reported OR for miscarriage in SEET compared with standard care as 0.86 (0.40–1.85) for maternal age < 37 years and 0.89 (0.44–1.78) for maternal age ≥ 37 years 9 . The OR for maternal age < 37 years in this study was similar to the age-adjusted OR. However, notably, the age-adjusted OR for maternal age ≥ 37 years in this study was higher than that reported in the Cochrane review, suggesting a predominantly negative effect on the miscarriage rate in FET cycles for maternal age ≥ 37 years. Subgroup analysis confirmed that the negative effects of endometrial injection of embryo culture supernatant were observed in both GQB and non-GQB embryos, suggesting that embryo quality alone does not fully account for the observed outcomes. The stronger adverse effects seen in patients aged 37 years and older may be explained by the higher baseline miscarriage risk in this age group, which amplifies the impact of SEET. In contrast, the lower miscarriage rate in younger patients reduces the absolute number of events, making it statistically more challenging to detect a significant effect. These findings highlight the age-dependent nature of SEET’s influence on miscarriage risk, emphasizing the need for careful patient selection. Further studies with larger sample sizes are warranted to validate these findings and clarify the underlying mechanisms. A limitation of this study was its retrospective design. The decision to perform endometrial injection of the embryo culture supernatant, based on information provided by the physician and the patient’s preference, represents a significant source of bias, as it may have introduced selection bias or influenced patient characteristics. Additionally, the absence of certain clinical information, such as the patient’s history of miscarriage or duration of infertility, may have contributed to unmeasured confounding factors, posing another limitation.　 The difference in the number of previous embryo transfer cycles between the SEET and BT groups also represents a limitation, as this discrepancy may reflect the tendency for patients who have experienced prior embryo transfer failures to consider endometrial injection of embryo culture supernatants as a treatment option. To mitigate potential sources of bias in ART outcomes, we selected only embryos that had reached the blastocyst stage on day five, thereby excluding cycles that might affect implantation outcomes. Furthermore, we sought to enhance the accuracy of assessing the impact of endometrial injection of embryo culture supernatants on ART outcomes by performing multivariate analyses to calculate aORs. These analyses accounted not only for maternal age differences between the SEET and BT groups but also for other potential confounding factors that could influence clinical pregnancy and miscarriage rates. The strength of this study lies in our ability to investigate the effects of endometrial injection of embryo culture supernatants in a substantial cohort of 996 FET cycles. This provides additional insights into the field, which has often been limited by studies with small sample sizes. In conclusion, we demonstrated that endometrial injection of the embryo culture supernatant has no significant positive effect on implantation and pregnancy during the FET cycle. In contrast, it has an adverse effect by increasing the risk of miscarriage. Consequently, this modality should be avoided, especially for mothers aged ≥ 37 years, as it may have a significant adverse effect on ART outcomes. However, the findings of this study were limited by its retrospective design. Hence, future studies should involve larger cohorts and employ prospective randomized trials with appropriate inclusion and exclusion criteria.

In assisted reproductive technology (ART), improving reproductive outcomes is of great concern to both patients with infertility and reproductive specialists. In recent years, many attempts have been made to improve ART outcomes, including endometrial injury 1 , the use of a hyaluronic acid-enriched transfer medium 2 , and the administration of granulocyte colony-stimulating growth factor 3 . In one of these attempts, the endometrial injection of the embryo culture supernatant was first reported by Goto et al. in 2007 4 , showing a significant increase in implantation and pregnancy rates during frozen-thawed embryo transfer (FET) cycles. The authors emphasized the critical role of the crosstalk between the developing embryo and the endometrium, wherein several factors released during embryonic development regulate endometrial receptivity, ultimately impacting implantation. They asserted that endometrial injection of the embryo culture supernatant facilitates this crosstalk 5 – 8 . This method, referred to as stimulation of endometrial embryo transfer (SEET) by Goto et al. 4 , is now widely used in ART in Japan. Several randomized trials have investigated the effect of the endometrial injection of embryo culture supernatants; however, they are all limited by small sample sizes. Furthermore, meta-analyses of these studies failed to demonstrate any significant effects on ART outcomes 9 . In addition, the use of endometrial injection of embryo culture supernatants has not been extensively reported in a substantial number of FET cycles 4 , 10 – 12 , resulting in limited evidence. Therefore, this retrospective study aimed to investigate the effect of endometrial injection of embryo culture supernatants in a large cohort of FET cycles on ART outcomes, aiming to contribute to the existing body of evidence by evaluating a substantial number of FET cycles.