When to test: defining the earliest reliable time for pregnancy detection and its prognostic value after frozen embryo transfer

Huiying Xiao: Writing – original draft, Visualization, Data curation. Xue Shang: Formal analysis, Data curation. Gege Ouyang: Validation, Investigation. Yuan Fang: Validation, Software. Dingying Zhao: Supervision, Methodology. Yue Niu: Visualization, Methodology. Yanran Liu: Methodology. Jialin Zou: Investigation. Ning Li: Validation. Ze Wang: Investigation. Lianlian Liu: Writing – review & editing, Data curation. Daimin Wei: Writing – review & editing, Funding acquisition, Conceptualization.

A total of 120 patients undergoing FET were recruited. The baseline characteristics of the enrolled patients were shown in Table 1 . Among them, 84 (70.0%) patients achieved pregnancy and 36 (30.0%) patients had negative serum hCG tests. Compared to the patients who failed to achieve pregnancy, the patients who achieved pregnancy had significantly higher percentages of embryos with an ICM of grade A (67.5% vs 42.9%, P =.013) and TC of grade A (51.8% vs 25.7%, P =.009). Additionally, a higher proportion of patients with pregnancy received a Day 5 blastocyst transfer (92.9% vs 75.0%, P =.007). No statistically significant differences were observed in other baseline characteristics. Table 1 Baseline characteristics of the included patients Table 1 dummy alt text Characteristics Total N =120 Patients with pregnancy N =84 Patients without pregnancy N =36 P value Age (y) 32.0 (28.0–34.0) 31.5 (27.3–34.0) 32.0 (29.0–34.0) .629 BMI (kg/m²) 24.07 (21.94–26.13) 23.93 (21.57–26.13) 24.72 (22.69–26.82) .272 Duration of infertility (y) 3.0 (1.5–4.5) 2.3 (1.1–4.5) 3.0 (2.0–4.0) .416 Primary infertility ( n , %) 73 (60.8) 54 (64.3) 19 (52.8) .237 Infertility causes ( n , %) .688  Tubal factor 63 (52.5) 42 (50.0) 21 (58.3)  Male factor 29 (24.2) 21 (25.0) 8 (22.2)  Other factors 28 (23.3) 21 (25.0) 7 (19.4) Gravidity ( n , %) .237  0 73 (60.8) 54 (64.3) 19 (52.8)  ≥1 47 (39.2) 30 (35.7) 17 (47.2) Parity ( n , %) .157  0 99 (82.5) 72 (85.7) 27 (75.0)  ≥1 21 (17.5) 12 (14.3) 9 (25.0) Abortion ( n , %) .211  0 83 (69.2) 61 (72.6) 22 (61.1)  ≥1 37 (30.8) 23 (27.4) 14 (38.9) Live ( n , %) .109  0 100 (83.3) 73 (86.9) 27 (75.0)  ≥1 20 (16.7) 11 (13.1) 9 (25.0) Baseline hormone levels  FSH (IU/L) 5.95 (5.15–6.86) 5.95 (5.14–6.96) 5.94 (5.18–6.68) .699  LH (IU/L) 6.43 (4.93–11.63) 6.90 (5.29–11.78) 5.63 (4.23–11.32) .161  E2 (pg/mL) 37.10 (27.20–48.40) 36.65 (28.43–46.96) 38.10 (26.40–53.30) .570  P (ng/mL) 0.24 (0.15–0.36) 0.24 (0.17–0.36) 0.24 (0.14–0.48) .969  PRL (ng/mL) 15.09 (10.91–20.15) 14.84 (10.01–18.69) 15.78 (11.96–24.87) .172  T (ng/dL) 28.77 (21.15–48.41) 29.30 (20.23–49.39) 28.46 (23.30–48.02) .647  AMH (ng/mL) 5.59 (3.39–8.22) 6.11 (3.42–8.76) 4.79 (3.25–7.09) .304  TSH (uIU/mL) 2.35 (1.65–3.11) 2.31 (1.65–2.95) 2.63 (1.62–3.16) .481 Fertilization method ( n , %) .922  IVF 70 (58.3) 48 (57.1) 22 (61.1)  ICSI 25 (20.8) 18 (21.4) 7 (19.4)  IVF+ICSI/PGT 25 (20.8) 18 (21.4) 7 (19.4) Blastocyst expansion ( n , %) .513  4 12/118 (10.2) 7/83 (8.4) 5/35 (14.3)  5 100/118 (84.7) 71/83 (85.5) 29/35 (82.9)  6 6/118 (5.1) 5/83 (6.0) 1/35 (2.9) ICM grade ( n , %) .013  A 71/118 (60.2) 56/83 (67.5) 15/35 (42.9)  B 47/118 (39.8) 27/83 (32.5) 20/35 (57.1) TC grade ( n , %) .009  A 52/118 (44.1) 43/83 (51.8) 9/35 (25.7)  B/C 66/118 (55.9) 40/83 (48.2) 26/35 (74.3) Endometrial thickness before frozen embryo transfer (mm) 9.0 (8.5–10.0) 9.0 (8.1–10.0) 9.0 (8.5–10.0) .633  Stage of embryo transferred ( n , %) .007  Day 5 blastocyst 105 (87.5) 78 (92.9) 27 (75.0)  Day 6 blastocyst 15 (12.5) 6 (7.1) 9 (25.0)  hCG injection before endometrial transformation ( n , %) 63 (52.5) 45 (53.6) 18 (50.0) .720  hCG injection dose (IU) 8000.0 (6000.0–8000.0) 6500.0 (6000.0–8000.0) 8000.0 (6000.0–8000.0) .360 Data are presented as median (interquartile range) and n (%). AMH , anti-Müllerian hormone; BMI , body mass index; E2 , estradiol; FSH , follicle stimulating hormone; GnRH , gonadotropin releasing hormone; hCG , human chorionic gonadotropin; ICM , inner cell mass; ICSI , intracytoplasmic sperm injection; IVF , in vitro fertilization; IVF+ICSI , IVF salvage ICSI; LH , luteinizing hormone; P , progesterone; PGT , preimplantation genetic testing; PRL , prolactin; T , testosterone; TC , trophectoderm; TSH , thyroid stimulating hormone. Source: Xiao. When to test: defining the earliest reliable time. AJOG Glob Rep 2026. Baseline characteristics of the included patients Data are presented as median (interquartile range) and n (%). AMH , anti-Müllerian hormone; BMI , body mass index; E2 , estradiol; FSH , follicle stimulating hormone; GnRH , gonadotropin releasing hormone; hCG , human chorionic gonadotropin; ICM , inner cell mass; ICSI , intracytoplasmic sperm injection; IVF , in vitro fertilization; IVF+ICSI , IVF salvage ICSI; LH , luteinizing hormone; P , progesterone; PGT , preimplantation genetic testing; PRL , prolactin; T , testosterone; TC , trophectoderm; TSH , thyroid stimulating hormone. Given that some patients received exogenous hCG administration before FET, we analyzed urinary hCG positive results separately in patients with and without exogenous hCG exposure. In patients without exogenous hCG administration (57/120, 47.5%), the earliest positive urinary hCG results were detected on the fourth day after FET ( Table 2 ). Among patients achieving pregnancy confirmed by serum hCG measurement (39/57, 68.4%), 61.5% had a positive urinary hCG test on the fifth day and 100% on the ninth day after FET. Among patients without pregnancy, false positive urinary hCG results were detected in 1 patient on the sixth day, and in 2 patients with persistent false positive results on the seventh to 12th days after FET. Table 2 Daily urinary hCG test positive results in patients without exogenous hCG administration Table 2 dummy alt text Days after transplantation Total no. of patients N =57 Patients with pregnancy N =39 Patients without pregnancy N =18 Day 3 a 0 0 0 Day 4 6 (10.5) 6 (15.4) 0 Day 5 24 (42.1) 24 (61.5) 0 Day 6 33 (57.9) 32 (82.1) 1 (5.6) Day 7 36 (63.2) 34 (87.2) 2 (11.1) Day 8 40 (70.2) 38 (97.4) 2 (11.1) Day 9 41 (71.9) 39 (100.0) 2 (11.1) Day 10 41 (71.9) 39 (100.0) 2 (11.1) Day 11 41 (71.9) 39 (100.0) 2 (11.1) Day 12 41 (71.9) 39 (100.0) 2 (11.1) a There were 4 patients who did not undergo testing on the third day after embryo transfer. Source: Xiao. When to test: defining the earliest reliable time. AJOG Glob Rep 2026. Daily urinary hCG test positive results in patients without exogenous hCG administration There were 4 patients who did not undergo testing on the third day after embryo transfer. In patients receiving exogenous hCG administration (63/120, 52.5%), 28.3% had a positive urinary hCG result on the third day after FET ( Table 3 ). Among patients who achieved pregnancy (45/63, 71.4%), the proportion with positive urinary hCG results rapidly increased between the third and seventh days after FET, and all pregnancies were detected on the ninth day after FET. In contrast, in patients without pregnancy, some patients showed false positive results as early as the third day after FET (5/18, 27.8%). The percentage of false positive urinary hCG results decreased to its lowest level on the fifth day after FET (2/18, 11.1%). Table 3 Daily urinary hCG test positive results in patients with exogenous hCG administration Table 3 dummy alt text Days after transplantation Total no. of patients N =63 Patients with pregnancy N =45 Patients without pregnancy N =18 Day 3 a 17 (28.3) 12 (28.6) 5 (27.8) Day 4 25 (39.7) 21 (46.7) 4 (22.2) Day 5 34 (54.0) 32 (71.1) 2 (11.1) Day 6 45 (71.4) 41 (91.1) 4 (22.2) Day 7 49 (77.8) 44 (97.8) 5 (27.8) Day 8 49 (77.8) 44 (97.8) 5 (27.8) Day 9 50 (79.4) 45 (100.0) 5 (27.8) Day 10 50 (79.4) 45 (100.0) 5 (27.8) Day 11 50 (79.4) 45 (100.0) 5 (27.8) Day 12 50 (79.4) 45 (100.0) 5 (27.8) a There were 4 patients who did not undergo testing on the third day after embryo transfer. Source: Xiao. When to test: defining the earliest reliable time. AJOG Glob Rep 2026. Daily urinary hCG test positive results in patients with exogenous hCG administration There were 4 patients who did not undergo testing on the third day after embryo transfer. Among patients with positive urinary hCG results ( n =95), we excluded those patients with false positive urinary hCG results ( n =11). A total of 27 patients were classified into the early positive group (initial urinary hCG positivity detected on the third to fourth days after FET), while 57 patients were in the late positive group (initial urinary hCG positivity detected on the fifth to ninth days after FET). A comparative analysis of baseline characteristics between groups and the results of univariable logistic regression were shown in Table 4 . There were no significant differences in maternal age, duration of infertility, baseline hormone levels, or blastocyst scores between the 2 groups. However, the early positive group was associated with a lower BMI (22.95 ± 3.38 vs 24.58 ± 3.97, P =.069; Crude OR: 0.88; 95% CI: 0.77–1.01), higher proportions of male infertility (37.0% vs 19.3%; P =.136; Crude OR: 3.86; 95% CI: 0.97–15.44) and ICSI fertilization (29.6% vs 17.5%; P =.077; Crude OR: 6.40; 95% CI: 1.12–36.44), although these differences did not reach statistical significance. Table 4 Baseline characteristics of patients with early or late urinary hCG positive results Table 4 dummy alt text Characteristics Early positive group a N =27 Late positive group b N =57 P value Crude OR (95% CI) P value Age (y) 29.0 (27.0–34.0) 32.0 (28.0–34.0) .227 0.92 (0.82–1.04) .185 BMI (kg/m²) 22.95 (3.38) 24.58 (3.97) .069 0.88 (0.77–1.01) .074 Duration of infertility (y) 2.0 (1.0–4.0) 3.0 (1.5–5.3) .181 0.85 (0.70–1.05) .129 Primary infertility ( n , %) 19 (70.4) 35 (61.4) .423 1.49 (0.56–3.99) .424 Infertility causes ( n , %) .136  Tubal factor 13 (48.1) 29 (50.9) 1.91 (0.54–6.79) .320  Male factor 10 (37.0) 11 (19.3) 3.86 (0.97–15.44) .056  Other factors 4 (14.8) 17 (29.8) Gravidity ( n , %) .423  0 19 (70.4) 35 (61.4) 1.49 (0.56–3.99) .424  ≥1 8 (29.6) 22 (38.6) Parity ( n , %) .215  0 25 (92.6) 47 (82.5) 2.66 (0.54–13.09) .229  ≥1 2 (7.4) 10 (17.5) Abortion ( n , %) .750  0 19 (70.4) 42 (73.7) 0.85 (0.31–2.34) .751  ≥1 8 (29.6) 15 (26.3) Live ( n , %) .288  0 25 (92.6) 48 (84.2) 0.43 (0.09–2.13) .299  ≥1 2 (7.4) 9 (15.8) Baseline hormone levels  FSH (IU/L) 5.93 (5.28–6.47) 5.98 (5.07–7.14) .709 0.92 (0.72–1.17) .487  LH (IU/L) 6.85 (5.60–11.40) 6.98 (5.00–12.03) .670 1.02 (0.95–1.08) .643  E2 (pg/mL) 34.80 (24.50–40.30) 38.10 (31.45–50.20) .108 0.98 (0.95–1.01) .148  P (ng/mL) 0.25 (0.17–0.36) 0.24 (0.17–0.36) .943 1.64 (0.21–13.03) .641  PRL (ng/mL) 14.28 (11.04–18.40) 14.97 (9.86–18.88) .905 1.00 (0.94–1.05) .857  T (ng/dL) 30.97 (22.26–46.40) 26.80 (18.80–52.10) .803 1.00 (0.98–1.02) .895  AMH (ng/mL) 6.17 (4.74–7.58) 5.97 (2.97–9.37) .720 0.97 (0.87–1.09) .624  TSH (uIU/mL) 2.22 (1.25–2.51) 2.37 (1.82–3.29) .050 0.71 (0.45–1.13) .147 Fertilization method ( n , %) .077  IVF 17 (63.0) 31 (54.4) 4.39 (0.90–21.40) .067  ICSI 8 (29.6) 10 (17.5) 6.40 (1.12–36.44) .036  IVF+ICSI/PGT 2 (7.4) 16 (28.1) Blastocyst expansion ( n , %) .076  4 0 7/56 (12.5) 0  5 24 (88.9) 47/56 (83.9) 0.34 (0.05–2.18) .255  6 3 (11.1) 2/56 (3.6) ICM grade ( n , %) .695  A 19 (70.4) 37/56 (66.1) 1.22 (0.45–3.30) .696  B 8 (29.6) 19/56 (33.9) TC grade ( n , %) .995  A 14 (51.9) 29/56 (51.8) 1.00 (0.40–2.51) .995  B/C 13 (48.1) 27/56 (48.2) Endometrial thickness before frozen embryo transfer (mm) 9.5 (8.5–10.0) 9.0 (8.0–10.0) .620 1.01 (0.74–1.37) .953 Stage of embryo transferred ( n , %) .948  Day 5 blastocyst 25 (92.6) 53 (93.0) 0.94 (0.16–5.50) .948  Day 6 blastocyst 2 (7.4) 4 (7.0) hCG injection before endometrial transformation ( n , %) 21 (77.8) 24 (42.1) .002 4.81 (1.69–13.73) .003  hCG injection dose (IU) 8000.0 (7250.0–8000.0) 6000.0 (6000.0–7625.0) <.001 1.00 (1.00–1.00) .001 Data are presented as median (interquartile range) and n (%). AMH, anti-Müllerian hormone; BMI , body mass index; E2 , estradiol; FSH , follicle stimulating hormone; hCG , human chorionic gonadotropin; ICM , inner cell mass; ICSI , intracytoplasmic sperm injection; IVF , in vitro fertilization; IVF+ICSI , IVF salvage ICSI; LH , luteinizing hormone; P , progesterone; PGT , preimplantation genetic testing; PRL , prolactin; T , testosterone; TC , trophectoderm; TSH , thyroid stimulating hormone. a Early positive group was defined as having a positive urinary hCG test on the third to fourth days after embryo transfer. b Late positive group was defined as having a positive urinary hCG test on the fifth to ninth days after embryo transfer. Source: Xiao. When to test: defining the earliest reliable time. AJOG Glob Rep 2026. Baseline characteristics of patients with early or late urinary hCG positive results Data are presented as median (interquartile range) and n (%). AMH, anti-Müllerian hormone; BMI , body mass index; E2 , estradiol; FSH , follicle stimulating hormone; hCG , human chorionic gonadotropin; ICM , inner cell mass; ICSI , intracytoplasmic sperm injection; IVF , in vitro fertilization; IVF+ICSI , IVF salvage ICSI; LH , luteinizing hormone; P , progesterone; PGT , preimplantation genetic testing; PRL , prolactin; T , testosterone; TC , trophectoderm; TSH , thyroid stimulating hormone. Early positive group was defined as having a positive urinary hCG test on the third to fourth days after embryo transfer. Late positive group was defined as having a positive urinary hCG test on the fifth to ninth days after embryo transfer. We further compared early pregnancy outcomes between the early positive group and the late positive group, as detailed in Table 5 . The early positive group demonstrated a higher rate of ongoing pregnancy (92.6% vs 68.4%, P =.015) than the late positive group, as well as lower rates of overall pregnancy loss (7.4% vs 31.6%, P =.015) and clinical pregnancy loss (3.8% vs 22.0%, P =.039). Table 5 Pregnancy outcomes of patients with early or late urinary hCG positive results Table 5 dummy alt text Outcomes Total N =84 Early positive group a N =27 Late positive group b N =57 P value Clinical pregnancy 76 (90.5) 26 (96.3) 50 (87.7) .211 Pregnancy loss 20 (23.8) 2 (7.4) 18 (31.6) .015 Biochemical pregnancy 8 (9.5) 1 (3.7) 7 (12.3) .211 Clinical pregnancy loss 12/76 (15.8) 1/26 (3.8) 11/50 (22.0) .039 Ongoing pregnancy 64 (76.2) 25 (92.6) 39 (68.4) .015 Data are presented as n / N (%). a Early positive group was defined as having a positive urinary hCG test on the third to fourth days after embryo transfer. b Late positive group was defined as having a positive urinary hCG test on the fifth to ninth days after embryo transfer. Source: Xiao. When to test: defining the earliest reliable time. AJOG Glob Rep 2026. Pregnancy outcomes of patients with early or late urinary hCG positive results Data are presented as n / N (%). Early positive group was defined as having a positive urinary hCG test on the third to fourth days after embryo transfer. Late positive group was defined as having a positive urinary hCG test on the fifth to ninth days after embryo transfer.

In this prospective cohort study, we recruited patients who underwent frozen single blastocyst transfer from September 2024 to May 2025 at the Reproductive Medicine Center of Shandong University. This study was approved by the Ethics Committee of Reproductive Medicine Center of Shandong University. Written informed consent was obtained from all participants. Women aged ≤38 years who underwent their first or second cycle of FET were included. Additional inclusion criteria were women who had at least 1 frozen blastocyst (morphological score of 4BC or better) left that was derived from in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) cycle, or at least 1 euploid blastocyst (morphological score of 4BC or better) in preimplantation genetic testing (PGT) cycle. Blastocysts were scored according to Gardner morphological criteria based on the extent of expansion and development of the inner cell mass (ICM) and trophoblast cells (TC). 13 Blastocysts were vitrified at Day 5 or Day 6 depending on embryo development. Women with a history of recurrent miscarriages (defined as 3 or more continuous miscarriages), who had failed to achieve a clinical pregnancy after 2 or more ET cycles were excluded. We also excluded women with abnormal intrauterine cavity (such as adenomyosis, submucous myoma, or intrauterine adhesion), ultrasound-confirmed hydrosalpinx that had not received treatment, previous cancellation of FET cycle for endometrial thickness <7 mm, a history of autoimmune diseases (such as antiphospholipid syndrome, systemic lupus erythematosus, rheumatic immunodeficiency, and Hashimoto thyroiditis), or a history of breast cancer or thrombosis. All patients underwent a programmed regimen with hormone replacement for endometrial preparation. Oral estradiol valerate was administered at a daily dose of 4 to 6 mg starting on days 1 to 3 of the menstrual cycle. When endometrial thickness was at least 7 mm, 90 mg daily vaginal progesterone gel was added, along with oral dydrogesterone at a dose of 10 mg twice daily. Some patients were prescribed intramuscular injection of hCG (6000–10,000 IU) prior to endometrial transformation (6–7 days before ET) to improve endometrial receptivity at the discretion of their physicians, which was also reported in previous studies. 14 , 15 , 16 The frozen single blastocyst was then transferred on the sixth day of progesterone administration. Frozen blastocysts were thawed using a graded sucrose dilution method on the day of transfer, reassessed for post-thaw morphology and developmental stage, and incubated in culture medium until transfer. If pregnancy was achieved, oral estradiol valerate was continued until 8 weeks of gestation, and vaginal progesterone gel and oral dydrogesterone were continued until 10 to 11 weeks of gestation. Urinary hCG test was performed daily from the third day after FET until the 11th to 14th days when serum hCG levels were measured. The first-void urine on the testing day was used for urinary hCG test. A commercially available urine pregnancy test strip (David Early Pregnancy Test Strip; Guangdong Yikangnas Biopharmaceutical Technology Co., Ltd, Guangzhou, China) was utilized according to the manufacturer’s instructions. The test was considered positive when the urine hCG concentration was ≥25 mIU/mL, which is the analytical sensitivity threshold of the assay. Photographs of the test results were uploaded to the research nurse via WeChat and interpreted within 15 minutes. Urinary hCG results (positive/negative) were recorded daily until the serum hCG was measured, and the results of serum hCG measurement were documented. Serum hCG levels ≥25 IU/L were considered indicative of conception. Since there was no consensus on the cutoff day for the early urinary hCG positivity after FET, patients were categorized using a tertile-based approach according to the observed distribution of the timing of first urinary hCG positivity in the cohort. Patients with first urinary hCG positivity on Days 3 to 4 after FET (the first third) were assigned to the early positive group, whereas those with positivity on Days 5 to 9 (the remaining two thirds) were assigned to the late positive group. The outcomes of this study included the daily results of urinary hCG test after FET, conception (serum hCG level ≥25 IU/L), clinical pregnancy (detection of a gestational sac in the uterine cavity), ongoing pregnancy (detection of a viable fetus with fetal heartbeat at 11–12 weeks’ gestation), and pregnancy loss. Pregnancy loss included both biochemical pregnancy and clinical pregnancy loss. Biochemical pregnancy was defined as the absence of an identifiable pregnancy on ultrasound examination despite a positive hCG pregnancy test which was eventually followed by a decline. Clinical pregnancy loss was defined as the spontaneous miscarriage or therapeutic abortion of clinical pregnancy before 28 weeks of gestation. Normality of continuous variables was tested by using histograms and Shapiro–Wilk test. If data were normally distributed, they were described as mean ± standard deviation (SD), and the between-group difference was tested by the Student’s t test. If data were non-normally distributed, they were described as median and interquartile range (IQR), and tested by the Mann–Whitney U test. Categorical variables were expressed as frequency and percentage, and tested by the chi-square test. In consideration of the residual effect of the exogenous hCG administration, we stratified the analysis by patients with and without exogenous hCG administration. Univariable analysis was used to estimate the association between each clinical factor and the time to early urinary hCG positivity after FET. The magnitude of the association was assessed using odds ratio (OR) and 95% confidence interval (95% CI). All statistical analyses were performed using IBM SPSS Statistics for Windows, Version 27.0. A P value <.05 was considered statistically significant.

In this prospective cohort study, we found that pregnancy can be reliably detected using a urinary hCG test on the ninth day after FET, with a negative urinary hCG result on that day effectively excluding pregnancy. The earliest positive urinary hCG result was observed on the fourth day after FET. Furthermore, we found that patients with early positive hCG results had higher ongoing pregnancy rates and a lower pregnancy loss rate than those with late positive hCG results. These patients with early positive hCG results also tended to have a lower BMI and higher proportions of both male infertility and fertilization by ICSI. hCG is recognized as the first hormonal signal of conception, and its mRNA transcription can be detected as early as the 8-cell stage. The blastocyst expresses hCG prior to implantation. 17 , 18 Following implantation, the syncytiotrophoblast secretes increasing amounts of hCG, supporting endometrial maintenance and angiogenesis. 19 Serum hCG levels increase as the trophoblast mass expands 20 and are positively associated with the quantity and function of trophoblast cells, 2 , 21 as well as with pregnancy outcomes. 20 , 22 In natural pregnancies, serum hCG can be detected at 6 to 8 days after fertilization and urinary hCG can be detected at 3 to 4 days after implantation. 23 For patients who underwent FET, we found urinary hCG could be detected as early as the fourth day after FET, which was generally consistent with natural pregnancy. Current clinical practice relies on serum hCG measurement approximately 12 to 15 days after ET to confirm pregnancy, leaving patients with a prolonged period of uncertainty. Our prospective daily monitoring showed that urinary hCG testing on the ninth day after FET provided a practical and reliable time point at which a negative urinary hCG result effectively ruled out pregnancy, regardless of whether exogenous hCG was used for luteal support. Earlier reassurance following a negative test at this time point may reduce unnecessary repeated testing, facilitate subsequent clinical management, and alleviate patient anxiety during the waiting period. Serum hCG levels after ET could be affected by embryo-related factors, such as the number of embryos transferred (single vs twin), cryopreservation method (frozen vs fresh), embryo developmental stage (cleavage-stage vs blastocyst), and embryo morphology grade. 24 , 25 , 26 Therefore, in order to minimize potential confounding, we selected the cycles of single frozen embryo transfer to determine the earliest reliable time for pregnancy detection after FET. By controlling for embryonic developmental speed and avoiding the influence of ovarian hyperstimulation on endometrial receptivity, we ensured that the observed differences in urinary hCG detection timing more directly reflected the synchrony between the embryo’s intrinsic implantation potential and the endometrial receptivity window. Previous evidence indicated that embryo implantation timing determined the earliest time of urinary hCG positivity and affected the kinetic profile of urinary hCG. Embryos implanting early exhibited lower initial urinary hCG levels but reached detectable levels sooner, followed by a rapid rise within the first week after implantation. 27 Our analysis showed that compared with patients who had late positive urinary hCG results, those with early positive results had a higher rate of ongoing pregnancy and a decreased risk of pregnancy loss. Wilcox et al. 28 demonstrated that blastocyst implantation occurred 8 to 10 days after ovulation in natural pregnancy, and that delayed implantation, as determined by serial quantitative hCG measurements, was associated with a progressively increased risk of pregnancy loss. 28 Although implantation timing could not be directly determined in our study, the association between later first urinary hCG positivity and increased pregnancy loss observed in our cohort was consistent with these previous findings. The underlying biological mechanisms primarily involved 2 aspects: First, embryonic quality was a key determinant of implantation timing. Early urinary hCG positivity correlated with euploid blastocysts exhibiting robust trophoblastic activity. These trophoblasts acquired invasiveness earlier and secreted sufficient hCG sooner, capturing the optimal endometrial receptivity window. 27 , 29 Conversely, delayed implantation may stem from subclinical metabolic or functional defects in the blastocysts, postponing the initiation of embryo-endometrial dialogue. Even after eventual implantation, this compromised developmental trajectory predisposed embryos to higher rates of subsequent developmental arrest and pregnancy loss. Second, precise synchrony of the implantation window was critical. Successful implantation required meticulous molecular crosstalk between the embryo and endometrium. 30 Early urinary hCG positivity suggested that embryo implantation occurred in synchrony with the endometrial receptivity window. 19 Conversely, delayed implantation reflected embryo–endometrium asynchrony or endometrial milieu dysregulation, leading to compromised placental establishment and an elevated risk of early pregnancy loss. 31 We also observed that patients with earlier urinary hCG positivity had a lower BMI and higher proportions of male-factor infertility and ICSI cycles than those with later urinary hCG positivity. As a core indicator of metabolic health, elevated BMI is associated with pathophysiological changes, including insulin resistance, chronic inflammatory status, and dysregulated adipokine secretion. Previous studies have demonstrated that obesity-related abnormalities can compromise oocyte quality and impair embryonic developmental competence. 32 , 33 These abnormalities can also affect endometrial receptivity by interfering with stromal cell decidualization, 34 altering the formation and distribution of pinopodes, and disrupting the temporal expression of adhesion molecules and cytokines. 35 , 36 These changes ultimately delay embryo implantation and early trophoblast development. Zhang et al. 37 reported that in FET cycles, clinical pregnancy and live birth rates decreased progressively with increasing BMI. 37 Consequently, patients with lower BMI exhibited accelerated embryo development and timely implantation due to optimal embryo quality and endometrial receptivity, resulting in earlier and adequate hCG secretion. Additionally, our findings indicated a higher prevalence of male-factor infertility in the early positive group, resulting in a greater proportion of ICSI cycles, which were typically performed only in couples with severe male-factor infertility. This suggested more favorable maternal conditions in these couples, which may facilitate earlier embryo implantation. The use of exogenous hCG may represent an important confounding factor in the interpretation of early urinary hCG results. In our stratified analysis, we observed that patients receiving exogenous hCG support could have positive urinary hCG results as early as Day 3 after FET, while some nonpregnant patients also exhibited transient false-positive results. These findings indicated that exogenous hCG could interfere with early urinary hCG testing. However, this interference gradually diminished over time. By Day 9 after FET, all pregnant patients were correctly identified by urinary hCG testing, and the false-positive rate had decreased substantially. Therefore, even in the presence of exogenous hCG exposure, Day 9 remained a robust and reliable time point for determining pregnancy status. Accordingly, when interpreting urinary hCG results—particularly within the first 3 to 5 days after transfer—the patient's medication history should be carefully considered. The strengths of this study include its prospective design and close follow-up, ensuring the reliability of our findings. Moreover, the inclusion of both patients receiving exogenous hCG support and those without such support enhanced the generalizability and real-world applicability of the results. In addition, beyond evaluating the diagnostic value of urinary hCG testing, we also assessed its prognostic implications, further extending its clinical utility. Nevertheless, several limitations should be acknowledged. First, because we enrolled only FET cycles, our findings may not be directly generalizable to fresh embryo transfer cycles or those involving cleavage-stage embryos. Second, urinary hCG was measured using a qualitative assay, which precluded analysis of quantitative kinetic characteristics. Third, although differences in certain parameters, such as maternal age and BMI, were observed between groups, these did not reach statistical significance and thus require further investigation. Future validation in larger cohorts is warranted to draw more definitive conclusions.

Urinary hCG testing could reliably detect pregnancy as early as the ninth day after FET. Notably, a positive urinary hCG result appeared as early as the fourth day after FET. Patients with early positive hCG results demonstrated a significantly higher rate of ongoing pregnancy and a lower rate of pregnancy loss compared to those with late positive hCG results. These findings established the timing of urinary hCG positivity as a useful clinical marker for predicting early pregnancy outcomes after FET.

Assisted reproductive technologies (ART) have enabled over 8 million births globally, profoundly addressing infertility challenges for millions of families. 1 Despite continuous advancements in ART efficiency and safety, the success rate remains modest, imposing substantial emotional and psychological burdens on couples. 2 The uncertainty of cycle outcomes, particularly during the waiting period after embryo transfer (ET), exacerbates patient anxiety. 3 Early prediction of pregnancy is therefore critical not only for alleviating psychological stress but also for guiding clinicians in subsequent monitoring and clinical interventions. 1 , 4 Nonetheless, the earliest time to reliably detect pregnancy after ET remains uncertain. Serum biomarkers such as human chorionic gonadotropin (hCG), estradiol (E2), progesterone (P), and kisspeptin have been investigated for predicting pregnancy outcomes, and hCG has been established as the most reliable marker. 5 Synthesized by embryonic syncytiotrophoblast cells, 6 hCG represents the earliest detectable trophoblastic signal in maternal circulation post-implantation. 7 Serum hCG levels are usually measured at 12 to 15 days after ET to confirm pregnancy status. 8 , 9 It was reported that a serum hCG level exceeding 200 mIU/mL at 14 days after ET was associated with a higher rate of ongoing pregnancy. 10 Recently, some studies have attempted to advance the serum hCG test to days 5 to 6 after transfer and observed that hCG levels measured at this time point showed high predictive value for successful pregnancy in both fresh and frozen transfer cycles. 11 However, the earliest time for hCG measurement to reliably diagnose pregnancy and predict pregnancy outcomes remains unclear and warrants further studies. Urinary hCG test using pregnancy test strips offers a noninvasive and convenient alternative for continuous monitoring after ET. 12 We conducted a prospective cohort study to explore the earliest positive day of urinary hCG detection after frozen embryo transfer (FET) and to evaluate its predictive value for subsequent pregnancy outcomes.