Euploid sporadic miscarriage in advanced-age women is associated with reduced live birth rates in subsequent pregnancies.

For women with < 35 years, 513 women underwent subsequent transfers after euploid SM and 274 women underwent subsequent transfers after aneuploid SM. The age of couples, reproductive history, causes of infertility, AMH, endometrial thickness on the transfer day, the number of embryos transferred were comparable between groups. The euploid SM group had a significantly higher proportion of HRT cycles (81.3% vs. 68.2%; P <0.001). As shown in Table  1 , the proportion of day 5 blastocysts was significantly higher in the euploid SM group (55.8% vs. 38.7%; P <0.001). Among women aged 35–37 years, 95 transfers occurred after euploid SM and 73 after aneuploid SM. All baseline parameters were comparable between the two groups (Table  2 ). In women aged ≥ 38 years, 75 women underwent subsequent transfers followed euploid SM and 63 followed aneuploid SM. The AMH was significantly higher in the euploid SM group (3.2 ± 3.1 vs. 2.4 ± 1.6; P  = 0.037, Table  3 ). The other baseline characteristics were comparable between groups. The detailed embryo information was provided in Supplementary Table 1. Table 1 Baseline characteristics of patients < 35 years with prior sporadic miscarriage Euploid SM Aneuploid SM P value N  = 513 N  = 274 Female age (years) 30.3 ± 2.7 30.4 ± 2.9 0.581 Male age (years) 32.4 ± 4.0 32.9 ± 3.8 0.114 Reproductive history (%)  Nulligravida 381 (74.3) 187 (68.2) 0.073  Prior induced abortion 50 (9.7) 30 (10.9) 0.595  Prior live birth 82 (16.0) 57 (20.8) 0.091  AMH (ng/ml) 6.2 ± 4.7 5.5 ± 4.1 0.032  BMI (kg/m 2 ) 21.9 ± 2.8 22.0 ± 2.8 0.396 Fertility cause (%)  Tubal 193 (37.6) 121 (44.2) 0.069  Ovulation disorder 87 (17.0) 39 (14.2) 0.290  Tubal and ovulation disorder 75 (14.6) 30 (10.9) 0.132  Male factors 80 (15.6) 41 (15.0) 0.823  Other 78 (15.2) 43(15.7) 0.851 FET cycle regimen ( %)  Natural 96 (18.7) 87 (31.8) < 0.001  HRT 417 (81.3) 187 (68.2)  Endometrium thickness (mm) 11.1 ± 1.6 11.4 ± 1.7 0.017  No. Embryo transferred 1.4 ± 0.7 1.4 ± 0.5 0.477 Protocol for embryo origin (%)  Agonist protocol 263 (51.3) 142 (51.8) 0.881  Antagonist protocol 136 (26.5) 78 (28.4) 0.557  Mild stimulation protocol 114 (22.2) 54 (19.7) 0.412 Embryo quality (%)  High 200 (39.0) 104 (38.0) 0.777  Low 313 (61.0) 170 (62.0)  Day 5 embryo 286 (55.8) 106 (38.7) < 0.001  Day 6 embryo 227 (44.2) 168 (61.3) BMI body mass index, AMH anti-Müllerian hormone, ART assisted reproductive technology, FET frozen-thawed embryo transfer, HRT hormone replacement therapy Baseline characteristics of patients < 35 years with prior sporadic miscarriage BMI body mass index, AMH anti-Müllerian hormone, ART assisted reproductive technology, FET frozen-thawed embryo transfer, HRT hormone replacement therapy Table 2 Baseline characteristics of patients aged 35–37 years with prior sporadic miscarriage Euploid SM Aneuploid SM P value N  = 131 N  = 95 Female age (years) 35.8 ± 0.8 35.7 ± 0.7 0.390 Male age (years) 37.4 ± 3.4 37.3 ± 4.6 0.905 Reproductive History (%)  Nulligravida 62 (47.3) 50 (52.6) 0.431  Prior induced abortion 28 (21.4) 17 (17.9) 0.518  Prior live birth 41 (31.3) 28 (29.5) 0.769  AMH (ng/ml) 4.2 ± 3.5 4.3 ± 3.6 0.847  BMI (kg/m 2 ) 22.2 ± 2.5 22.3 ± 2.6 0.827 Fertility cause (%)  Tubal 72 (55.0) 47 (49.4) 0.414  Ovulation disorder 9 (6.9) 8 (8.4) 0.686  Tubal and ovulation disorder 19 (14.5) 11 (11.6) 0.517  Male factors 21 (16.0) 19 (20.0) 0.429  Other 10 (7.6) 10 (10.5) 0.449 FET cycle regimen ( %)  Natural 49 (37.4) 37 (38.9) 0.814  HRT 82 (62.6) 58 (61.1)  Endometrium thickness 11.1 ± 1.7 11.0 ± 1.8 0.640  No. Embryo transferred 1.3 ± 0.5 1.5 ± 0.5 0.083 Protocol for embryo origin (%)  Agonist protocol 54 (41.2) 37 (38.9) 0.814  Antagonist protocol 39 (29.8) 36 (37.9) 0.200  Mild stimulation protocol 38 (29.0) 22 (23.2) 0.326 Embryo quality (%)  High 54 (41.2) 34 (35.8) 0.408  Low 77 (58.8) 61 (64.2)  Day 5 embryo 55 (42.0) 46 (48.4) 0.337  Day 6 embryo 76 (58.0) 49 (51.6) BMI body mass index, AMH anti-Müllerian hormone, ART assisted reproductive technology, FET frozen-thawed embryo transfer, HRT hormone replacement therapy Baseline characteristics of patients aged 35–37 years with prior sporadic miscarriage BMI body mass index, AMH anti-Müllerian hormone, ART assisted reproductive technology, FET frozen-thawed embryo transfer, HRT hormone replacement therapy Table 3 Baseline characteristics of patients ≥ 38 years with prior sporadic miscarriage Euploid SM Aneuploid SM P value N  = 75 N  = 98 Female age (years) 39.5 ± 1.5 40.0 ± 1.8 0.060 Male age (years) 40.6 ± 4.5 40.5 ± 4.9 0.932 Reproductive History (%)  Nulligravida 30 (40.0) 46 (46.9) 0.362  Prior induced abortion 11 (14.7) 14 (14.3) 0.944  Prior live birth 34(45.3) 38 (38.8) 0.386  AMH (ng/ml) 3.2 ± 3.1 2.4 ± 1.6 0.037  BMI (kg/m 2 ) 22.4 ± 2.3 23.0 ± 2.4 0.106 Fertility cause (%)  Tubal 39 (52.0) 61 (62.2) 0.175  Ovulation disorder 2 (2.7) 2 (2.0) 1.000  Tubal and ovulation disorder 6 (8.0) 3 (3.1) 0.313  Male factors 14 (18.7) 17 (17.3) 0.803  Other 14 (18.7) 15 (15.3) 0.561 FET cycle regimen ( %)  Natural 23 (30.7) 29 (29.6) 0.879  HRT 52 (69.3) 69 (70.4)  Endometrium thickness 11.0 ± 1.8 11.2 ± 1.5 0.358  No. Embryo transferred 1.5 ± 0.5 1.4 ± 0.5 0.078 Protocol for embryo origin (%)  Agonist protocol 13 ( 17.3) 24 (24.5) 0.255  Antagonist protocol 30 (40.0) 35 (35.7) 0.564  Mild stimulation protocol 32 (42.7) 39 (39.8) 0.704 Embryo quality (%)  High 19 (25.3) 34 (34.7) 0.186  Low 56 (74.7) 64 (65.3)  Day 5 embryo 32 (42.7) 35 (35.7) 0.352  Day 6 embryo 43 (57.3) 63 (64.3) BMI body mass index, AMH anti-Müllerian hormone, ART assisted reproductive technology, FET frozen-thawed embryo transfer, HRT hormone replacement therapy Baseline characteristics of patients ≥ 38 years with prior sporadic miscarriage BMI body mass index, AMH anti-Müllerian hormone, ART assisted reproductive technology, FET frozen-thawed embryo transfer, HRT hormone replacement therapy The abnormal karyotype distribution was shown in Fig.  2 for those with aneuploid miscarriages. In women <35 years, the incidence of autosomal trisomy was 76.6% (210/274), autosomal monosomy was 0.4% (1/274) and monosomy X was 15.0% (41/274). The incidence of multiple aneuploidy and triploidy were 3.6% (10/274) and 4.4% (12/274). In women with 35–37 years, the incidence of autosomal trisomy was 86.3% (82/95) while incidence of autosomal monosomy was 2.1% (2/95). Monosomy X accounted for 6/95(6.3%). The incidence of multiple aneuploidy and triploidy were 4.2% (4/95) and 1.1% (1/95) respectively. In women ≥ 38 years, autosomal trisomy accounted for 90.8% (89/98) of cases, followed by multiple aneuploidy (4.1%, 4/98), triploidy (2.0%, 2/98), autosomal monosomy (2.0%, 2/98), and monosomy X (1.0%, 1/98). Fig. 2 Distribution of abnormal karyotypes of POCs in different age groups Distribution of abnormal karyotypes of POCs in different age groups Among all 1,186 women who underwent blastocyst transfers, the biochemical pregnancy rate (positive hCG) on the transfer day 12 was comparable between the euploid and aneuploid SM groups among all age groups ( 0.05). The biochemical pregnancy loss rate was higher in women with euploid miscarriages (< 35 years: 16.0% vs. 13.1%; 35–37 years: 24.4% vs. 15.8%; ≥38 years: 26.6% vs. 16.3%), but did not reach statistical significance. The early and late miscarriage rate were also comparable between the euploid and aneuploid SM groups. The live birth rate (55.2% vs. 60.2%; P  = 0.194) were comparable between groups with age< 35 years. In women aged 35–37 years, the live birth rate was significantly higher in patients with aneuploid SM than in those with euploid SM (52.6% vs.38.2% ; P  = 0.038). For women aged ≥ 38 years, the live birth rate was higher in patients with aneuploid SM than in those with euploid SM (39.8% vs. 26.7% ; P  = 0.072),without reaching statistic significance (Table  4 ). Table 4 Subsequent pregnancy outcomes after sporadic miscarriage <35 years 35–37 years ≥ 38 years Euploid SM Aneuploid SM P value Euploid SM Aneuploid SM P value Euploid SM Aneuploid SM P value N  = 513 N  = 274 N  = 131 N  = 95 N  = 75 N  = 98 Pregnancy outcomes (%)  hCG positive rate 403 (78.6) 223 (81.3) 0.397 95 (72.5) 73 (76.8) 0.482 49 (65.3) 63 (64.3) 0.899  Biochemical pregnancy loss 82 (16.0) 36 (13.1) 0.297 32 (24.4) 15 (15.8) 0.121 20 (26.6) 16 (16.3) 0.098  Early miscarriage rate 28 (5.5) 16 (5.8) 0.839 12 (9.1) 7 (7.4) 0.621 8 (10.7) 8 (8.2) 0.576  Late miscarriage rate 10 (1.9) 6 (2.2) 0.804 1 (0.8) 1 (1.1) 1.000 1 (1.3) 0 (0.0) 0.457  Live birth rate 283 (55.2) 165 (60.2) 0.194 50 (38.2) 50 (52.6) 0.038 20 (26.7) 39 (39.8) 0.072 SM sporadic miscarriage Subsequent pregnancy outcomes after sporadic miscarriage SM sporadic miscarriage After adjusting for confounders (including maternal age, reproductive history, BMI, AMH, treatment protocol, embryo parameters, and endometrial thickness), the live birth rate remained comparable (OR 1.209, 95% CI 0.828–1.764; P  = 0.326). In addition, the live birth rate remained statistically significant higher in aneuploid SM group with an OR 1.861; 95% CI, 1.030–3.361; P  = 0.040 in patients 35–37 years and with an OR of 2.251; 95% CI, 0.977–5.190; P  = 0.057 in patients ≥ 38 years (Table  5 ). Table 5 Live birth rate after multivariable logistic regression analysis OR for live birth rate Unadjusted OR P value Adjusted OR P value 95% CI 95% CI <35 years  Euploid SM 1 1  Aneuploid SM 1.206[0.835;1.742] 0.317 1.209[0.828;1.764] 0.326 35–37 years  Euploid SM 1 1  Aneuploid SM 1.800[1.054;3.074] 0.031 1.861[1.030;3.361] 0.040 ≥ 38 years  Euploid SM 1 1  Aneuploid SM 2.356[1.098;5.057] 0.028 2.251[0.977;5.190] 0.057 SM sporadic miscarriage Live birth rate after multivariable logistic regression analysis SM sporadic miscarriage Evidence-based maternal factors were evaluated in women who experienced subsequent pregnancy loss following their initial SM. All women who experienced a subsequent pregnancy loss underwent this full panel of tests. Among women aged < 35 years, positive maternal factors were identified in 49.2% (59/120) of those with prior euploid SM and 37.9% (22/58) of those with prior aneuploid SM ( P  = 0.189). In the 35–37 years group, the rates were 48.9% (22/45) and 52.1% (12/23), respectively ( P  = 0.798). For women ≥ 38 years, the rates were 55.2% (16/29) in the euploid SM group and 45.8% (11/24) in the aneuploid SM group ( P  = 0.498). In addition, screening for maternal factors (including uterine anatomy, adenomyosis, thyroid dysfunction, antiphospholipid antibodies and obesity) demonstrated no significant differences between euploid and aneuploid SM groups among three age groups (all P  > 0.05; see Table  6 ). Table 6 Prevalence of maternal factors in women with subsequent pregnancy loss <35 years 35–37 years ≥ 38 years Euploid SM Aneuploid SM P value Euploid SM Aneuploid SM P value Euploid SM Aneuploid SM P value N  = 120 N  = 58 N  = 45 N  = 23 N  = 29 N  = 24 Positive cases (%) 59 (49.2) 22 (37.9) 0.211 22 (48.9) 12 (52.1) 0.798 16 (55.2) 11 (45.8) 0.498 Maternal factors (%)  Uterine anatomy 6 (5.0) 0 (0.0) 0.182 2 (4.4) 0 (0.0) 0.531 0 (0.0) 0 (0.0) N/A  Adenomyosis 24 (20.0) 13 (22.4) 0.671 11 (24.4) 6 (26.1) 0.882 13 (44.8) 6 (25.0) 0.160  Thyroid dysfunction 20 (16.7) 5 (8.6) 0.159 8 (17.8) 5 (21.7) 0.750 3 (10.3) 3 (12.5) 1.000  Antiphospholipid antibodies 5 (4.2) 3 (5.2) 0.836 2 (4.4) 1 (4.3) 1.000 2 (6.9) 3 (12.5) 0.649  Obesity 6 (5.0) 2 (3.4) 0.517 0 (0.0) 0 (0.0) N/A 0 (0.0) 0 (0.0) N/A Some cases had more than one maternal factor; pregnancy loss included biochemical pregnancy loss N/A: Not applicable due to zero incidence in two groups. P value was not calculated Prevalence of maternal factors in women with subsequent pregnancy loss Some cases had more than one maternal factor; pregnancy loss included biochemical pregnancy loss N/A: Not applicable due to zero incidence in two groups. P value was not calculated

From August 2019 to May 2024, a total of 4,029 women in ART presenting with clinical miscarriage and undergoing copy number variation sequencing (CNV-seq) analysis were initially identified. Informed consent for genetic testing was obtained from all participants. This study retrospectively enrolled women experiencing their first clinical pregnancy loss before 12 weeks of gestation, with no history of miscarriage prior to the index pregnancy loss. To simulate natural conception, we excluded preimplantation genetic testing for aneuploidy (PGT-A) from all subsequent cycles. Patients with a history of autoimmune diseases, diabetes mellitus, cases lost to follow-up and samples with maternal cell contamination were also excluded. Moreover, cases with findings of small chromosomal duplications/micro-deletions or mosaicism were excluded from the final analysis due to uncertain clinical significance regarding pregnancy loss [ 11 , 12 ]. To minimize variability from multiple stimulation cycles, only frozen-thawed cycles and sibling blastocyst transfers were enrolled. Thus, 1,186 eligible blastocyst transfer cycles were included for the final analysis of pregnancy outcomes. Patients were stratified into three age groups (< 35, 35–37, and ≥ 38 years) based on the well-established age-related increase in embryonic aneuploidy risk [ 13 ]. The patient enrollment process is detailed in Fig. 1 . Fig. 1 Flow chart of case selection. ART = assisted reproductive technology; CNV = copy number variation sequencing；PGT-A = preimplantation genetic testing analysis, SM = sporadic miscarriage Flow chart of case selection. ART = assisted reproductive technology; CNV = copy number variation sequencing；PGT-A = preimplantation genetic testing analysis, SM = sporadic miscarriage The Institutional Review Board at Citic Xiangya Reproductive and Genetic hospital approved this retrospective cohort study (No.LL-SC-2025-003). All experiments were performed in accordance with the Helsinki Declaration ethical principles for medical research. All the patients signed informed consent forms and all data were collected and analyzed anonymously. Pituitary desensitization was performed using either a long luteal gonadotropin-releasing hormone (GnRH) agonist protocol or an antagonist protocol. In the agonist protocol, 1.5 mg of the GnRH analog, triptorelin (Decapeptyl; Ferring, Malmo, Sweden), was administered in the mid-luteal phase. After full desensitization was achieved, 112.5–375 IU recombinant follicle-stimulating hormone (FSH) (Gonal-F, Merck-Serono, Geneva, Switzerland; Puregon, NV Organon, Oss, The Netherlands) and/or human menopausal gonadotrophins (hMG, Lizhu, China) were used daily until the day of human chorionic gonadotropin (hCG) administration. In the antagonist protocol group, recombinant FSH or HMG with a fixed dose of 150–225 IU/ day was used from Day 2–3. When the leading follicle reached a diameter of 12 mm, 0.25 mg of the GnRH antagonist (cetrotide; Merck-Serono, Geneva, Switzerland) was administered until hCG was administered. The starting dose of gonadotrophins was based on patient age, body weight, AMH and previous response to ovarian stimulation. HCG (5000– 10 000 IU, Pregnyl; Merck) was injected when at least three follicles reach the size of 17 mm. For the mild stimulation protocol, 100 mg of clomiphene citrate was administered starting on day 2 up to day 6 of the cycle; 150 IU of gonadotrophin (r-FSH) was initiated as soon as the leading follicle reached a diameter of ≥ 14 mm on average and continued up to the day of hCG administration. Oocyte retrieval (OR) was performed 34–36 h later under general anesthesia using intravenous propofol (AstraZeneca UK Ltd). All oocytes were fertilized 4–6 h after OR, and normal fertilization was identified at 16–18 h by the presence of two pronuclei and two polar bodies. For fresh embryo transfer, luteal support was provided by administering 90 mg of vaginal progesterone gel (Crinone gel 8%; Merck Serono SA) once daily from the day of oocyte retrieval. For frozen-thawed embryo transfers, two endometrial preparation protocols were used: the hormone replacement therapy (HRT) cycle and the natural cycle. In HRT cycles, patients received with 17β-estradiol starting on day 2 or 3 of the menstrual cycle for at least 10 days before embryo transfer until the endometrial thickness reached at least 8 mm. Blastocyst transfer was performed five days after the initiation of progesterone administration for endometrial transformation. The luteal support of estradiol valerate 3 mg twice daily and oral dydrogesterone twice daily as well as vaginal progesterone 200 mg twice daily were administered continuously. Natural cycles were used for patients with regular menstrual cycles. The blastocyst transfer was performed five days after follicular rupture. The luteal support was initiated with oral dydrogesterone twice daily on the ovulation day. Cycles were canceled if endometrial thickness < 8 mm on the transfer day. Blastocysts were graded using the Gardner system [ 14 ], which assesses the degree of blastocoel expansion and the morphology of the inner cell mass (ICM) and trophectoderm (TE). The ICM and TE were each assigned a quality grade of A, B, or C. The overall blastocyst grade was a composite of its developmental stage and these ICM/TE scores. We defined blastocysts with grades of AA, AB, BA, or BB as high-quality, indicating the absence of a ‘C’ grade in either lineage. Those with a ‘C’ in either the ICM or TE (e.g., AC, BC, CA, CB) were classified as low-quality. Blastocysts of CC grade were designated as very low-quality and were excluded from transfer. CNV for POCs was carried out in accordance with the manufacturer’s instructions. In brief, total genomic DNA was extracted from tissue samples using the Amp Genomic DNA Kit (TIANGEN Biotech, Beijing, China). After shearing the genomic DNA to an average size of 200 bp, 2.5 ng of the fragmented DNA was used to create the sequencing library. 8-bp bar-coded sequencing adaptors were ligated to the DNA fragments, and PCR was performed to amplify the ligation products. The generated libraries were then pooled and sequenced on a NextSeq CN 500 high-throughput platform at approximately 1× depth after purification of the PCR product using magnetic beads. For each sample, 8–10 million of 35-bp single-end raw reads were produced. Short reads were aligned to the human reference genome (hg19) using the BWA aligner after sequencing quality control and trimming. Each reference chromosome was divided equally by a 100-kb window and the number of uniquely mapped reads in each window of each chromosome was counted. The LOWESS model was used to adjust the GC-bias of per window read counts. The corrected read counts were contrasted with an internal reference database created from a collection of 100 samples with a normal karyotype that was verified using G-banded karyotype analysis. For biochemical pregnancy loss, serum human chorionic gonadotropin (hCG) level temporarily increased and then gradually decreased, and there was no gestational sac visualized intrauterinely or extrauterinely under ultrasound examination. For early miscarriage, serum hCG level increased, there was a gestational sac inside uterus, but no fetal heart beat was detected or the heart beat had stopped within 12 weeks of pregnancy. For late miscarriage, fetal heart beat stopped between 12 and 28 weeks. Live birth was defined as the delivery of a fetus with signs of life after 28 completed weeks of the gestational age. Evidence-based diagnostic tests were performed and criteria for abnormal test results were based on current ESHRE recommendations [ 7 ]. Uterine anatomic defects were identified by hysterosalpingogram (HSG), ultrasound or sonohysterography (SHG). Uterine anomalies include unicornuate, bicornuate uteri and uterine septa. Adenomyosis is also considered a risk factor [ 15 ]. Antiphospholipid antibodies: Lupus anticoagulant was evaluated using the dilute Russell viper venom test and PTT-LA. Results greater than 42 s that were not corrected with a 1:1 mix with normal serum were considered abnormal if confirmed by a hexagonal phase phospholipid test. Serum levels of anticardiolipin (aCL) IgG and IgM were measured by enzyme-linked immunoassay, ELISA, with abnormal levels exceeding 20 phospholipid units. All positive tests were confirmed by repeat testing at least 12 weeks later. Thyroid dysfunction: Serum levels of thyroid stimulating hormone less than 0.45 µIU/mL or greater than 4.0 µIU/mL were considered abnormal. Obesity: A female body mass index (BMI)>28 kg/m 2 was considered abnormal based on Asian standard [ 16 ]. All statistical analyses were performed by using the IBM SPSS Statistics Version 25.0 (IBM Corp., USA). Means and standard deviations were calculated for continuous variables and Student’s t-test was used for comparisons. We used the Chi-square test or Fisher’s exact test to compare the frequencies and proportions. Fisher’s exact test was employed instead of the Chi-square test when the total sample size was < 40, or when any expected cell count was < 1, or when any observed cell count was ≤ 1. Differences in continuous parametric data among groups were assessed using one-way analysis of variance followed by a post hoc pairwise comparison in case of a statistical difference. Binary logistic regression was employed to assess the association between embryonic chromosomal abnormalities and reproductive outcomes. Both unadjusted and adjusted models were constructed. The regression analysis was performed after adjusting for confounders: maternal age, live birth history, body mass index (BMI), AMH (Anti-Müllerian Hormone), ART technology, endometrial thickness on the day of transfer, number of embryos transferred and embryo quality. A two-sided P value < 0.05 was considered statistically significant.

In conclusion, a single euploid SM in advanced-age women might be associated with reduced live birth rates in their subsequent pregnancies. In this population, karyotype analysis of POCs may help to identify a high-risk subgroup that could benefit from earlier and more personalized interventions.

This study demonstrated that the influence of SM karyotype is age-dependent. For women with advanced maternal age (≥ 35 years), a single euploid miscarriage might be associated with a reduced live birth rate compared to those with a previous aneuploid miscarriage. Embryonic aneuploidy is a well-established cause in half of SMs [ 17 ]. Although genetic testing of POCs is not routinely suggested for SM, evidence from recurrent pregnancy loss cohorts indicates that older women with recent euploid miscarriage are associated with a poorer prognosis [ 9 , 18 ]. However, two previous studies reported no association between the POC karyotype of SM and subsequent live birth [ 19 , 20 ], which is inconsistent with our findings. It should be noted that one of the studies did not perform age-stratified analysis [ 19 ]. In another study, although age-specific evaluation performed, the subgroup of advanced-age women with euploid miscarriage included only 30 cases [ 20 ], which may limit the statistical power to detect clinically meaningful differences. The reduced pregnancy outcomes in our study coincided with an elevated incidence of biochemical pregnancy loss in euploid SM group rather than clinical miscarriages. In fact, an increased rate of biochemical pregnancy loss has already been observed in populations with recurrent pregnancy loss [ 21 ], among whom euploid miscarriages account for nearly half of the cases [ 17 ]. As PGT-A was excluded in subsequent cycles, embryonic aneuploidy cannot be ruled out as a contributor to biochemical pregnancy loss. Nevertheless, previous evidence suggests that biochemical pregnancy loss may reflect maternal or endometrial pathology rather than embryonic aneuploidy [ 22 ]. In this study, the maternal factors were comparable among subgroups, suggesting that other undefined etiologies might play roles to affect reproductive outcomes. A growing body of literature suggests that impaired decidualization and dysregulation of key signaling molecules like leukemia inhibitory factor (LIF), epidermal growth factor (EGF), and specific microRNAs, might lead to early biochemical pregnancy loss [ 23 , 24 ]. Transcriptomic research further indicates that certain endometrial subtypes with aberrant gene expression profiles involved in cell cycle, vascular development, and metabolism, correlating with a high biochemical pregnancy rate [ 25 ]. Clinically, insufficient endometrial thickness has been identified as a significant risk factor for biochemical pregnancy loss [ 26 ], while chronic endometritis is also consistently associated with an elevated risk [ 27 ]. In our study, euploid SM did not lead to significantly different subsequent pregnancy outcomes in younger women, demonstrating an age-dependent heterogeneity. It has been reported that advanced maternal age, besides aneuploid embryos, is unequivocally associated with a decline in endometrial receptivity [ 28 ]. This age-related impairment is driven by many factors, including endometrial thinning, hormonal dysregulation and a state of chronic inflammation [ 29 – 34 ]. The initial euploid SM might suggest a possible link to an underlying impairment in endometrial receptivity, potentially leading to the higher rate of biochemical pregnancy loss observed in subsequent cycles. It is possible that younger women may have more robust compensatory capacity to resolve transient disturbances, making SM more likely to be an isolated event. However, this hypothesis requires direct validation in future studies. Indeed, aneuploid embryo is an obvious risk factor in older women. Nevertheless, several studies have indicated that PGT-A for aneuploidy selection might not improve live birth rates for women with a history of aneuploid miscarriage in contrast to non-PGT-A cycles [ 35 , 36 ]. Even for euploid embryo transfer, increasing maternal age is independently associated with a decline in ART success [ 37 ]. Therefore, improving endometrial receptivity may be a more promising strategy for older women with euploid miscarriages. It has been reported that low-dose aspirin administration might benefit women with biochemical pregnancy loss by improving endometrial blood perfusion [ 38 ]. This study has several limitations, including its retrospective, single-center design and limited sample size in certain age subgroups. Moreover, PGT-A cycles were excluded in this study. Consequently, we cannot rule out the potential influence of undetected aneuploid embryos in subsequent transfer. In addition, non-visualized ectopic pregnancies could also affect the interpretation of biochemical pregnancy loss. Moreover, only a single subsequent pregnancy outcome was analyzed, indicating that the present study could not address the potential differences when cumulative live birth rates were accounted. Future prospective studies with larger sample sizes are needed to validate our findings.

Sporadic miscarriage (SM) is one of the most common complications in early pregnancy, affecting approximately 10–15% of clinically recognized pregnancies [ 1 , 2 ]. It is estimated that fewer than 5% of couples will experience two consecutive miscarriages and only about 1% will have three or more recurrent miscarriages [ 3 ]. Embryonic chromosomal abnormalities, particularly aneuploidies, are the leading cause of sporadic pregnancy loss and are strongly associated with advanced maternal age [ 4 , 5 ]. However, genetic testing of products of conception (POCs) has not been routinely recommended for SM unless performed for explanatory purposes or to validate treatment in cases with potentially remediable causes [ 6 , 7 ]. Current evidence suggests that copy number variation (CNV) testing of POCs is cost-effective and provides valuable prognostic information specifically for couples with recurrent pregnancy loss [ 8 ]. The euploid miscarriages in recurrent pregnancy loss have been identified as a negative prognostic indicator for subsequent pregnancies [ 9 ]. A study by Popescu et al. demonstrated that 84.8% of euploid miscarriages were associated with positive findings of maternal factors, compared to only 25.4% in aneuploid losses [ 10 ]. Consequently, euploid SM not only impose a profound psychological burden on couples but also present a clinical dilemma for physicians due to their uncertain prognosis. To address this problem, the objective of this cohort study was to investigate subsequent reproductive outcomes in women following their first SM. This research aims to elucidate the prognostic significance of euploid and aneuploid SM and their impact on future pregnancy success in ART cycles.

Supplementary Material 1. Supplementary Material 1.