Impact of antinuclear and antiphospholipid antibodies on ART outcomes in women with recurrent reproductive failure.

As illustrated in the study flow chart ( Fig.  1 ) , 896 women with RRF were initially screened. Following the exclusion of 129 women with identifiable etiologies and 85 with incomplete data, the final cohort included 682 participants. Fig. 1 Flow chart of study participants. RIF, recurrent implantation failure; RPL, recurrent pregnancy loss; RRF, recurrent reproductive failure; ANA, antinuclear antibody; aPL, antiphospholipid antibody Flow chart of study participants. RIF, recurrent implantation failure; RPL, recurrent pregnancy loss; RRF, recurrent reproductive failure; ANA, antinuclear antibody; aPL, antiphospholipid antibody Baseline characteristics were compared among the four groups defined by ANA and aPL status: ANA-/aPL- ( n  = 466), ANA-/aPL+ ( n  = 29), ANA+/aPL- ( n  = 151), and ANA+/aPL+ ( n  = 36). Significant differences were observed for female age ( p  = 0.040), with ANA+/aPL- women being slightly older (34.5 ± 4.2 years) compared to other groups. The duration of infertility showed marked variation ( p  < 0.001), being longest in the ANA+/aPL+ group (4.40 ± 4.22 years). Reproductive history variables, including gravidity and number of abortions showed significant differences among the groups (all p  < 0.001), with the ANA-/aPL- group consistently exhibiting higher values. In contrast, no significant differences were found in BMI ( p  = 0.810), parity ( p  = 0.407), or any hormonal parameters, including basal FSH ( p  = 0.273), LH ( p  = 0.200), PRL ( p  = 0.773), E2 ( p  = 0.334), testosterone (T) ( p  = 0.911), or AMH levels ( p  = 0.763), indicating similar endocrine profiles across all antibody status groups. Ovarian stimulation parameters, including the total duration of Gn stimulation and total Gn dose were not significantly different among the groups. The mean endometrial thickness measured on the day of HCG administration ranged from 10.16 ± 2.62 mm to 10.97 ± 2.50 mm across the groups. Detailed data are summarized in Table  1 . Table 1 Baseline characteristics by ANA and aPL status Variable Group p -value ANA-/aPL- N  = 466 ANA-/aPL+ N  = 29 ANA+/aPL- N  = 151 ANA+/aPL+ N  = 36 Age (years) 33.5 ± 4.2 33.9 ± 4.2 34.5 ± 4.2 33.0 ± 4.9 0.040 BMI (kg/m²) 21.63 ± 2.80 21.70 ± 2.95 21.45 ± 2.91 21.26 ± 2.66 0.810 Infertility duration (years) 2.37 ± 2.61 3.15 ± 2.61 2.72 ± 2.92 4.40 ± 4.22 < 0.001 Gravidity (G) 2.44 ± 1.59 1.52 ± 1.50 1.81 ± 1.64 1.06 ± 1.24 < 0.001 Parity (P) 0.21 ± 0.46 0.34 ± 0.48 0.21 ± 0.44 0.17 ± 0.38 0.407 Number of abortions (A) 2.04 ± 1.39 1.07 ± 1.22 1.45 ± 1.52 0.67 ± 1.07 < 0.001 Platelet count (×10⁹/L) 267 ± 65 269 ± 60 273 ± 69 264 ± 54 0.774 Basal FSH (IU/L) 5.99 ± 1.99 5.54 ± 1.61 6.19 ± 2.35 6.41 ± 1.73 0.273 Basal LH (IU/L) 3.34 ± 1.71 3.15 ± 1.97 3.32 ± 1.55 3.93 ± 1.80 0.200 Basal PRL (ng/mL) 19 ± 19 16 ± 9 18 ± 10 19 ± 10 0.773 Basal E2 (pg/mL) 36 ± 27 29 ± 12 38 ± 20 36 ± 21 0.334 Basal T (ng/mL) 0.44 ± 2.88 0.32 ± 0.17 0.28 ± 0.11 0.30 ± 0.12 0.911 AMH (ng/mL) 3.80 ± 3.04 3.76 ± 3.45 3.51 ± 3.13 4.03 ± 2.75 0.763 Initial Gn dose (IU/day) 250.43 ± 60.31 244.83 ± 53.19 250.67 ± 57.38 232.64 ± 59.18 0.355 Total Gn dose (IU) 2685.57 ± 896.68 2588.79 ± 752.51 2674.92 ± 891.50 2588.19 ± 976.35 0.880 Total duration of Gn stimulation (days) 10.44 ± 2.11 10.31 ± 1.73 10.51 ± 2.28 10.67 ± 2.23 0.892 Endometrial thickness on HCG day (mm) 10.16 ± 2.62 10.97 ± 2.50 10.88 ± 3.95 10.89 ± 2.93 0.035 Data are presented as mean ± SD. ANA, antinuclear antibody; aPL, antiphospholipid antibody; BMI, body mass index; FSH, follicle-stimulating hormone; LH, luteinizing hormone; PRL, prolactin; E2, estradiol; T, testosterone; AMH, anti-Müllerian hormone; Gn, gonadotropin; hCG, human chorionic gonadotropin. Baseline characteristics by ANA and aPL status Endometrial thickness on HCG day (mm) Live birth rates differed significantly among the groups ( p  = 0.001). The ANA−/aPL− group achieved the highest live birth rate (36.3%), which was substantially higher than rates observed in the ANA−/aPL+ (17.2%), ANA+/aPL− (23.2%), and ANA+/aPL+ (16.7%) groups. Clinical pregnancy rates showed a trend toward significance ( p  = 0.050), with the ANA+/aPL+ group exhibiting the lowest rate (30.6%) compared to the ANA−/aPL− group (49.8%). In contrast, the mean number of implanted embryos was comparable across the four groups, as were the total number of retrieved oocytes ( p  = 0.357), the number of normally fertilized oocytes (2PN) ( p  = 0.128), the number of cleaved embryos ( p  = 0.134), the number of usable embryos ( p  = 0.354), and the number of high-quality embryos ( p  = 0.121). Biochemical pregnancy rates did not differ significantly among the groups ( p  = 0.106), and both birth weight and miscarriage rates were similarly comparable ( p  = 0.762). Detailed outcomes are summarized in Table  2 . Table 2 Embryological and reproduction outcomes according to ANA and aPL status Variable Group p -value ANA-/aPL- N  = 466 ANA-/aPL+ N  = 29 ANA+/aPL- N  = 151 ANA+/aPL+ N  = 36 Total number of retrieved oocytes 14.85 ± 8.00 14.54 ± 7.90 13.37 ± 8.30 14.31 ± 8.50 0.357 Number of normally fertilized oocytes (2PN) 9.04 ± 5.70 9.76 ± 5.28 8.09 ± 5.48 10.11 ± 5.62 0.128 Number of cleaved embryos 9.85 ± 6.17 10.76 ± 5.75 9.16 ± 6.20 11.69 ± 6.24 0.134 Number of usable embryos 4.93 ± 3.61 5.31 ± 3.69 4.57 ± 3.36 5.66 ± 4.09 0.354 Number of high-quality embryos 3.74 ± 3.26 3.90 ± 2.85 3.35 ± 2.90 4.77 ± 3.90 0.121 Number of embryos transferred 0.52 ± 0.54 0.45 ± 0.63 0.42 ± 0.52 0.33 ± 0.53 0.081 Biochemical pregnancy, n (%) 60 (12.9%) 4 (13.8%) 10 (6.6%) 6 (16.7%) 0.106 Miscarriage, n (%) 48 (10.3%) 3 (10.3%) 20 (13.2%) 3 (8.3%) 0.762 Clinical pregnancy, n (%) 232 (49.8%) 12 (41.4%) 62 (41.1%) 11 (30.6%) 0.050 Live birth, n (%) 169 (36.3%) 5 (17.2%) 35 (23.2%) 6 (16.7%) 0.001 Birth weight (g) 3,111 ± 507 2,788 ± 550 3,010 ± 413 2,758 ± 468 0.132 Data are presented as mean ± SD or n (%)ANA, antinuclear antibody; aPL, antiphospholipid antibody; 2PN, two-pronuclei Embryological and reproduction outcomes according to ANA and aPL status Data are presented as mean ± SD or n (%)ANA, antinuclear antibody; aPL, antiphospholipid antibody; 2PN, two-pronuclei Univariable logistic regression analysis was performed to identify factors associated with live birth. Higher age (OR 0.96, 95% CI 0.92–1.00; p  = 0.031), longer infertility duration (OR 0.90, 95% CI 0.84–0.96; p  = 0.001), higher basal FSH (OR 0.92, 95% CI 0.84–1.00; p  = 0.048), ANA positivity (OR 0.49, 95% CI 0.33–0.73; p  < 0.001), and LAC positivity (OR 0.36, 95% CI 0.16–0.82; p  = 0.014) were each associated with lower odds of live birth. Other variables, including BMI, platelet count, basal LH, basal PRL, basal E2, basal testosterone, AMH, aCL positivity, and anti-β2GPI positivity, showed no significant associations (all p  > 0.05). Backward stepwise regression was applied to select the lowest AIC model for multivariable analysis. In this adjusted analysis, ANA positivity remained independently associated with reduced odds of live birth (adjusted OR 0.57, 95% CI 0.38–0.86; p  = 0.008), and longer infertility duration continued to be inversely associated with live birth (adjusted OR 0.92, 95% CI 0.86–0.99; p  = 0.018). Basal PRL showed a borderline inverse association (adjusted OR 0.98, 95% CI 0.97–1.00; p  = 0.058). No significant associations were observed for age (adjusted OR 0.97, 95% CI 0.93–1.01; p  = 0.138), aCL positivity (adjusted OR 0.69, 95% CI 0.18–2.59; p  = 0.584), anti-β2GPI positivity (adjusted OR 0.32, 95% CI 0.04–2.68; p  = 0.296), lupus anticoagulant (adjusted OR 0.47, 95% CI 0.20–1.10; p  = 0.080), or basal FSH (adjusted OR 0.94, 95% CI 0.86–1.02; p  = 0.150) ( Table  3 ) . Table 3 Univariable and multivariable logistic regression analyses of factors associated with live birth Characteristic Univariable Multivariable N Event N OR 95% CI p -value N Event N OR 95% CI p -value Age (years) 682 215 0.96 (0.92, 1.00) 0.031 682 215 0.97 (0.93, 1.01) 0.138 Infertility duration (years) 682 215 0.90 (0.84, 0.96) 0.001 682 215 0.92 (0.86, 0.99) 0.018 BMI 682 215 1.00 (0.94, 1.06) 0.985 Platelet Count (×10⁹/L) 678 214 1.00 (1.00, 1.00) 0.734 Basal FSH (IU/L) 682 215 0.92 (0.84, 1.00) 0.048 682 215 0.94 (0.86, 1.02) 0.150 Basal LH (IU/L) 682 215 0.94 (0.85, 1.04) 0.241 Basal PRL (ng/mL) 682 215 0.99 (0.97, 1.00) 0.056 682 215 0.98 (0.97, 1.00) 0.058 Basal E2 (pg/mL) 682 215 1.00 (0.99, 1.00) 0.505 Basal T (ng/mL) 682 215 0.61 (0.17, 2.17) 0.449 AMH (ng/mL) 682 215 1.04 (0.98, 1.09) 0.166 ANA positivity  Negative 502 177 — — 502 177 — —  Positive 180 38 0.49 (0.33, 0.73) < 0.001 180 38 0.57 (0.38, 0.86) 0.008 aCL positivity  Negative 666 212 — — 666 212 — —  Positive 16 3 0.49 (0.14, 1.75) 0.275 16 3 0.69 (0.18, 2.59) 0.584 Anti-β2GPI positivity  Negative 672 214 — — 672 214 — —  Positive 10 1 0.24 (0.03, 1.89) 0.174 10 1 0.32 (0.04, 2.68) 0.296 Lupus anticoagulant positivity  Negative 635 208 — — 635 208 — —  Positive 47 7 0.36 (0.16, 0.82) 0.014 47 7 0.47 (0.20, 1.10) 0.080 OR, odds ratio; CI, confidence interval; BMI, body mass index; FSH, follicle-stimulating hormone; LH, luteinizing hormone; PRL, prolactin; E2, estradiol; T, testosterone; AMH, anti-Müllerian hormone; ANA, antinuclear antibody; aCL, anticardiolipin antibody; anti-β2GPI, anti-β2 glycoprotein I antibody Univariable and multivariable logistic regression analyses of factors associated with live birth OR, odds ratio; CI, confidence interval; BMI, body mass index; FSH, follicle-stimulating hormone; LH, luteinizing hormone; PRL, prolactin; E2, estradiol; T, testosterone; AMH, anti-Müllerian hormone; ANA, antinuclear antibody; aCL, anticardiolipin antibody; anti-β2GPI, anti-β2 glycoprotein I antibody Among the 187 ANA positive women, 113 (60.4%) had single ANA specificity and 74 (39.6%) had multiple ANA specificities. Baseline characteristics including age, body mass index, infertility duration, and ovarian reserve markers (basal FSH, LH, E2, AMH) were comparable between the single and multiple ANA groups (all p  > 0.05), except for a modestly higher basal LH in the multiple ANA cohort (3.9 ± 2.1 vs. 3.1 ± 1.2 IU/L; p  = 0.005). There were no significant differences in phospholipid antibody positivity (17.6% vs. 18.6%, p  = 0.860). Regarding reproductive outcomes, no significant differences were observed between the single and multiple ANA groups in biochemical pregnancy rates (9.5% vs. 8.0%, p  = 0.721), miscarriage rates (10.8% vs. 13.3%, p  = 0.616), clinical pregnancy rates (36.5% vs. 40.7%, p  = 0.563), or live birth rates (24.3% vs. 20.4%, p  = 0.521) ( Table  4 ) . Table 4 Baseline characteristics and reproductive outcomes in ANA-positive participants Variable Multiple ANA+ ( N  = 74) Single ANA+ ( N  = 113) p -value Age (years) 34.1 ± 4.4 34.3 ± 4.4 0.785 Infertility duration (years) 3.1 ± 3.0 3.0 ± 3.4 0.905 Basal FSH (IU/L) 6.31 ± 2.67 6.18 ± 1.92 0.724 Basal LH (IU/L) 3.89 ± 2.06 3.14 ± 1.15 0.005 Basal PRL (ng/mL) 18 ± 9 19 ± 11 0.622 Basal E2 (pg/mL) 41 ± 24 36 ± 17 0.143 Basal T (ng/mL) 0.29 ± 0.14 0.28 ± 0.09 0.603 AMH (ng/mL) 4.07 ± 3.83 3.33 ± 2.45 0.183 aPL positivity, n (%) 13 (17.6%) 21 (18.6%) 0.860 Biochemical pregnancy, n (%) 7 (9.5%) 9 (8.0%) 0.721 Miscarriage, n (%) 8 (10.8%) 15 (13.3%) 0.616 Clinical pregnancy, n (%) 27 (36.5%) 46 (40.7%) 0.563 Live birth, n (%) 18 (24.3%) 23 (20.4%) 0.521 Data are presented as mean ± SD or n (%)ANA, antinuclear antibody; aPL, antiphospholipid antibody; FSH, follicle-stimulating hormone; LH, luteinizing hormone; PRL, prolactin; E2, estradiol; T, testosterone; AMH, anti-Müllerian hormone Baseline characteristics and reproductive outcomes in ANA-positive participants Data are presented as mean ± SD or n (%)ANA, antinuclear antibody; aPL, antiphospholipid antibody; FSH, follicle-stimulating hormone; LH, luteinizing hormone; PRL, prolactin; E2, estradiol; T, testosterone; AMH, anti-Müllerian hormone

This retrospective cohort study included 682 women with RRF who underwent IVF/ICSI at the First Affiliated Hospital of Sun Yat-sen University between April 2019 and April 2024. All eligible women were identified consecutively from the institutional ART database. At their initial visit, all patients were systematically screened for conventional etiologies of reproductive failure, including genetic abnormalities, uterine structural anomalies, endocrine disorders, and established autoimmune diseases. Women without identifiable causes were classified as having unexplained RRF, comprising recurrent implantation failure (RIF) and recurrent pregnancy loss (RPL). RIF was defined as the failure to achieve a clinical pregnancy (presence of a gestational sac on transvaginal ultrasound) after at least three transfers of high-quality embryos or after the transfer of ≥ 4 blastocysts. RPL was defined as two or more pregnancy losses before 20 weeks of gestation, irrespective of whether conception was spontaneous or ART-assisted. Testing for ANA and aPL was primarily indicated by a history of RIF or RPL in the absence of other identified causes. Autoantibody testing was performed before the initiation of the index IVF/ICSI cycle, and only women with complete autoantibody results were included in the final analysis. Inclusion criteria were female age < 38 years, basal FSH < 10 IU/L measured on day 2–4 of a spontaneous menstrual cycle, and no prior history of ovarian surgery, chemotherapy, or autoimmune diseases. Only women with unexplained RRF fulfilling all of the above criteria were included. Exclusion criteria included chromosomal abnormalities or a family history of genetic disorders, moderate-to-severe endometriosis, adenomyosis, congenital reproductive tract anomalies, chronic endometritis, polycystic ovary syndrome, thyroid dysfunction, severe male factor infertility, reproductive system malignancies, and acute reproductive tract infections. Patients with incomplete clinical, laboratory, or follow-up outcome data were also excluded. Ovarian stimulation protocols were individualized based on patient characteristics and included gonadotropin regimens with gonadotropin-releasing hormone (GnRH) agonist protocol or GnRH antagonist protocol. The human chorionic gonadotropin (hCG) trigger was administered when at least two to three follicles reached 17–18 mm in diameter, and oocyte retrieval was conducted within 36–38 h later through transvaginal ultrasonography-guided aspiration. Transfer of fresh embryos was performed 3 or 5 days after oocyte collection, depending on embryo quality. For frozen embryos transfer, embryos were thawed and incubated in a series of commercially available solutions. Cleavage-stage embryos or blastocysts were transferred 3 or 5 days after ovulation in a natural cycle, or 4 or 6 days after progesterone supplementation in a hormone replacement treatment cycle. Fourteen days post-transfer, urinary and serum hCG levels were assessed; if positive, an ultrasound was performed 3 weeks later to confirm a clinical pregnancy. All ovarian stimulation, laboratory, and embryo transfer procedures were conducted according to the standard operating protocols of the reproductive center. Serum autoantibody testing was performed using standardized, validated clinical laboratory techniques. The following autoantibodies were measured: antinuclear antibodies, and aPL, which included anti-cardiolipin antibodies (aCL), anti-β2 glycoprotein I antibodies (anti-β2GPI), and lupus anticoagulant (LAC). ANA status was determined using indirect immunofluorescence (IIF) on HEp-2 cells and/or an equivalent test by solid-phase immunoassays. ANA positivity was defined as an IIF titer ≥ 1:80 and/or a positive result for specific extractable nuclear antigens (ENAs) based on the laboratory’s reference cut-off values. Identified specificities included anti-dsDNA, anti-C1q, anti-histone, anti-Sm, anti-SSA/Ro52, anti-SSA/Ro60, anti-SSB, anti-Scl-70, anti-Jo-1, anti-centromere, and anti-RNP antibodies. Anti-β2GPI (IgG/IgM) and anti-cardiolipin (IgG/IgM) antibodies were measured using enzyme-linked immunosorbent assay (ELISA). LAC was detected using a simplified dilute Russell’s viper venom time (dRVVT) assay with screening and confirmatory steps. Positivity for aPL was defined as aCL ≥ 20 U/mL, anti-β2GPI ≥ 20 RU/mL, or a positive LAC test. Participants were categorized into four groups based on ANA and aPL status (ANA−/aPL−, ANA−/aPL+, ANA+/aPL−, and ANA+/aPL+). Women with positive ANA were further categorized based on the number of specific antibodies detected: single positivity, defined as one specific ANA antibody; and multiple positivity, defined as ≥ 2 different ANA specificities. Clinical and demographic data were retrieved from patients’ medical records and the ART laboratory database. Collected variables included age, body mass index (BMI), duration of infertility, and reproductive history, encompassing gravidity (G), parity (P), and number of abortions (A). Ovarian reserve markers were documented, including basal levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), and anti-Müllerian hormone (AMH). Ovarian stimulation parameters were recorded for all participants, including the initial gonadotropin (Gn) dose (IU/day), the total Gn dose administered during the stimulation cycle (IU), the total duration of Gn stimulation (days), and endometrial thickness measured on the day of hCG administration (mm). Cycle-level embryological data were extracted from the electronic ART database using a standardized data collection form. Embryological outcomes included the total number of retrieved oocytes, number of normally fertilized oocytes (2PN), number of cleaved embryos, number of usable embryos (available for fresh transfer or cryopreservation), and number of high-quality embryos, all defined according to routine morphological criteria at the center. The implantation rate was calculated as the number of gestational sacs observed on transvaginal ultrasound divided by the number of embryos transferred. The primary outcome was the live birth rate, defined as the delivery of at least one living infant beyond 24 weeks of gestation, expressed as the number and percentage of live births per embryo transfer cycle. Secondary outcomes included biochemical pregnancy, defined as a positive serum β-hCG test approximately 14 days after embryo transfer without subsequent ultrasound confirmation of a gestational sac; miscarriage, defined as spontaneous pregnancy loss before 20 completed weeks of gestation among women who achieved a clinical pregnancy; clinical pregnancy, defined as the presence of at least one intrauterine gestational sac detected by transvaginal ultrasound at 4–5 weeks after embryo transfer (corresponding to approximately 6–7 weeks of gestation), with or without fetal cardiac activity; and birth weight, recorded in grams immediately after delivery. All secondary outcomes were reported as number and percentage where applicable. Continuous variables were assessed for normality. Normally distributed data are presented as mean ± SD and were compared using one-way ANOVA. Non-normally distributed data are presented as median and were compared using the Kruskal–Wallis test. Categorical variables are expressed as frequencies (percentages) and were compared using the Chi-square test or Fisher’s exact test. Univariate logistic regression analysis was performed to evaluate the association between individual factors and live birth outcomes. A backward stepwise selection approach based on AIC (using the stepAIC function in the MASS package in R) was employed to identify independent predictors. Results are presented as odds ratios (ORs) with 95% confidence intervals (CIs). Statistical significance was defined as a two-sided p-value < 0.05. All statistical analyses were performed using R (version 4.2.2).

Infertility, defined as the inability to conceive after 12 months of unprotected intercourse, is a multifactorial condition that affects millions of couples worldwide. Its etiologies range from ovulatory dysfunction and male factor infertility to tubal disease and uterine abnormalities [ 1 ]. In cases of infertility where conventional treatments such as ovarian stimulation and intrauterine insemination (OS-IUI) fail, in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) often become the primary treatment options [ 2 – 4 ]. Despite advancements in assisted reproductive technology (ART), the success rates remain suboptimal, with approximately 50–60% of IVF cycles failing to achieve implantation [ 3 , 5 , 6 ]. Recurrent reproductive failure (RRF), defined as multiple failed IVF cycles or pregnancy losses, is influenced by a variety of factors, including genetic abnormalities, endocrine dysfunction, uterine defects, and immune dysregulation [ 7 ]. Among these contributors, immune dysregulation has emerged as an important yet insufficiently characterized determinant of ART outcomes [ 8 , 9 ]. Autoimmune diseases represent a significant and often underrecognized cause of RRF. While conditions such as antiphospholipid syndrome (APS) and systemic lupus erythematosus (SLE) have well-established links with adverse pregnancy outcomes, the role of serum autoantibodies in the absence of overt autoimmune disease remains an area of ongoing investigation. Notably, a substantial proportion of women with RRF exhibit nonspecific autoimmune features, including serum autoantibody positivity, without meeting the diagnostic criteria for a defined autoimmune disease [ 10 ]. Among these autoantibodies, antiphospholipid antibodies (aPL) and antinuclear antibodies (ANA) have received particular attention due to their elevated prevalence in women with reproductive failure. aPL, a diagnostic hallmark of APS, has been strongly associated with adverse reproductive outcomes, including recurrent pregnancy loss (RPL), implantation failure, and preterm birth [ 11 ]. Similarly, ANAs, which encompass a diverse family of autoantibodies targeting nuclear components such as DNA, RNA, and proteins, have been implicated in RPL and infertility [ 12 , 13 ]. ANA-positive women undergoing IVF/ICSI exhibit lower clinical pregnancy and implantation rates, alongside higher miscarriage rates, compared with ANA-negative counterparts [ 14 ]. Despite these associations, the clinical management of women who test positive for ANA and/or aPL but do not meet criteria for a defined autoimmune disease remains challenging. These women constitute a substantial proportion of the ART population, yet clinicians lack evidence-based guidance regarding their prognosis, therapy, or risk stratification. Moreover, the combined presence of ANA and aPL may reflect a more complex immune phenotype that could carry greater reproductive risk, potentially amplifying complement-mediated inflammation at the maternal-fetal interface [ 15 ]; however, its impact on ART outcomes remains insufficiently characterized. In particular, the diversity of autoantibody profiles and the overlapping nature of aPL further complicate efforts to clarify their precise roles in infertility and reproductive failure. Given these clinical and knowledge gaps, this study systematically evaluates the reproductive outcomes associated with ANA and aPL positivity, both individually and in combination, among women with RRF undergoing ART. By clarifying the reproductive significance of serum autoantibodies, we aim to provide evidence that can guide clinical counseling and improve therapeutic decision-making in this population.

Our findings contribute to the growing body of evidence on the differential impacts of autoantibodies on ART outcomes in RRF patients. The data suggest that the presence of ANA and aPL is associated with a lower likelihood of achieving a live birth. While these results point to the potential utility of immune profiling in this high-risk population, further prospective research is warranted to validate the clinical value of screening and to determine whether targeted interventions can improve reproductive outcomes.

ANA and aPL are hallmark immunologic markers frequently associated with systemic autoimmune diseases. Their presence often reflects subclinical immune dysregulation, even in women without a formal autoimmune diagnosis [ 16 ]. In the context of ART, increasing evidence points to the potential impact of autoantibodies on reproductive success; however, most previous studies have focused on either ANA or aPL alone, and few have quantified their combined impact in women with RRF. Our study expands upon existing literature by demonstrating that the coexistence of ANA and aPL exerts a more pronounced detrimental effect on ART outcomes compared with isolated antibody positivity. In our cohort, women positive for both ANA and aPL had a clinical pregnancy rate of 30.6% and a live birth rate of 16.7%, compared with 49.8% and 36.3% in ANA−/aPL− women, with a reduction in live birth of nearly 20% points relative to ANA−/aPL− patients. By contrast, parameters reflecting ovarian response and embryo development, including the number of usable and high-quality embryos, embryos transferred, and birth weight, were comparable across groups (all p > 0.05). Taken together, these associations suggest that dual antibody positivity primarily affects events after implantation, impairing early pregnancy maintenance rather than intrinsic oocyte competence or fetal growth. Both thrombotic and non-thrombotic mechanisms may underlie this vulnerability. Thrombotic mechanisms include aPL-mediated activation of the coagulation cascade, endothelial injury, and impaired spiral artery remodeling, which can restrict uteroplacental blood flow and compromise placental development [ 17 ]. Non-thrombotic mechanisms include complement activation, microvascular inflammation, and disruption of maternal–fetal immune tolerance, collectively inducing a hostile inflammatory microenvironment at the implantation site [ 18 – 20 ]. Experimental and clinical studies have demonstrated aPL-induced complement activation, endothelial dysfunction, and immune complex deposition in placental tissue, supporting these mechanisms as potential therapeutic targets [ 21 – 23 ]. Immunopathological studies have revealed complement C3 and C4 deposition in placental tissues from individuals with SLE, suggesting local immune complex-mediated inflammation [ 24 ]. In vitro evidence of embryotoxic effects of immunoglobulins from ANA-positive women further indicates that autoantibodies can damage embryos at very early stages [ 25 ]. These immune-mediated pathways provide a biologically plausible explanation for the impaired implantation, placentation, and early pregnancy maintenance observed in the ANA+/aPL+ group. A novel and clinically relevant finding of this study is the lack of association between the number of ANA specificities and reproductive outcomes, suggesting that the qualitative presence of autoimmunity may be more critical than the quantitative burden. Among ANA-positive women, live birth rates were 24.3% in those with multiple ANA specificities and 20.4% in those with a single specificity ( p  = 0.521), and clinical pregnancy rates were likewise similar. These differences were small in absolute terms, but this interpretation should be tempered by the possibility that our subgroup sample sizes were insufficient to detect modest differences. Thus, the clinical relevance of ANA multiplicity remains uncertain and warrants further investigation. Accordingly, it may be more appropriate for treatment decisions to prioritize the overall clinical phenotype, reproductive history, and functional immune markers rather than autoantibody count alone. Consistent with prior studies, ANA positivity alone remained an independent predictor of reduced live birth rates in our multivariable model. After adjustment for age, infertility duration, hormonal parameters and other autoantibodies, ANA-positive women had 43% lower odds of live birth compared with ANA-negative women. This finding aligns with the pooled estimates from a meta-analysis of 11 studies, which reported a significantly lower clinical pregnancy rate (RR 0.66, 95% CI 0.56–0.79) in ANA-positive women undergoing IVF/ICSI [ 14 ]. Our results reinforce ANA’s clinical significance and suggest that pre-ART immune screening may be considered in selected populations, especially those with unexplained infertility or recurrent implantation failure, although its clinical utility requires further prospective evaluation. Additionally, we observed that longer infertility duration was independently associated with lower live birth rates (adjusted OR 0.92, 95% CI 0.86–0.99; p = 0.017), consistent with prior reports emphasizing the detrimental impact of delayed intervention. This likely reflects the cumulative impact of prolonged ART exposure, chronic low-grade inflammation, or persistent endometrial dysfunction, all of which may exacerbate immunological barriers to successful pregnancy [ 26 ]. This study has several limitations. Its retrospective nature precludes causal inference, and the sample size in specific autoantibody subgroups was limited, possibly underpowering some comparisons. Moreover, we did not analyze antibody titers, immunofluorescence patterns, or other immune parameters such as regulatory T-cell activity or natural killer cell profiles. Future prospective studies should incorporate comprehensive immunophenotyping to better delineate pathogenic subsets. In parallel, leveraging large-scale clinical datasets with modern analytical methods, such as ensemble-based learning or integrative genotype-phenotype correlation analyses [ 27 , 28 ], could further enhance our ability to model reproductive risk and treatment response in this population. Additionally, interventional trials evaluating targeted immunotherapies such as low-dose steroids, heparin, hydroxychloroquine, or IVIG could consider stratifying participants based on precise immune profiles rather than broad serological classifications. Such trial designs may help determine whether tailored immunomodulatory strategies can optimize treatment, minimize overtreatment, and ultimately improve ART outcomes for women with immune-mediated reproductive failure.