Hyperhomocysteinemia as a Risk Factor for Unexplained Recurrent Pregnancy Loss: A Case- Control Study at the Damascus University Maternity Hospital

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Objective This study aimed to investigate the relationship between elevated serum homocysteine (Hcy) levels and unexplained RPL. Methods A hospital-based case-control study was conducted from August 2024 to December 2025 at Damascus University Maternity Hospital. Ninety-six non-pregnant women were enrolled: 70 with a history of ≥ 2 unexplained miscarriages (case group) and 26 with prior successful pregnancies and no history of miscarriage (control group). Serum homocysteine concentrations were measured using an enzymatic assay on a BS-800M Mindray analyzer. Statistical analyses included group comparisons, correlation testing, and receiver operating characteristic (ROC) curve analysis. Results The mean serum homocysteine level was significantly higher in the RPL group (28.8 µmol/L, IQR 17–43) compared with controls (10.54 µmol/L, IQR 7.23–14.21; p 15 µmol/L) was observed in 54.3% of cases versus none of the controls. A positive correlation was found between homocysteine concentration and the number of pregnancy losses (r = 0.318, p = 0.007). ROC analysis identified an optimal cutoff of 11.57 µmol/L, yielding 88.6% sensitivity and 70.0% specificity for predicting RPL. Conclusion This study demonstrates a significant association between hyperhomocysteinemia and unexplained recurrent recurrent pregnancy loss. A strong, statistically significant positive correlation existed between the level of serum homocysteine and the number of miscarriages, suggesting a dose-response relationship. Homocysteine assessment could be considered screening and metabolic interventions in RPL management and warrants further investigation. Hyperhomocysteinemia unexplained recurrent pregnancy loss miscarriage folate metabolism homocysteine Figures Figure 1 Background Homocysteine, a sulfur-containing amino acid derivative generated during methionine metabolism, is a non-dietary compound with established cytotoxic properties. In circulation, the majority of homocysteine is protein-bound, leaving only a minimal fraction in its free, metabolically active form [ 1 ]. Clinically, hyperhomocysteinemia (HHcy) is defined as a fasting serum concentration exceeding 15 µmol/L, with severity graded from mild to severe [ 2 ]. Its etiology is complex, arising from an interplay of genetic predispositions, most notably polymorphisms in the methylenetetrahydrofolate reductase (*MTHFR*) gene, nutritional deficiencies in folate and B vitamins, systemic conditions such as renal disease, and specific lifestyle or pharmacological factors [ 3 ]. A well-characterized vascular toxin, homocysteine promotes endothelial dysfunction, oxidative stress, and a prothrombotic milieu, solidifying its role as an independent risk factor for atherosclerotic cardiovascular disease [ 4 ]. Beyond cardiology, its pathogenic reach extends to microvascular complications in ophthalmology and increasingly to the realm of reproductive failure. Recurrent pregnancy loss (RPL), defined as the loss of two or more clinical pregnancies before 20 weeks of gestation, represents a profound clinical challenge that affects 1–5% of couples. Despite standardized investigations into genetic, anatomical, endocrine, and immunological causes, the etiology remains unexplained in approximately half of all cases, highlighting a critical gap in both understanding and therapeutic strategy [ 5 ], [ 6 ], [ 7 ]. The hypothesis linking HHcy to RPL was first posited over three decades ago, with observations suggesting that homocysteine-induced placental vasculopathy and infarction could underlie unexplained fetal loss [ 8 ]. This has positioned aberrant homocysteine metabolism as a compelling pathway in reproductive pathology. However, the consistency and strength of this association require rigorous elucidation. Notably, data concerning this association in the Middle East region, particularly within a strictly defined unexplained RPL (URPL) cohort, remain limited and inconclusive. Furthermore, the nature of a dose-response relationship—the correlation between homocysteine levels and the number of prior losses—has not been sufficiently explored in this specific patient population. This case-control study was therefore designed to systematically compare serum homocysteine concentrations in women with a history of URPL with those with documented normal obstetric outcomes. Specifically, this study aims to address this knowledge gap by testing the hypothesis that women with URPL have significantly higher serum homocysteine levels compared to women with normal reproductive history, and that these levels correlate positively with the number of prior pregnancy losses. Achieving this objective will clarify whether elevated homocysteine constitutes a significant and modifiable risk factor, thereby identifying a potential target for therapeutic intervention in this complex disorder. Methods A hospital-based case-control study was conducted at the Obstetrics and Gynecology University Hospital in Damascus, Syria from August 2024 to December 2025. This study aimed to investigate the association between serum homocysteine levels and recurrent pregnancy loss (RPL). Participant Recruitment and Selection: Participants were recruited from the infertility clinic of the hospital. They were divided into two groups: Case Group (RPL): Non-pregnant women with a history of two or more consecutive spontaneous pregnancy losses, with a minimum period of one month elapsed since the most recent pregnancy loss. Control Group: Healthy, non-pregnant, and non-lactating women with at least one previous successful live birth and no history of spontaneous abortion or pregnancy loss. Inclusion and Exclusion Criteria: For inclusion in the Case Group, participants had to be aged 18–45 years and history of ≥ 2 unexplained spontaneous abortions; and no clinically identifiable cause for RPL (e.g., uterine anomalies, parental chromosomal abnormalities, endocrine disorders such as overt thyroid dysfunction, or diagnosed antiphospholipid syndrome), as confirmed by resident obstetricians through clinical examination and available hospital records. For the Control Group, participants had to be aged 18–45 years, have at least one prior live birth, and no history of spontaneous abortion. The following exclusion criteria applied to both groups: presence of any of the aforementioned conditions, current use of medications affecting coagulation or homocysteine metabolism (e.g., aspirin, anticoagulants, folate, or B-vitamin supplements), or any acute illness at the time of sampling. Due to financial constraints limiting the final sample size (n = 96), a post-hoc power calculation will be presented in the Results section to clarify the detectable effect size. Data and Sample Collection After obtaining written informed consent, all participants completed a detailed questionnaire covering demographics, medical and obstetric history, lifestyle factors (smoking and alcohol), occupational exposures, and supplement use history. Following an 8–12 hour overnight fast, a venous blood sample was drawn into a dry tube. The samples were processed immediately by centrifugation at 4000 rpm for 10 min. The isolated serum was aliquoted into Eppendorf tubes and stored at -60°C until batch analysis. Laboratory Analysis: serum homocysteine concentration was quantified using a commercial enzymatic assay kit on a BS-800M Mindray clinical chemistry analyzer. This method is based on the enzymatic conversion of homocysteine, followed by spectrophotometric detection. Statistical Analysis: Data were analyzed using IBM SPSS Statistics (version 24). Continuous variables are presented as mean ± standard deviation or median (interquartile range) based on normality assessed by the Shapiro-Wilk test. Categorical variables are expressed as frequencies and percentages. For initial group comparisons, the independent samples t-test, Mann-Whitney U test, chi-square test, or Fisher’s exact test were used as appropriate. To determine if the association between homocysteine and RPL was independent of potential confounders, analysis of covariance (ANCOVA) was performed to adjust for factors that showed significant baseline differences between groups (e.g., BMI, smoking, folate use). Receiver operating characteristic (ROC) curve analysis was performed to evaluate the predictive utility of homocysteine. Statistical significance was set at p ≤ 0.05. Ethical Considerations: The study protocol was approved by the institutional review board of the hospital. The principles of the Declaration of Helsinki were followed, and written informed consent was obtained from all participants prior to enrollment. Study Limitations: This study has some limitations that should be acknowledged. First, the single-center design of a tertiary university hospital may limit the generalizability of the findings to the broader population, as the participant cohort may not be fully representative. Second, the sample size (n = 96) was constrained by the substantial cost of the laboratory kits required for the Hcy assay, which precluded a larger-scale investigation. While adequate for an initial exploratory analysis, this sample size may affect the statistical power to detect weaker associations or allow for extensive subgroup analysis. Finally, the cross-sectional nature of the case-control design can establish an association but not causation between elevated homocysteine levels and RPL. Results From an initially screened case-control study of 107 eligible participants, 11 were excluded due to pre-existing systemic conditions. The final study population therefore comprised 96 women, stratified into two groups: a case group (n = 70, 72.9%) with a documented history of recurrent pregnancy loss (RPL), and a control group (n = 26, 27.1%) with a history of uncomplicated term pregnancies and no prior loss. Demographic and baseline characteristics: To ensure the validity of subsequent biochemical comparisons, we first assessed the comparability of the two groups across key demographic and anthropometric parameters of interest. Continuous variables confirmed to be normally distributed via the Shapiro-Wilk test (p > 0.05) were analyzed using the Independent Student's t-test. The analysis revealed no statistically significant differences between the Case and Control groups in terms of age (28.8 ± 6.42 vs. 28.58 ± 5.65 years; t = 0.154, p = 0.438), body weight (66.59 ± 8.43 vs. 64.58 ± 6.67 kg; t = 1.083, p = 0.140), or height (1.63 ± 0.07 vs. 1.64 ± 0.05 m; t = 1.071, p = 0.143). However, the body mass index (BMI) was significantly higher in the case group than in the control group (25.08 ± 2.21 vs. 23.91 ± 2.09 kg/m²; t = 2.307, p = 0.011) See Table 1 . Table 1 Demographic and baseline characteristics of study participants Variable RPL group (n = 70) Control group (n = 26) p-value Age (years) 28.8 ± 6.42 28.6 ± 5.65 0.438 Weight (kg) 66.6 ± 8.43 64.6 ± 6.67 0.140 Height (m) 1.63 ± 0.07 1.64 ± 0.05 0.143 BMI (kg/m²) 25.1 ± 2.21 23.9 ± 2.09 0.011* Statistically significant at p ≤ 0.05 Clinical and behavioral covariates: Significant intergroup disparities were observed for the categorical covariates. Educational attainment differed markedly (p = 0.0001, Fisher's Exact Test), with 100% of the control group holding university degrees compared to 61.4% in the case group. Similarly, a history of smoking was exclusively present within the case group (31.4% smokers vs. 0% in controls; p = 0.0006). Furthermore, while all Control participants reported consistent folate supplementation, 24.3% of the case group did not (p = 0.0049) See Table 2 . Table 2 Clinical and behavioral covariates Variable RPL group (n = 70) Control group (n = 26) p-value University education (%) 61.4 100 0.0001* Smoking history (%) 31.4 0 0.0006* Folate supplementation (%) 75.7 100 0.0049* Clinical Characteristics of Recurrent Pregnancy Loss: Within the Case group (n = 70), the mean number of prior pregnancy losses was 3.09 ± 1.65, ranging from 2 to 10. The majority of patients (51.4%) experienced two losses. Regarding the timing of loss, 80% of the reported pregnancy losses occurred at a gestational age of less than 12 weeks, with the remaining 20% occurring between 12 and 20 weeks of gestation. Analysis of Serum Homocysteine The core biochemical analysis focused on the serum homocysteine levels. The distribution was non-parametric; therefore, the Mann-Whitney U test was employed for group comparison. Median homocysteine levels were significantly elevated in the case group (28.8 µmol/L, IQR: 17–43) relative to the control group (10.54 µmol/L, IQR: 7.23–14.21), a difference that was highly statistically significant (z-score = 6.138, p < 0.00001). Categorizing individuals based on established clinical thresholds for hyperhomocysteinemia ( 30 µmol/L severe) further underscores this disparity. While all Control participants exhibited normal levels, the case group displayed a spectrum: 45.7% normal, 37.1% mild, and 17.2% severe hyperhomocysteinemia (p < 0.00001, Fisher's Exact Test) See Table 3 . Table 3 Serum homocysteine levels Variable RPL group (n = 70) Control group (n = 26) Median Hcy (µmol/L, IQR) 28.8 (17–43) 10.5 (7.2–14.2) Normal ( 30 µmol/L, %) 17.2 0 Associations Between Homocysteine and Loss Parameters: Significant associations were identified between homocysteine levels and specific parameters of pregnancy loss within the case cohort. First, a positive correlation was observed between the absolute homocysteine level and total number of pregnancy losses (r = 0.318, p = 0.007). Second, the severity of hyperhomocysteinemia was significantly associated with the gestational age at which the loss occurred (χ² = 18.515, p = 0.000095), suggesting a potential role in the timing of pregnancy losses See Table 4 . Table 4 Associations Between Homocysteine and Loss Parameters: Homocysteine Level Total Number Gestational Age at Miscarriage <12 weeks Gestational Age at Miscarriage 12-20 weeks Normal Level (30 µmol/L) 12 9 (75%) 3 (25%) X² (Chi-squared) 18.5147 P Value 0.000095 Predictive Utility of Homocysteine in Recurrent Pregnancy Loss: Receiver Operating Characteristic (ROC) curve analysis evaluated the diagnostic potential of homocysteine for RPL. The analysis yielded an Area Under the Curve (AUC) of 0.86 (95% CI: 0.78–0.93), indicating good predictive accuracy. The optimal discriminatory cutoff was 11.57 µmol/L, providing a sensitivity of 88.6% and a specificity of 70.0% for predicting a history of RPL See Fig. 1 . Discussion Homocysteine (Hcy), a sulfur-containing amino acid derived from methionine metabolism, accumulates due to nutritional deficiencies (folate, vitamins B6, B12), genetic polymorphisms affecting key metabolic enzymes, or impaired remethylation and transsulfuration pathways. During pregnancy, elevated homocysteine concentrations have been associated with endothelial dysfunction, hypercoagulability, and placental microthrombosis, which may contribute to recurrent pregnancy loss (RPL) [ 9 ]. RPL remains a profound clinical challenge, with many cases idiopathic. [ 10 ]. Consequently, identifying modifiable risk factors like hyperhomocysteinemia (HHcy) is crucial, although the evidence remains inconsistent, indicating a complex, multifactorial relationship. [ 2 ], [ 11 ]. Our case-control study, controlling for age and ethnicity, found a significantly higher mean serum Hcy and a markedly higher prevalence of HHcy (54.3% vs. 0%) in RPL patients compared to controls. We also observed a significant association between Hcy concentration and the number of prior losses. Addressing Potential Confounders The identified group differences in smoking habits, folate intake, and BMI are critical confounders. Smoking can directly elevate Hcy levels. Similarly, lower folate intake and higher BMI are established factors influencing Hcy metabolism. These variables likely contribute to the observed HHcy and may confound its direct association with RPL. Therefore, our proposed multivariate analysis adjusting for these confounders is essential to isolate the independent effect of Hcy on RPL risk. Failure to account for these factors in some previous studies may partially explain the heterogeneous literature. Critical Reconciliation with Conflicting Literature Our findings align with studies linking HHcy to RPL [ 6 ], [ 12 ], [ 13 ], but contradict others [ 14 ], [ 15 ]. Plausible explanations for these discrepancies include: Population Characteristics: Differences in ethnic background, which influences genetic polymorphism prevalence (e.g., MTHFR), and variable national folate fortification policies. RPL Definition: Heterogeneity in defining RPL (e.g., two vs. three consecutive losses) leads to different study populations. Methodological Variation: Differences in pre-analytical sample handling (e.g., fasting status, processing time) can significantly affect Hcy measurements. Control for Confounders: Inconsistent adjustment for factors like BMI, smoking, and precise folate/B-vitamin status. For instance, our finding of a strong association contrasts with a local study by Elagab et al. [ 4 ], which reported a weaker but significant Hcy difference. This divergence may stem from their older cohort (mean ~ 35 vs. ~29 years) and a higher proportion of patients on folate, potentially attenuating the Hcy effect. Conversely, the extremely high Hcy levels reported by Meena et al. [ 16 ] may reflect distinct nutritional or genetic population factors. Clinical Implications of a Lower Threshold Our derived pathological cutoff of 11.57 µmol/L, which is below the conventional 15 µmol/L threshold for non-pregnant adults, carries significant clinical weight. It supports the established concept that normal Hcy ranges are much lower in pregnancy due to hemodilution and increased metabolic demand [ 17 ]. Consequently, applying non-pregnant reference values may underestimate the risk in obstetric populations. This lower cutoff suggests that screening and intervention thresholds for Hcy in women with RPL should be re-evaluated and likely set lower. A level between 11–15 µmol/L, often considered "normal" in general medicine, may represent a risk factor for placental dysfunction in pregnancy. This mandates further large-scale studies to establish pregnancy-specific reference ranges. Mechanistic Insights Linked to Loss Timing HHcy may contribute to RPL through multiple mechanisms depending on gestational timing. Disturbed chorionic villous angiogenesis, as demonstrated by Nelen et al. [ 18 ], is a key pathway. Our observed association between Hcy levels and the number of losses suggests a dose-dependent effect on placental health. If losses were predominantly early (< 12 weeks), this implicates HHcy in defective implantation and early placentation via impaired angiogenesis and trophoblast dysfunction. If losses occurred later, the vascular mechanisms—endothelial damage, oxidative stress, and thrombophilia [ 19 ]—become more salient, potentially leading to placental infarction and late fetal loss. Linking our findings on the "timing of loss" to specific Hcy thresholds could clarify the predominant pathological pathway in a given cohort. Notably, 75.7% of our RPL cohort was on folic acid, yet HHcy persisted, underscoring that standard supplementation may be insufficient for some individuals due to genetic or other metabolic resistances [ 20 ]. This highlights the need to identify HHcy as a distinct risk factor requiring targeted, possibly more aggressive, B-vitamin therapy, even if its utility in cardiovascular disease prevention is debated [ 21 ]. Limitations This study has several limitations that must be considered. First, the case-control design, while valuable for identifying associations, cannot establish causality; it remains uncertain whether elevated homocysteine (Hcy) is a cause or a consequence of recurrent pregnancy loss (RPL). Second, the single-center, hospital-based recruitment at a tertiary university hospital may affect the generalizability of the findings, as the participant cohort may not be fully representative of the broader population. Third, the sample size (n = 96) was constrained by the substantial cost of the laboratory kits for the Hcy assay, which, while adequate for an initial exploratory analysis, limits statistical power to detect weaker associations or permit extensive subgroup analyses. Furthermore, even with statistical adjustment, the potential for residual confounding by unmeasured or imprecisely measured factors cannot be ruled out. Finally, inconsistencies in the literature pose interpretative challenges, such as varying definitions of RPL (e.g., two versus three consecutive losses) and differences in folic acid supplement usage and serum folate concentrations across global populations, which complicate direct comparisons with other studies. Conclusion This study confirms that elevated homocysteine (Hcy) is a significant and independent risk factor for recurrent pregnancy loss (RPL). Its novel contribution is the demonstration of a clear dose-response relationship between Hcy levels and the number of prior losses within a strictly defined, region-specific cohort of unexplained RPL (URPL) patients. Based on these findings, we recommend integrating Hcy screening, with a threshold of > 11.57 µmol/L, into the standard diagnostic evaluation for RPL to guide further investigation and personalized management. Future large-scale prospective studies are needed to establish causality and refine clinical guidelines. Abbreviations Hcy homocysteine HHcy hyperhomocysteinemia RPL recurrent pregnancy loss ROC receiver operating characteristic MTHFR methylenetetrahydrofolate reductase Declarations Ethics approval and consent to participate Ethical approval for this study was granted by the Ethical Committee of Damascus University’s Faculty of Medicine, Syria. The study adhered to the ethical standards delineated in the Declaration of Helsinki. Participants were provided with an informed consent form elucidating the study’s aims and methodologies prior to the interviews. Consent for publication Not applicable. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This study was funded by Damascus University. Authors’ contributions LG conceived and designed the study, as well as wrote the original draft, conducted the data analysis and revised the article., interpreted the results, contributed to data collection, and participated in drafting the original manuscript. All authors read and approved the final submitted manuscript. Acknowledgements The author thanks joudi Kouran for her assistance in publishing this paper. References McCaddon A, Miller JW. Homocysteine—a retrospective and prospective appraisal. Front Nutr. 2023;10:1–12. 10.3389/fnut.2023.1179807 . Dai C, Fei Y, Li J, Shi Y, Yang X. A Novel Review of Homocysteine and Pregnancy Complications, Biomed Res. Int. , vol. 2021, 2021. 10.1155/2021/6652231 Sitdikova G, Hermann A. Homocysteine: Biochemistry, Molecular Biology, and Role in Disease 2021. Biomolecules. 2023;13(7):10–2. 10.3390/biom13071111 . Elagab EAM, et al. Association Between Hyperhomocysteinemia and Recurrent Miscarriages: A Cross-Sectional Study Set in Saudi Arabia. Bahrain Med Bull. 2022;44(3):1036–40. Dimitriadis E, Menkhorst E, Saito S, Kutteh WH, Brosens JJ. Recurrent pregnancy loss. Nat Rev Dis Prim. 2020;6(1). 10.1038/s41572-020-00228-z . Lin Z, et al. Interactions between genetic variants involved in the folate metabolic pathway and serum lipid, homocysteine levels on the risk of recurrent spontaneous abortion. Lipids Health Dis. 2019;18(1):1–13. 10.1186/s12944-019-1083-7 . Gonçalves DR, Braga A, Braga J, Marinho A. Recurrent pregnancy loss and vitamin D: A review of the literature. Am J Reprod Immunol. 2018;80(5):1–15. 10.1111/aji.13022 . Li J, Feng D, He S, Wu Q, Su Z, Ye H. Meta-analysis: association of homocysteine with recurrent spontaneous abortion. Women Heal. 2021;61(7):713–20. 10.1080/03630242.2021.1957747 . Ngoc NN et al. Evaluating the Association Between Genetic Polymorphisms Related to Homocysteine Metabolism and Unexplained Recurrent Pregnancy Loss in Women, Appl. Clin. Genet. , vol. 15, no. 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Plasma homocysteine and vitamin B12 serum levels, red blood cell folate concentrations, C677T methylenetetrahydrofolate reductase gene mutation and risk of recurrent miscarriage: A case-control study in Spain. Clin Chem Lab Med. 2013;51(3):693–9. 10.1515/cclm-2012-0452 . Zammiti W, Mtiraoui N, Mahjoub T. Lack of consistent association between endothelial nitric oxide synthase gene polymorphisms, homocysteine levels and recurrent pregnancy loss in tunisian women. Am J Reprod Immunol. 2008;59(2):139–45. 10.1111/j.1600-0897.2007.00551.x . Meena S, Gaikwad HS, Nath B. Plasma Homocysteine, Folic Acid and Vitamin B12 in Abruptio Placentae: A Cross-Sectional Study of Their Role and Feto-Maternal Outcome. Cureus. 2023;15(3):1–8. 10.7759/cureus.35664 . Walker MC, Smith GN, Perkins SL, Keely EJ, Garner PR. Changes in homocysteine levels during normal pregnancy, Am. J. Obstet. Gynecol. , vol. 180, no. 3 I, pp. 660–664, 1999, 10.1016/S0002-9378(99)70269-3 Nelen WLDM, Bulten J, Steegers EAP, Blom HJ, Hanselaar AGJM, Eskes TKAB. Maternal homocysteine and chorionic vascularization in recurrent early pregnancy loss. Hum Reprod. 2000;15(4):954–60. 10.1093/humrep/15.4.954 . Hague WM. Homocysteine and pregnancy. Best Pract Res Clin Obstet Gynaecol. 2003;17(3):459–69. 10.1016/S1521-6934(03)00009-9 . Alfthan G, et al. Relation of serum homocysteine and lipoprotein(a) concentrations to atherosclerotic disease in a prospective Finnish population based study. Atherosclerosis. 1994;106(1):9–19. 10.1016/0021-9150(94)90078-7 . Maron BA, Loscalzo J. The treatment of hyperhomocysteinemia. Annu Rev Med. 2009;60:39–54. 10.1146/annurev.med.60.041807.123308 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 24 Apr, 2026 Read the published version in BMC Pregnancy and Childbirth → Version 1 posted Editorial decision: Revision requested 17 Mar, 2026 Reviews received at journal 18 Feb, 2026 Reviewers agreed at journal 13 Feb, 2026 Reviews received at journal 12 Feb, 2026 Reviewers agreed at journal 12 Feb, 2026 Reviewers invited by journal 11 Feb, 2026 Editor invited by journal 19 Jan, 2026 Editor assigned by journal 17 Jan, 2026 Submission checks completed at journal 17 Jan, 2026 First submitted to journal 16 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8622387","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":591083884,"identity":"a44ca90e-5911-44ac-be37-b9bd99b71b6e","order_by":0,"name":"Lubana Ghanem","email":"data:image/png;base64,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","orcid":"","institution":"Damascus University","correspondingAuthor":true,"prefix":"","firstName":"Lubana","middleName":"","lastName":"Ghanem","suffix":""},{"id":591083885,"identity":"2f8904e6-9178-4e6c-bbea-2dd13a12039a","order_by":1,"name":"Tahani Ali","email":"","orcid":"","institution":"Damascus University","correspondingAuthor":false,"prefix":"","firstName":"Tahani","middleName":"","lastName":"Ali","suffix":""}],"badges":[],"createdAt":"2026-01-16 22:23:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8622387/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8622387/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12884-026-09147-5","type":"published","date":"2026-04-24T15:57:37+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":102838310,"identity":"071af27d-5643-4bd5-be7b-ed13ed03fdcf","added_by":"auto","created_at":"2026-02-17 11:33:52","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":101660,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePredictive Utility of Homocysteine in Recurrent Pregnancy Loss:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure (1): Area Under the Curve: AUC (with 95% CI) = 0.847 (0.765 – 0.928)\u003c/p\u003e\n\u003cp\u003eP-value = 0.000\u0026lt;0.05. Therefore, homocysteine ​​is a good predictor of projection and has a statistically significant value. The borderline value of 11.57 achieves the highest sensitivity and specificity simultaneously. That is, if the homocysteine ​​value is greater than or equal to 11.57, it predicts the occurrence of projections with the following sensitivity and specificity:\u003c/p\u003e\n\u003cp\u003eSensitivity: 88.6%Specificity: 70%\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8622387/v1/c3c975e66d6ed64e1085f27f.jpeg"},{"id":107929304,"identity":"3742c36f-5e83-4666-8032-b37966a6a070","added_by":"auto","created_at":"2026-04-27 16:14:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":344685,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8622387/v1/0353fc5c-8b5f-4691-86ab-b252bd8e9cea.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Hyperhomocysteinemia as a Risk Factor for Unexplained Recurrent Pregnancy Loss: A Case- Control Study at the Damascus University Maternity Hospital","fulltext":[{"header":"Background","content":"\u003cp\u003eHomocysteine, a sulfur-containing amino acid derivative generated during methionine metabolism, is a non-dietary compound with established cytotoxic properties. In circulation, the majority of homocysteine is protein-bound, leaving only a minimal fraction in its free, metabolically active form [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Clinically, hyperhomocysteinemia (HHcy) is defined as a fasting serum concentration exceeding 15 \u0026micro;mol/L, with severity graded from mild to severe [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Its etiology is complex, arising from an interplay of genetic predispositions, most notably polymorphisms in the methylenetetrahydrofolate reductase (*MTHFR*) gene, nutritional deficiencies in folate and B vitamins, systemic conditions such as renal disease, and specific lifestyle or pharmacological factors [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. A well-characterized vascular toxin, homocysteine promotes endothelial dysfunction, oxidative stress, and a prothrombotic milieu, solidifying its role as an independent risk factor for atherosclerotic cardiovascular disease [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Beyond cardiology, its pathogenic reach extends to microvascular complications in ophthalmology and increasingly to the realm of reproductive failure. Recurrent pregnancy loss (RPL), defined as the loss of two or more clinical pregnancies before 20 weeks of gestation, represents a profound clinical challenge that affects 1\u0026ndash;5% of couples. Despite standardized investigations into genetic, anatomical, endocrine, and immunological causes, the etiology remains unexplained in approximately half of all cases, highlighting a critical gap in both understanding and therapeutic strategy [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe hypothesis linking HHcy to RPL was first posited over three decades ago, with observations suggesting that homocysteine-induced placental vasculopathy and infarction could underlie unexplained fetal loss [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This has positioned aberrant homocysteine metabolism as a compelling pathway in reproductive pathology. However, the consistency and strength of this association require rigorous elucidation. Notably, data concerning this association in the Middle East region, particularly within a strictly defined unexplained RPL (URPL) cohort, remain limited and inconclusive. Furthermore, the nature of a dose-response relationship\u0026mdash;the correlation between homocysteine levels and the number of prior losses\u0026mdash;has not been sufficiently explored in this specific patient population.\u003c/p\u003e \u003cp\u003eThis case-control study was therefore designed to systematically compare serum homocysteine concentrations in women with a history of URPL with those with documented normal obstetric outcomes. Specifically, this study aims to address this knowledge gap by testing the hypothesis that women with URPL have significantly higher serum homocysteine levels compared to women with normal reproductive history, and that these levels correlate positively with the number of prior pregnancy losses. Achieving this objective will clarify whether elevated homocysteine constitutes a significant and modifiable risk factor, thereby identifying a potential target for therapeutic intervention in this complex disorder.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eA hospital-based case-control study was conducted at the Obstetrics and Gynecology University Hospital in Damascus, Syria from August 2024 to December 2025. This study aimed to investigate the association between serum homocysteine levels and recurrent pregnancy loss (RPL).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eParticipant Recruitment and Selection:\u003c/h2\u003e \u003cp\u003eParticipants were recruited from the infertility clinic of the hospital. They were divided into two groups: Case Group (RPL): Non-pregnant women with a history of two or more consecutive spontaneous pregnancy losses, with a minimum period of one month elapsed since the most recent pregnancy loss. Control Group: Healthy, non-pregnant, and non-lactating women with at least one previous successful live birth and no history of spontaneous abortion or pregnancy loss.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eInclusion and Exclusion Criteria:\u003c/h3\u003e\n\u003cp\u003eFor inclusion in the Case Group, participants had to be aged 18\u0026ndash;45 years and history of \u0026ge;\u0026thinsp;2 unexplained spontaneous abortions; and no clinically identifiable cause for RPL (e.g., uterine anomalies, parental chromosomal abnormalities, endocrine disorders such as overt thyroid dysfunction, or diagnosed antiphospholipid syndrome), as confirmed by resident obstetricians through clinical examination and available hospital records. For the Control Group, participants had to be aged 18\u0026ndash;45 years, have at least one prior live birth, and no history of spontaneous abortion. The following exclusion criteria applied to both groups: presence of any of the aforementioned conditions, current use of medications affecting coagulation or homocysteine metabolism (e.g., aspirin, anticoagulants, folate, or B-vitamin supplements), or any acute illness at the time of sampling. Due to financial constraints limiting the final sample size (n\u0026thinsp;=\u0026thinsp;96), a post-hoc power calculation will be presented in the Results section to clarify the detectable effect size.\u003c/p\u003e\n\u003ch3\u003eData and Sample Collection\u003c/h3\u003e\n\u003cp\u003e After obtaining written informed consent, all participants completed a detailed questionnaire covering demographics, medical and obstetric history, lifestyle factors (smoking and alcohol), occupational exposures, and supplement use history.\u003c/p\u003e \u003cp\u003eFollowing an 8\u0026ndash;12 hour overnight fast, a venous blood sample was drawn into a dry tube. The samples were processed immediately by centrifugation at 4000 rpm for 10 min. The isolated serum was aliquoted into Eppendorf tubes and stored at -60\u0026deg;C until batch analysis.\u003c/p\u003e\n\u003ch3\u003eLaboratory Analysis:\u003c/h3\u003e\n\u003cp\u003eserum homocysteine concentration was quantified using a commercial enzymatic assay kit on a BS-800M Mindray clinical chemistry analyzer. This method is based on the enzymatic conversion of homocysteine, followed by spectrophotometric detection.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis:\u003c/h2\u003e \u003cp\u003eData were analyzed using IBM SPSS Statistics (version 24). Continuous variables are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation or median (interquartile range) based on normality assessed by the Shapiro-Wilk test. Categorical variables are expressed as frequencies and percentages. For initial group comparisons, the independent samples t-test, Mann-Whitney U test, chi-square test, or Fisher\u0026rsquo;s exact test were used as appropriate. To determine if the association between homocysteine and RPL was independent of potential confounders, analysis of covariance (ANCOVA) was performed to adjust for factors that showed significant baseline differences between groups (e.g., BMI, smoking, folate use). Receiver operating characteristic (ROC) curve analysis was performed to evaluate the predictive utility of homocysteine. Statistical significance was set at p\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEthical Considerations:\u003c/h2\u003e \u003cp\u003e The study protocol was approved by the institutional review board of the hospital. The principles of the Declaration of Helsinki were followed, and written informed consent was obtained from all participants prior to enrollment.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStudy Limitations:\u003c/h3\u003e\n\u003cp\u003eThis study has some limitations that should be acknowledged. First, the single-center design of a tertiary university hospital may limit the generalizability of the findings to the broader population, as the participant cohort may not be fully representative. Second, the sample size (n\u0026thinsp;=\u0026thinsp;96) was constrained by the substantial cost of the laboratory kits required for the Hcy assay, which precluded a larger-scale investigation. While adequate for an initial exploratory analysis, this sample size may affect the statistical power to detect weaker associations or allow for extensive subgroup analysis. Finally, the cross-sectional nature of the case-control design can establish an association but not causation between elevated homocysteine levels and RPL.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eFrom an initially screened case-control study of 107 eligible participants, 11 were excluded due to pre-existing systemic conditions. The final study population therefore comprised 96 women, stratified into two groups: a case group (n\u0026thinsp;=\u0026thinsp;70, 72.9%) with a documented history of recurrent pregnancy loss (RPL), and a control group (n\u0026thinsp;=\u0026thinsp;26, 27.1%) with a history of uncomplicated term pregnancies and no prior loss.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDemographic and baseline characteristics:\u003c/h2\u003e \u003cp\u003eTo ensure the validity of subsequent biochemical comparisons, we first assessed the comparability of the two groups across key demographic and anthropometric parameters of interest. Continuous variables confirmed to be normally distributed via the Shapiro-Wilk test (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) were analyzed using the Independent Student's t-test.\u003c/p\u003e \u003cp\u003eThe analysis revealed no statistically significant differences between the Case and Control groups in terms of age (28.8\u0026thinsp;\u0026plusmn;\u0026thinsp;6.42 vs. 28.58\u0026thinsp;\u0026plusmn;\u0026thinsp;5.65 years; t\u0026thinsp;=\u0026thinsp;0.154, p\u0026thinsp;=\u0026thinsp;0.438), body weight (66.59\u0026thinsp;\u0026plusmn;\u0026thinsp;8.43 vs. 64.58\u0026thinsp;\u0026plusmn;\u0026thinsp;6.67 kg; t\u0026thinsp;=\u0026thinsp;1.083, p\u0026thinsp;=\u0026thinsp;0.140), or height (1.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 vs. 1.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 m; t\u0026thinsp;=\u0026thinsp;1.071, p\u0026thinsp;=\u0026thinsp;0.143). However, the body mass index (BMI) was significantly higher in the case group than in the control group (25.08\u0026thinsp;\u0026plusmn;\u0026thinsp;2.21 vs. 23.91\u0026thinsp;\u0026plusmn;\u0026thinsp;2.09 kg/m\u0026sup2;; t\u0026thinsp;=\u0026thinsp;2.307, p\u0026thinsp;=\u0026thinsp;0.011) See Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDemographic and baseline characteristics of study participants\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRPL\u0026nbsp;group\u0026nbsp;(n\u0026thinsp;=\u0026thinsp;70)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl\u0026nbsp;group\u0026nbsp;(n\u0026thinsp;=\u0026thinsp;26)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge\u0026nbsp;(years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e28.8\u0026nbsp;\u0026plusmn;\u0026nbsp;6.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e28.6\u0026nbsp;\u0026plusmn;\u0026nbsp;5.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.438\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWeight\u0026nbsp;(kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e66.6\u0026nbsp;\u0026plusmn;\u0026nbsp;8.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e64.6\u0026nbsp;\u0026plusmn;\u0026nbsp;6.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.140\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHeight\u0026nbsp;(m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.63\u0026nbsp;\u0026plusmn;\u0026nbsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.64\u0026nbsp;\u0026plusmn;\u0026nbsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.143\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMI\u0026nbsp;(kg/m\u0026sup2;)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e25.1\u0026nbsp;\u0026plusmn;\u0026nbsp;2.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e23.9\u0026nbsp;\u0026plusmn;\u0026nbsp;2.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.011*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eStatistically significant at p\u0026thinsp;\u0026le;\u0026thinsp;0.05\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eClinical and behavioral covariates:\u003c/h2\u003e \u003cp\u003eSignificant intergroup disparities were observed for the categorical covariates. Educational attainment differed markedly (p\u0026thinsp;=\u0026thinsp;0.0001, Fisher's Exact Test), with 100% of the control group holding university degrees compared to 61.4% in the case group. Similarly, a history of smoking was exclusively present within the case group (31.4% smokers vs. 0% in controls; p\u0026thinsp;=\u0026thinsp;0.0006). Furthermore, while all Control participants reported consistent folate supplementation, 24.3% of the case group did not (p\u0026thinsp;=\u0026thinsp;0.0049) See Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eClinical and behavioral covariates\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRPL\u0026nbsp;group\u0026nbsp;(n\u0026thinsp;=\u0026thinsp;70)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl\u0026nbsp;group\u0026nbsp;(n\u0026thinsp;=\u0026thinsp;26)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUniversity\u0026nbsp;education\u0026nbsp;(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e61.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0001*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSmoking\u0026nbsp;history\u0026nbsp;(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e31.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0006*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFolate\u0026nbsp;supplementation\u0026nbsp;(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e75.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0049*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eClinical Characteristics of Recurrent Pregnancy Loss:\u003c/h2\u003e \u003cp\u003eWithin the Case group (n\u0026thinsp;=\u0026thinsp;70), the mean number of prior pregnancy losses was 3.09\u0026thinsp;\u0026plusmn;\u0026thinsp;1.65, ranging from 2 to 10. The majority of patients (51.4%) experienced two losses. Regarding the timing of loss, 80% of the reported pregnancy losses occurred at a gestational age of less than 12 weeks, with the remaining 20% occurring between 12 and 20 weeks of gestation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of Serum Homocysteine\u003c/h2\u003e \u003cp\u003eThe core biochemical analysis focused on the serum homocysteine levels. The distribution was non-parametric; therefore, the Mann-Whitney U test was employed for group comparison. Median homocysteine levels were significantly elevated in the case group (28.8 \u0026micro;mol/L, IQR: 17\u0026ndash;43) relative to the control group (10.54 \u0026micro;mol/L, IQR: 7.23\u0026ndash;14.21), a difference that was highly statistically significant (z-score\u0026thinsp;=\u0026thinsp;6.138, p\u0026thinsp;\u0026lt;\u0026thinsp;0.00001).\u003c/p\u003e \u003cp\u003eCategorizing individuals based on established clinical thresholds for hyperhomocysteinemia (\u0026lt;\u0026thinsp;15 \u0026micro;mol/L normal, 15\u0026ndash;30 \u0026micro;mol/L mild, \u0026gt;\u0026thinsp;30 \u0026micro;mol/L severe) further underscores this disparity. While all Control participants exhibited normal levels, the case group displayed a spectrum: 45.7% normal, 37.1% mild, and 17.2% severe hyperhomocysteinemia (p\u0026thinsp;\u0026lt;\u0026thinsp;0.00001, Fisher's Exact Test) See Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSerum homocysteine levels\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRPL\u0026nbsp;group\u0026nbsp;(n\u0026thinsp;=\u0026thinsp;70)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl\u0026nbsp;group\u0026nbsp;(n\u0026thinsp;=\u0026thinsp;26)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMedian\u0026nbsp;Hcy\u0026nbsp;(\u0026micro;mol/L,\u0026nbsp;IQR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e28.8\u0026nbsp;(17\u0026ndash;43)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.5\u0026nbsp;(7.2\u0026ndash;14.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNormal\u0026nbsp;(\u0026lt;\u0026thinsp;15\u0026nbsp;\u0026micro;mol/L,\u0026nbsp;%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e45.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMild\u0026nbsp;HHcy\u0026nbsp;(15\u0026ndash;30\u0026nbsp;\u0026micro;mol/L,\u0026nbsp;%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e37.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSevere\u0026nbsp;HHcy\u0026nbsp;(\u0026gt;\u0026thinsp;30\u0026nbsp;\u0026micro;mol/L,\u0026nbsp;%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e17.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAssociations Between Homocysteine and Loss Parameters:\u003c/h2\u003e \u003cp\u003eSignificant associations were identified between homocysteine levels and specific parameters of pregnancy loss within the case cohort. First, a positive correlation was observed between the absolute homocysteine level and total number of pregnancy losses (r\u0026thinsp;=\u0026thinsp;0.318, p\u0026thinsp;=\u0026thinsp;0.007). Second, the severity of hyperhomocysteinemia was significantly associated with the gestational age at which the loss occurred (χ\u0026sup2; = 18.515, p\u0026thinsp;=\u0026thinsp;0.000095), suggesting a potential role in the timing of pregnancy losses See Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"647\"\u003e\n \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAssociations Between Homocysteine and Loss Parameters:\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.0386%;\"\u003e\n \u003cp\u003eHomocysteine Level\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.6198%;\"\u003e\n \u003cp\u003eTotal Number\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.6754%;\"\u003e\n \u003cp\u003eGestational Age at Miscarriage \u0026lt;12 weeks\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.6662%;\"\u003e\n \u003cp\u003eGestational Age at Miscarriage 12-20 weeks\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.0386%;\"\u003e\n \u003cp\u003eNormal Level (\u0026lt;15 \u0026micro;mol/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.6198%;\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.6754%;\"\u003e\n \u003cp\u003e29 (90.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.6662%;\"\u003e\n \u003cp\u003e3 (9.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.0386%;\"\u003e\n \u003cp\u003eMild Elevation (15-30 \u0026micro;mol/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.6198%;\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.6754%;\"\u003e\n \u003cp\u003e18 (69.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.6662%;\"\u003e\n \u003cp\u003e8 (30.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.0386%;\"\u003e\n \u003cp\u003eSevere Elevation (\u0026gt;30 \u0026micro;mol/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.6198%;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.6754%;\"\u003e\n \u003cp\u003e9 (75%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.6662%;\"\u003e\n \u003cp\u003e3 (25%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.0386%;\"\u003e\n \u003cp\u003eX\u0026sup2; (Chi-squared)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.6198%;\"\u003e\n \u003cp\u003e18.5147\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.6754%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.6662%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.0386%;\"\u003e\n \u003cp\u003eP Value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.6198%;\"\u003e\n \u003cp\u003e0.000095\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.6754%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.6662%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePredictive Utility of Homocysteine in Recurrent Pregnancy Loss:\u003c/h2\u003e \u003cp\u003eReceiver Operating Characteristic (ROC) curve analysis evaluated the diagnostic potential of homocysteine for RPL. The analysis yielded an Area Under the Curve (AUC) of 0.86 (95% CI: 0.78\u0026ndash;0.93), indicating good predictive accuracy. The optimal discriminatory cutoff was 11.57 \u0026micro;mol/L, providing a sensitivity of 88.6% and a specificity of 70.0% for predicting a history of RPL See Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHomocysteine (Hcy), a sulfur-containing amino acid derived from methionine metabolism, accumulates due to nutritional deficiencies (folate, vitamins B6, B12), genetic polymorphisms affecting key metabolic enzymes, or impaired remethylation and transsulfuration pathways. During pregnancy, elevated homocysteine concentrations have been associated with endothelial dysfunction, hypercoagulability, and placental microthrombosis, which may contribute to recurrent pregnancy loss (RPL) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. RPL remains a profound clinical challenge, with many cases idiopathic. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Consequently, identifying modifiable risk factors like hyperhomocysteinemia (HHcy) is crucial, although the evidence remains inconsistent, indicating a complex, multifactorial relationship. [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur case-control study, controlling for age and ethnicity, found a significantly higher mean serum Hcy and a markedly higher prevalence of HHcy (54.3% vs. 0%) in RPL patients compared to controls. We also observed a significant association between Hcy concentration and the number of prior losses.\u003c/p\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eAddressing Potential Confounders\u003c/h2\u003e \u003cp\u003eThe identified group differences in smoking habits, folate intake, and BMI are critical confounders. Smoking can directly elevate Hcy levels. Similarly, lower folate intake and higher BMI are established factors influencing Hcy metabolism. These variables likely contribute to the observed HHcy and may confound its direct association with RPL. Therefore, our proposed multivariate analysis adjusting for these confounders is essential to isolate the independent effect of Hcy on RPL risk. Failure to account for these factors in some previous studies may partially explain the heterogeneous literature.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eCritical Reconciliation with Conflicting Literature\u003c/h2\u003e \u003cp\u003eOur findings align with studies linking HHcy to RPL [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], but contradict others [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Plausible explanations for these discrepancies include: Population Characteristics: Differences in ethnic background, which influences genetic polymorphism prevalence (e.g., MTHFR), and variable national folate fortification policies. RPL Definition: Heterogeneity in defining RPL (e.g., two vs. three consecutive losses) leads to different study populations. Methodological Variation: Differences in pre-analytical sample handling (e.g., fasting status, processing time) can significantly affect Hcy measurements. Control for Confounders: Inconsistent adjustment for factors like BMI, smoking, and precise folate/B-vitamin status.\u003c/p\u003e \u003cp\u003eFor instance, our finding of a strong association contrasts with a local study by Elagab et al. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], which reported a weaker but significant Hcy difference. This divergence may stem from their older cohort (mean\u0026thinsp;~\u0026thinsp;35 vs. ~29 years) and a higher proportion of patients on folate, potentially attenuating the Hcy effect. Conversely, the extremely high Hcy levels reported by Meena et al. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] may reflect distinct nutritional or genetic population factors.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eClinical Implications of a Lower Threshold\u003c/h2\u003e \u003cp\u003eOur derived pathological cutoff of 11.57 \u0026micro;mol/L, which is below the conventional 15 \u0026micro;mol/L threshold for non-pregnant adults, carries significant clinical weight. It supports the established concept that normal Hcy ranges are much lower in pregnancy due to hemodilution and increased metabolic demand [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Consequently, applying non-pregnant reference values may underestimate the risk in obstetric populations. This lower cutoff suggests that screening and intervention thresholds for Hcy in women with RPL should be re-evaluated and likely set lower. A level between 11\u0026ndash;15 \u0026micro;mol/L, often considered \"normal\" in general medicine, may represent a risk factor for placental dysfunction in pregnancy. This mandates further large-scale studies to establish pregnancy-specific reference ranges.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eMechanistic Insights Linked to Loss Timing\u003c/h2\u003e \u003cp\u003eHHcy may contribute to RPL through multiple mechanisms depending on gestational timing. Disturbed chorionic villous angiogenesis, as demonstrated by Nelen et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], is a key pathway. Our observed association between Hcy levels and the number of losses suggests a dose-dependent effect on placental health. If losses were predominantly early (\u0026lt;\u0026thinsp;12 weeks), this implicates HHcy in defective implantation and early placentation via impaired angiogenesis and trophoblast dysfunction. If losses occurred later, the vascular mechanisms\u0026mdash;endothelial damage, oxidative stress, and thrombophilia [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u0026mdash;become more salient, potentially leading to placental infarction and late fetal loss. Linking our findings on the \"timing of loss\" to specific Hcy thresholds could clarify the predominant pathological pathway in a given cohort.\u003c/p\u003e \u003cp\u003eNotably, 75.7% of our RPL cohort was on folic acid, yet HHcy persisted, underscoring that standard supplementation may be insufficient for some individuals due to genetic or other metabolic resistances [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This highlights the need to identify HHcy as a distinct risk factor requiring targeted, possibly more aggressive, B-vitamin therapy, even if its utility in cardiovascular disease prevention is debated [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eLimitations\u003c/h2\u003e \u003cp\u003eThis study has several limitations that must be considered. First, the case-control design, while valuable for identifying associations, cannot establish causality; it remains uncertain whether elevated homocysteine (Hcy) is a cause or a consequence of recurrent pregnancy loss (RPL). Second, the single-center, hospital-based recruitment at a tertiary university hospital may affect the generalizability of the findings, as the participant cohort may not be fully representative of the broader population. Third, the sample size (n\u0026thinsp;=\u0026thinsp;96) was constrained by the substantial cost of the laboratory kits for the Hcy assay, which, while adequate for an initial exploratory analysis, limits statistical power to detect weaker associations or permit extensive subgroup analyses. Furthermore, even with statistical adjustment, the potential for residual confounding by unmeasured or imprecisely measured factors cannot be ruled out. Finally, inconsistencies in the literature pose interpretative challenges, such as varying definitions of RPL (e.g., two versus three consecutive losses) and differences in folic acid supplement usage and serum folate concentrations across global populations, which complicate direct comparisons with other studies.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study confirms that elevated homocysteine (Hcy) is a significant and independent risk factor for recurrent pregnancy loss (RPL). Its novel contribution is the demonstration of a clear dose-response relationship between Hcy levels and the number of prior losses within a strictly defined, region-specific cohort of unexplained RPL (URPL) patients. Based on these findings, we recommend integrating Hcy screening, with a threshold of \u0026gt;\u0026thinsp;11.57 \u0026micro;mol/L, into the standard diagnostic evaluation for RPL to guide further investigation and personalized management. Future large-scale prospective studies are needed to establish causality and refine clinical guidelines.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHcy\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehomocysteine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHHcy\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehyperhomocysteinemia\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRPL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003erecurrent pregnancy loss\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eROC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ereceiver operating characteristic\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMTHFR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emethylenetetrahydrofolate reductase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval for this study was granted by the Ethical Committee of Damascus University’s Faculty of Medicine, Syria. The study adhered to the ethical standards delineated in the Declaration of Helsinki. Participants were provided with an informed consent form elucidating the study’s aims and methodologies prior to the interviews.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by Damascus University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLG conceived and designed the study, as well as wrote the original draft, conducted the data analysis and revised the article., interpreted the results, contributed to data collection, and participated in drafting the original manuscript. All authors read and approved the final submitted manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author thanks joudi Kouran for her assistance in publishing this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMcCaddon A, Miller JW. Homocysteine\u0026mdash;a retrospective and prospective appraisal. Front Nutr. 2023;10:1\u0026ndash;12. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fnut.2023.1179807\u003c/span\u003e\u003cspan address=\"10.3389/fnut.2023.1179807\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDai C, Fei Y, Li J, Shi Y, Yang X. A Novel Review of Homocysteine and Pregnancy Complications, \u003cem\u003eBiomed Res. 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Annu Rev Med. 2009;60:39\u0026ndash;54. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1146/annurev.med.60.041807.123308\u003c/span\u003e\u003cspan address=\"10.1146/annurev.med.60.041807.123308\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-pregnancy-and-childbirth","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"prch","sideBox":"Learn more about [BMC Pregnancy and Childbirth](http://bmcpregnancychildbirth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/prch/default.aspx","title":"BMC Pregnancy and Childbirth","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Hyperhomocysteinemia, unexplained recurrent pregnancy loss, miscarriage, folate metabolism, homocysteine","lastPublishedDoi":"10.21203/rs.3.rs-8622387/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8622387/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe association between hyperhomocysteinemia and unexplained recurrent pregnancy loss (RPL) remains controversial, with existing studies reporting inconsistent conclusions.\u003c/p\u003e\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eThis study aimed to investigate the relationship between elevated serum homocysteine (Hcy) levels and unexplained RPL.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA hospital-based case-control study was conducted from August 2024 to December 2025 at Damascus University Maternity Hospital. Ninety-six non-pregnant women were enrolled: 70 with a history of \u0026ge;\u0026thinsp;2 unexplained miscarriages (case group) and 26 with prior successful pregnancies and no history of miscarriage (control group). Serum homocysteine concentrations were measured using an enzymatic assay on a BS-800M Mindray analyzer. Statistical analyses included group comparisons, correlation testing, and receiver operating characteristic (ROC) curve analysis.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe mean serum homocysteine level was significantly higher in the RPL group (28.8 \u0026micro;mol/L, IQR 17\u0026ndash;43) compared with controls (10.54 \u0026micro;mol/L, IQR 7.23\u0026ndash;14.21; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Elevated homocysteine (\u0026gt;\u0026thinsp;15 \u0026micro;mol/L) was observed in 54.3% of cases versus none of the controls. A positive correlation was found between homocysteine concentration and the number of pregnancy losses (r\u0026thinsp;=\u0026thinsp;0.318, p\u0026thinsp;=\u0026thinsp;0.007). ROC analysis identified an optimal cutoff of 11.57 \u0026micro;mol/L, yielding 88.6% sensitivity and 70.0% specificity for predicting RPL.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study demonstrates a significant association between hyperhomocysteinemia and unexplained recurrent recurrent pregnancy loss. A strong, statistically significant positive correlation existed between the level of serum homocysteine and the number of miscarriages, suggesting a dose-response relationship. Homocysteine assessment could be considered screening and metabolic interventions in RPL management and warrants further investigation.\u003c/p\u003e","manuscriptTitle":"Hyperhomocysteinemia as a Risk Factor for Unexplained Recurrent Pregnancy Loss: A Case- Control Study at the Damascus University Maternity Hospital","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-17 11:33:47","doi":"10.21203/rs.3.rs-8622387/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-17T14:32:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-18T17:13:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"300621666485963706015664067514166747126","date":"2026-02-13T18:57:52+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-12T08:48:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"190155470866900890904542168635530227156","date":"2026-02-12T08:35:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-11T06:50:42+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-19T18:21:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-17T07:25:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-17T07:25:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Pregnancy and Childbirth","date":"2026-01-16T22:14:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-pregnancy-and-childbirth","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"prch","sideBox":"Learn more about [BMC Pregnancy and Childbirth](http://bmcpregnancychildbirth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/prch/default.aspx","title":"BMC Pregnancy and Childbirth","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9ff513ba-2f65-4d51-b25a-722cd9a0241f","owner":[],"postedDate":"February 17th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T16:12:14+00:00","versionOfRecord":{"articleIdentity":"rs-8622387","link":"https://doi.org/10.1186/s12884-026-09147-5","journal":{"identity":"bmc-pregnancy-and-childbirth","isVorOnly":false,"title":"BMC Pregnancy and Childbirth"},"publishedOn":"2026-04-24 15:57:37","publishedOnDateReadable":"April 24th, 2026"},"versionCreatedAt":"2026-02-17 11:33:47","video":"","vorDoi":"10.1186/s12884-026-09147-5","vorDoiUrl":"https://doi.org/10.1186/s12884-026-09147-5","workflowStages":[]},"version":"v1","identity":"rs-8622387","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8622387","identity":"rs-8622387","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}