Free 25(Oh)d3 Levels in Follicular Ovarian Fluid Top Quality Embryo is Higher Than Non- Top Quality Embryo in Normoresponders Group

Free 25(Oh)d3 Levels in Follicular Ovarian Fluid Top Quality Embryo is Higher Than Non- Top Quality Embryo in Normoresponders Group | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Free 25(Oh)d3 Levels in Follicular Ovarian Fluid Top Quality Embryo is Higher Than Non- Top Quality Embryo in Normoresponders Group Artha Falentin Putri Susilo, Hanom Husni Syam, Hartanto Bayuaji, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4375279/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Nov, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Introduction: Vitamin D and calcium in follicular fluid play essential roles in modulating steroidogenesis, folliculogenesis, and oocyte quality determination. Both collaborate to produce high-quality embryos (TQE) during in vitro fertilization. This study aimed to compare free 25(OH)D3 and calcium levels in follicular fluid between TQE and non-TQE groups (non-TQE). Methods This cross-sectional study included women who underwent IVF (In Vitro Fertilization) procedures at tertiary hospitals in Bandung, Indonesia. Ovarian follicular fluid was collected during the ovum pick-up procedure. Examination of 25(OH)D3 levels, vitamin D-binding protein and calcium in the follicles was performed using an enzyme-linked immunosorbent assay (ELISA). Free 25(OH)D3 levels were calculated using a method of Vermeulen formula. Results A total of 173 samples, consisting of 86 subjects in the TQE group and 87 subjects in the non-TQE group, met the study criteria. There was a significant difference in free 25(OH)D3 follicular fluid levels between the TQE and non-TQE groups (p = 0,017), there was no significant difference in calcium levels between the two groups (p = 0,805). This study also revealed that there was a significant association between free 25(OH)D3 follicular fluid level and embryo quality (OR 3.05, 95% CI, 1.46–6.38; p value = 0.002), but there was no significant association between follicular fluid calcium and embryo quality (p = 0.144 and OR, 1.74 (95% CI 0.82–3.68)). Conclusion These circumstances allow free 25(OH)D3 and calcium in the follicular fluid to act independently in the processes of steroidogenesis, folliculogenesis, and fertilization. Vitamin D Embryo In Vitro Fertilization Top-Quality Embryo Figures Figure 1 INTRODUCTION Vitamin D is a steroid hormone that plays essential roles in influencing several physiological processes, especially calcium and phosphorus homeostasis and bone mineralization.[ 1 – 7 ] Vitamin D also plays a pivotal role in human reproductive functions such as folliculogenesis, steroidogenesis, and successful embryo implantation.[ 3 , 4 ] Vitamin D is a steroid hormone that plays an essential role in influencing several physiological processes, especially calcium and phosphorus homeostasis and bone mineralization.[ 1 – 6 ] Thus, vitamin D levels could be a predictor of assisted reproductive technology success rate.[ 4 , 6 ] The effects of vitamin D and calcium on oocyte development have important clinical implications for the success of in vitro fertilization (IVF) programs. Vitamin D deficiency can lead to calcium deficiency, resulting in disturbances in oocyte maturation, development, and fertilization.[ 8 – 10 ] Vitamin D plays a role in follicle regulation and oocyte maturation through both direct and indirect pathways.[ 4 ] Vitamin D plays a role in embryogenesis and folliculogenesis. Vitamin D modulates primary follicle recruitment by regulating anti-Müllerian hormone (AMH) production.[ 11 ] Thus, intrafollicular vitamin D levels could be a potential marker of oocyte and embryo quality as well as a predictor of IVF outcomes.[ 4 ] Assessment of free vitamin D3 levels and bioavailable vitamin D3 could become a more accurate predictor in assessing vitamin D status.[ 12 ] Levels of free 25(OH)D3 and calcium in follicular fluid play a role in the final maturation of follicles and are thought to be related to the rate of fertilization, cleavage, and the quality of the embryo to increase the rate of pregnancy. However, their exact roles in stimulating oocyte and embryo quality have not been adequately reported. This study aimed to compare free 25(OH)D3 and calcium levels in follicular fluid between top-quality embryo (TQE) and non-top-quality embryo (non-TQE) groups and to evaluate the correlation between free 25(OH)D3 and calcium levels in follicular fluid and embryo quality in normoresponder patients who underwent IVF procedures. MATERIAL AND METHODS Study Design This observational cross-sectional study used secondary data from the ovarian follicular fluid of women who underwent IVF in tertiary hospitals in Bandung, Indonesia. This study was conducted by the Declaration of Helsinki of the World Medical Association Declaration. Helsinki. All methods were performed by relevant guidelines and regulations after obtaining approval and recommendations from the Ethics Committee Review Board of Hasan Sadikin General Hospital, Faculty of Medicine Universitas Padjadjaran (reference number LB.02.01/X.6.5/399/2022). Research Subjects The subjects of this study were women who underwent IVF procedures at the Aster Clinic, Dr. Hasan Sadikin General Hospital Bandung, and Bandung Fertility Center Limijati Hospital Bandung between November 2021-March 2022 that met the inclusion and exclusion criteria of this study. Women of reproductive age (20–35 years old) who belong to normoresponder group, with body mass index (BMI) varied between 18–25 kg/m 2 , participated in an IVF program for the first time, as well as women who were not exposed to toxic substances (cigarettes, alcohol), and neither had an infection nor previous ovarian surgery were included in this study. However, subjects in the hyper- and poor-responder groups, had no oocytes in the ovarian follicular fluid, had two oocytes found in one sample tube, had severe endometriosis, adenomyosis, or polycystic ovarian syndrome who fulfilled the Rotterdam criteria, and patients with severe sperm abnormalities were excluded. Sampling Sizes and Sampling Procedures Consecutive sampling was used in this study from November 2021 to March 2022. Samples were collected from the participants who met the study criteria. Determination of the sample size was carried out based on statistical calculations by setting a 95% confidence level and 80% power test. With a 95% confidence level and 80% power, this study required a minimum of 86 samples per group, equalling 172 samples in total. Data Collecting and Laboratory Examination The subjects followed an IVF program using a controlled ovarian stimulation protocol with either a short or a long protocol. A transvaginal ultrasound examination was performed before the start of the program. The short protocol began when the patient was on the 2nd day of menstruation, using recombinant FSH at a dose of 75–450 IU/day. On the 6th or 7th day of stimulation, or when follicles ≥ 14 mm were found, a GnRH antagonist (Cetrorelix 0.25 mg) was administered. Triggers using recombinant human chorionic gonadotropin (hCG) or a GnRH agonist were performed when at least three follicles with a diameter of 18 mm were found. Oocyte picking was carried out 34–36 hours after the triggering. The long protocol begins when the patient is on the 21st day of menstruation, down regulation was carried out using a 0.5 mg GnRH analog until the patient was menstruating. When the patient menstruated on the first day, endometrial thickness assessment and antral follicle counts were performed. If endometrial thickness was less than 5 mm, stimulation was performed using recombinant FSH at a dose of 75–450 IU/day and a GnRH analog of 0.2 mg. Triggers using recombinant human chorionic gonadotropin were used when at least three follicles with a diameter of 18 mm were found. The ovum pick-up procedure was performed 34–36 h after the triggering. Before the ovum pick-up procedure, the size of the follicles was examined by oocyte aspiration. Follicle size was measured using two diameter measurements and averaged. Oocytes were collected using a 17-gauge oocyte aspiration needle under transvaginal ultrasound guidance. The follicular fluid and oocytes were then aspirated without flushing and stored in a Falcon tube. In the present study, only one Falcon tube was used for each follicle. The tube containing follicular fluid was examined by an embryologist using a light microscope to identify oocytes and assess oocyte maturity. The follicular fluid in a tube containing oocytes was used as a research sample, and some codes were assigned to those tubes by the examiner. The tube containing the follicular fluid was stored at -85 o C until the levels of 25-OH-vitamin D3, vitamin D-binding protein, albumin, and calcium were examined. Each patient had a maximum of eight follicular fluid samples. Examination of 25-OH-vitamin D3, vitamin D-binding protein, albumin, and calcium levels was performed at the Clinical Pathology Laboratory of Dr. Hasan Sadikin General Hospital, Bandung. The tube containing follicular fluid would be centrifuged at 300 rpm, then they would be separated into 2 aliquots containing 0.5–1 mL in each aliquot. Examination of 25(OH)D3, vitamin D-binding protein, albumin, and calcium levels in the follicles was performed using ELISA. Free 25(OH)D3 levels were calculated using a method Vermeulen formula:[ 13 ] Study Outcomes The outcomes of this study were as follows: (1) Differences in 25(OH)D3 and calcium levels in ovarian follicular fluid between TQE and non-TQE normoresponder women who underwent IVF procedures. (2) Correlation between free 25(OH)D3 and calcium levels in ovarian follicular fluid and embryo quality in normoresponder patients undergoing IVF procedures. RESULTS During the study period, 173 follicular fluid samples were obtained, with 86 and 87 samples from the TQE and non-TQE groups, respectively. Table 1 presents the participants’ characteristics. The two groups (TQE and non-TQE) were comparable in terms of age, BMI, infertility duration, total gonadotropin dose, estradiol level, progesterone level, basal AMH level, AFC, sperm analysis, and sperm counts. The mean age of patients in the TQE group was 30 ± 2.9 and in the non-TQE group 30.6 ± 2.9 (p = 0.678). The mean value of estradiol levels on trigger day in the TQE group was 4645 ± 386.7 and in the non-TQE group was 4727.4 ± 2386.9 (p = 0.771), which means that statistically there was no difference (Table 1 ). Table 1. Comparison of patient baseline demographics and clinical parameters between *Notes: *) Chi-square test was performed on the Age, Type of Infertility, and Sperm Analysis variables; duration of infertility, total gonadotropin dose, and sperm count were analyzed with Mann-Whitney test; others were analyzed by t test. Comparison of various research variables of the two groups in Table 2 , The two groups differed significantly in total follicular vitamin D levels (p = 0.009), free follicular vitamin D levels (p = 0.017), and follicle sizes (p < 0.001). The levels of 25-OH-vitamin D3 total follicles, 25-OH-vitamin D3 free follicles, and follicular calcium, which can be used as predictors of TQE, were analyzed using the ROC curve to determine the cut-off point, AUC (area under the curve (AUC), sensitivity, and specificity (Table 3 ). The relationship between the cut-off value of total 25-OH-vitamin D3 levels and follicle-free 25-OH-vitamin D3 levels with embryo quality, which was divided into TQE and non-TQE groups, in normoresponder patients undergoing in vitro fertilization (FIV) procedures in Table 4 . Patients with total follicular 25(OH)D3 levels of > 12.1 ng/mL had a 2.38 times tendency to develop TQE. Patients with follicular-free 25(OH)D3 levels > 0.00175 ng/mL had a 3.05 times chance of developing TQE than those with 25(OH)D3 levels ≤ 0.00175 ng/mL. The cutoff value for follicular calcium levels was > 4.88 mg/dL (p = 0.144). There was no statistically significant correlation between the follicular calcium levels in the two study groups. Table 4. Correlation between Total 25(OH)D 3 and Free 25(OH)D 3 Levels in Follicular Fluid with Embryo Quality in Normoresponder Patients Underwent IVF Procedures The follicle-free 25-25-OH-vitamin D3 cut-off value that can be used as a predictor of TQE was > 0.00175 ng/mL, and the follicle calcium cutoff value that can be used as a predictor of TQE was > 4.88 mg/dL. Here, we suggest cutoff values for predicting top-quality embryos. A follicular total 25(OH)D3 level > 12.1 ng/mL (p = 0.0075), free follicular 25(OH)D3 level > 0.00175 (p = 0.0146), and follicle size > 19.5 mm (p < 0.001) were all suggestive of top quality embryos (Fig. 1 ). DISCUSSION Principal findings In this study, the total follicle 25(OH)D3 and follicular free 25(OH)D3 levels were higher in the TQE group than in the non-TQE group. The results of this study were consistent with those reported by Alawad et al. Intrafollicular vitamin D levels are positively correlated with oocyte maturity, fertilization rate, and top-quality embryos.[ 14 ] Arnanz et al found that total follicle 25(OH)D3 levels and follicular free 25(OH)D3 levels were predictors of euploid embryos in the vitamin D-deficient group.[ 13 ] However, a study conducted by Ciepiela et al. showed different results; the TQE group had lower follicular 25(OH)D3 levels than the non-TQE group [ 4 ]. Rudick et al. revealed that race had an effect on the relationship between vitamin D status and post-IVF pregnancy rates.[ 15 ] The results of this study were in line with those of Zainab M. Alawad and Arnanz et al. due to the same sample (Asian racial group), while in the study of Ciepiela et al. the study subjects were the Hispanic race group. A study conducted by Cai et al. showed that serum total 25(OH)D3 levels correlate with serum-free 25(OH)D3 levels.[ 16 ] In this study, total follicle 25(OH)D3 levels correlate with follicular free 25(OH)D3 levels. However, in this study, total follicular 25(OH)D3 levels were not correlated with total serum 25-(OH)-vitamin D3 levels, in contrast to the study by Liu et al., in which the levels of vitamin D in follicular fluid correlated with serum levels of vitamin D.[ 15 ] This is thought to be related to the pleiotropic effect of vitamin D, which is related to the vitamin D response element (VDRE) gene, which gives a different phenotype in each cell. The median follicular calcium level in the TQE group was higher than in the non-TQE group (6.30 (3.74–18.49) mg/dL vs 6.23 (3.60–17.82) mg/dL respectively), but not significantly different statistically (p = 0.805). Follicular calcium levels were positively correlated with serum 25(OH)D3 levels in the TQE group (r = 0.218, p = 0.046) and all study samples (r = 0.216, p = 0.004). However, calcium levels did not correlate with serum 25(OH)D3 levels in the non-TQE group (r = 0.051, p = 0.216). Follicular calcium levels were positively correlated with serum calcium levels in the TQE group (r = 0.222, p = 0.040), the non-TQE (r = 0.215, p = 0.046), and all study samples (r = 0.224, p = 0.003). The results of this study are in line with those of Ozdamar et al., in which follicular calcium levels in the IVF group in pregnant vs. non-pregnant patients were not significantly different.[ 17 ] It was hypothesized that during follicular fluid sampling, a specific calcium oscillation process did not occur, so there was no activation of intracellular calcium ions. Patients with total follicular 25(OH)D3 levels of > 12.1 ng/mL had a 2.38 times tendency to obtain high-quality embryos (TQE). Ciepiela et al. reported that the cut-off value of serum 25(OH)D3 as a predictor of top-quality embryos was > 12.7 ng/mL [ 4 ]. In a study by Liu et al., a cut-off value of 25(OH)D3 serum of > 14.1 ng/mL can predict a normal fertilization rate, pregnancy rate, and live birth rate.[ 15 ] The cut off value of follicular free 25(OH)D3, which can be used as a predictor of top quality embryo (TQE), is > 0.00175 ng/mL (95% CI 0.605 (0.528–0.678), p = 0.0146, sensitivity 34.88%, specificity 85.06%). The relationship between the cut off value of follicle-free 25(OH)D3 levels > 0.00175 ng/mL between the TQE and non-TQE groups differed statistically significantly (p = 0.002, OR (95% CI) 3.05(1.46–6) ,38)). Patients with follicle-free 25(OH)D3 levels > 0.00175 ng/mL showed a 3.05 times tendency to obtain top-quality embryos (TQE). The mechanism that could explain the results of this study is that follicular free 25(OH)D3 affects AMH signaling and steroidogenesis in granulosa cells; thus, good follicle development can occur. Modulation of steroidogenesis in granulosa cells through activation of the AMP-activated protein kinase (AMPK) pathway increases follicle maturation. Vitamin D also regulates oxidative stress at the cellular level, which plays an important role in follicular development. Mature oocytes can fertilize and form high-quality embryos. CONCLUSION Free 25(OH)D3 and calcium in follicular fluid act independently during steroidogenesis, folliculogenesis, and fertilization. Further studies regarding the correlation of free 25(OH)D3 levels in follicular fluid and serum with euploid embryo status, clinical pregnancy rate, and live birth rate are needed. Additional sequelae studies are needed to evaluate whether vitamin D3 supplementation improves oocyte and embryo quality, as well as the correlation of calcium ion levels in granulosa cells with embryo quality. Abbreviations 25(OH)D3 : 25-hidroxy-vitamin D3 AFC : Antral Follicle Count AMH : Anti-Mullerian hormone IVF : in vitro fertilization IGF-1 : insulin-like growth factor-1 IGFBP-1 : insulin-like growth factor-binding protein-1 VDBP : vitamin D-binding protein VDR : vitamin D receptors Declarations Data availability The dataset used and /or analysed during the current study available from the corresponding author on reasonable request Funding Source We declare that we do not receive external funding for this study. Conflict of Interest The authors declare that they have no conflict of interest. Data Sharing The author(s) declare that they have no conflict of interest in this study. Patients Permisssion Ethics Approval and Institutional Review Board Statement The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board (or Ethics Committee) of Ethics Committee Review Board of Hasan Sadikin General Hospital – Faculty of Medicine, Universitas Padjadjaran (protocol date LB.02.01/X.6.5/349/2022 and date of approval 06 October 2022). All study participants provided written informed consent prior to engaging in any study-related procedures. All authors hereby declare that all patients were examined in accordance with the ethical standards specified in the 1964 Declaration of Helsinki. Acknowledgment The authors would like to thank the Department of Obstetrics and Gynecology, Faculty of Medicine, University of Padjadjaran/Dr. Hasan Sadikin General Hospital, Padjadjaran University, Ministry of Research, Technology, and Higher Education, as well as all patients who participated in our study. 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Hosseinisadat, R., et al., Assessment of the effect of serum and follicular fluid vitamin D and glucose on assisted reproductive technique outcome: A cross-sectional study. International Journal of Reproductive BioMedicine, 2022. 20 (3): p. 221. Ko, J.K., et al., 100 YEARS OF VITAMIN D: Effect of serum vitamin D level before ovarian stimulation on the cumulative live birth rate of women undergoing in vitro fertilization: a retrospective analysis. Endocrine Connections, 2022. 11 (2). Thomson, R.L., S. Spedding, and J.D. Buckley, Vitamin D in the aetiology and management of polycystic ovary syndrome. Clinical endocrinology, 2012. 77 (3): p. 343-350. Jensen, M.B., Vitamin D and male reproduction. Nature Reviews Endocrinology, 2014. 10 (3): p. 175-186. Cozzolino, M., et al., How vitamin D level influences in vitro fertilization outcomes: results of a systematic review and meta-analysis. Fertility and Sterility, 2020. 114 (5): p. 1014-1025. Grzeczka, A., et al., Relevance of Vitamin D and Its Deficiency for the Ovarian Follicle and the Oocyte: An Update. Nutrients, 2022. 14 (18): p. 3712. Chun, R.F. and C.M. Nielson, Free Vitamin D: concepts, assays, outcomes, and prospects. Vitamin D, 2018: p. 925-937. Alawad, Z.M., Level of follicular fluid vitamin D and embryo quality in a sample of Iraqi women undergoing IVF. Journal of the Faculty of Medicine Baghdad, 2018. 60 (4): p. 215-221. Arnanz, A., et al., Vitamin D in follicular fluid correlates with the euploid status of blastocysts in a vitamin D deficient population. Frontiers in endocrinology, 2021. 11 : p. 609524. Monastra, G., et al., Vitamin D: a steroid hormone with progesterone-like activity. European Review for Medical & Pharmacological Sciences, 2018. 22 (8). Rudick, B., et al., Characterizing the influence of vitamin D levels on IVF outcomes. Human reproduction, 2012. 27 (11): p. 3321-3327. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 22 Nov, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 27 Jun, 2024 Reviews received at journal 23 Jun, 2024 Reviewers agreed at journal 12 Jun, 2024 Reviews received at journal 10 Jun, 2024 Reviewers agreed at journal 31 May, 2024 Reviewers invited by journal 30 May, 2024 Editor assigned by journal 30 May, 2024 Editor invited by journal 16 May, 2024 Submission checks completed at journal 15 May, 2024 First submitted to journal 06 May, 2024 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-4375279","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":305280067,"identity":"e1ed7901-9013-4c70-a2eb-766c38279a29","order_by":0,"name":"Artha Falentin Putri Susilo","email":"data:image/png;base64,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","orcid":"","institution":"Universitas Padjadjaran - Dr. Hasan Sadikin General Hospital","correspondingAuthor":true,"prefix":"","firstName":"Artha","middleName":"Falentin Putri","lastName":"Susilo","suffix":""},{"id":305280068,"identity":"202f0e7d-c42f-49c0-957b-1215daea46e5","order_by":1,"name":"Hanom Husni Syam","email":"","orcid":"","institution":"Universitas Padjadjaran - Dr. Hasan Sadikin General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hanom","middleName":"Husni","lastName":"Syam","suffix":""},{"id":305280069,"identity":"6513ce40-d1d5-40fd-8d9c-6c78187a1c97","order_by":2,"name":"Hartanto Bayuaji","email":"","orcid":"","institution":"Universitas Padjadjaran - Dr. Hasan Sadikin General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hartanto","middleName":"","lastName":"Bayuaji","suffix":""},{"id":305280070,"identity":"43b077ef-66fa-4740-8504-febae9f58183","order_by":3,"name":"Anita Rachmawati","email":"","orcid":"","institution":"Universitas Padjadjaran - Dr. Hasan Sadikin General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Anita","middleName":"","lastName":"Rachmawati","suffix":""},{"id":305280071,"identity":"565f29e5-c427-4b11-b87e-66f930e77cae","order_by":4,"name":"Binarwan Halim","email":"","orcid":"","institution":"Universitas Sumatera Utara – H Adam Malik Hospital","correspondingAuthor":false,"prefix":"","firstName":"Binarwan","middleName":"","lastName":"Halim","suffix":""},{"id":305280072,"identity":"293cb3a8-6c18-4866-a9f8-58bc2807e5da","order_by":5,"name":"Wiryawan Permadi","email":"","orcid":"","institution":"Universitas Padjadjaran - Dr. Hasan Sadikin General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Wiryawan","middleName":"","lastName":"Permadi","suffix":""},{"id":305280073,"identity":"0013000a-b5dc-4fac-855f-994f390e22c5","order_by":6,"name":"Tono Djuwantono","email":"","orcid":"","institution":"Universitas Padjadjaran - Dr. Hasan Sadikin General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Tono","middleName":"","lastName":"Djuwantono","suffix":""}],"badges":[],"createdAt":"2024-05-06 08:29:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4375279/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4375279/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-71769-6","type":"published","date":"2024-11-22T15:57:39+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":57021226,"identity":"01769a01-1761-4338-bf69-8ae1aa964a7d","added_by":"auto","created_at":"2024-05-23 13:59:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":466161,"visible":true,"origin":"","legend":"\u003cp\u003eROC Curves of Total Follicular 25(OH)D\u003csub\u003e3\u003c/sub\u003e, Follicular Free 25(OH)D\u003csub\u003e3\u003c/sub\u003e, and Follicle Calcium\u003c/p\u003e","description":"","filename":"944920Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4375279/v1/5fcc9c98c5d399c4023ab93d.png"},{"id":69835171,"identity":"6cdd82f9-2496-42fc-b290-0e4539c43d4f","added_by":"auto","created_at":"2024-11-25 16:12:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":936138,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4375279/v1/a1ce3c8e-c717-4279-b743-e48773aadd97.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eFree 25(Oh)d3 Levels in Follicular Ovarian Fluid Top Quality Embryo is Higher Than Non- Top Quality Embryo in Normoresponders Group\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eVitamin D is a steroid hormone that plays essential roles in influencing several physiological processes, especially calcium and phosphorus homeostasis and bone mineralization.[\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] Vitamin D also plays a pivotal role in human reproductive functions such as folliculogenesis, steroidogenesis, and successful embryo implantation.[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] Vitamin D is a steroid hormone that plays an essential role in influencing several physiological processes, especially calcium and phosphorus homeostasis and bone mineralization.[\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] Thus, vitamin D levels could be a predictor of assisted reproductive technology success rate.[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eThe effects of vitamin D and calcium on oocyte development have important clinical implications for the success of in vitro fertilization (IVF) programs. Vitamin D deficiency can lead to calcium deficiency, resulting in disturbances in oocyte maturation, development, and fertilization.[\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eVitamin D plays a role in follicle regulation and oocyte maturation through both direct and indirect pathways.[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] Vitamin D plays a role in embryogenesis and folliculogenesis. Vitamin D modulates primary follicle recruitment by regulating anti-M\u0026uuml;llerian hormone (AMH) production.[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eThus, intrafollicular vitamin D levels could be a potential marker of oocyte and embryo quality as well as a predictor of IVF outcomes.[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] Assessment of free vitamin D3 levels and bioavailable vitamin D3 could become a more accurate predictor in assessing vitamin D status.[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] Levels of free 25(OH)D3 and calcium in follicular fluid play a role in the final maturation of follicles and are thought to be related to the rate of fertilization, cleavage, and the quality of the embryo to increase the rate of pregnancy. However, their exact roles in stimulating oocyte and embryo quality have not been adequately reported.\u003c/p\u003e \u003cp\u003eThis study aimed to compare free 25(OH)D3 and calcium levels in follicular fluid between top-quality embryo (TQE) and non-top-quality embryo (non-TQE) groups and to evaluate the correlation between free 25(OH)D3 and calcium levels in follicular fluid and embryo quality in normoresponder patients who underwent IVF procedures.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy Design\u003c/h2\u003e \u003cp\u003eThis observational cross-sectional study used secondary data from the ovarian follicular fluid of women who underwent IVF in tertiary hospitals in Bandung, Indonesia. This study was conducted by the Declaration of Helsinki of the World Medical Association Declaration. Helsinki. All methods were performed by relevant guidelines and regulations after obtaining approval and recommendations from the Ethics Committee Review Board of Hasan Sadikin General Hospital, Faculty of Medicine Universitas Padjadjaran (reference number LB.02.01/X.6.5/399/2022).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eResearch Subjects\u003c/h2\u003e \u003cp\u003eThe subjects of this study were women who underwent IVF procedures at the Aster Clinic, Dr. Hasan Sadikin General Hospital Bandung, and Bandung Fertility Center Limijati Hospital Bandung between November 2021-March 2022 that met the inclusion and exclusion criteria of this study. Women of reproductive age (20\u0026ndash;35 years old) who belong to normoresponder group, with body mass index (BMI) varied between 18\u0026ndash;25 kg/m\u003csup\u003e2\u003c/sup\u003e, participated in an IVF program for the first time, as well as women who were not exposed to toxic substances (cigarettes, alcohol), and neither had an infection nor previous ovarian surgery were included in this study. However, subjects in the hyper- and poor-responder groups, had no oocytes in the ovarian follicular fluid, had two oocytes found in one sample tube, had severe endometriosis, adenomyosis, or polycystic ovarian syndrome who fulfilled the Rotterdam criteria, and patients with severe sperm abnormalities were excluded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSampling Sizes and Sampling Procedures\u003c/h2\u003e \u003cp\u003eConsecutive sampling was used in this study from November 2021 to March 2022. Samples were collected from the participants who met the study criteria. Determination of the sample size was carried out based on statistical calculations by setting a 95% confidence level and 80% power test. With a 95% confidence level and 80% power, this study required a minimum of 86 samples per group, equalling 172 samples in total.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eData Collecting and Laboratory Examination\u003c/h2\u003e \u003cp\u003eThe subjects followed an IVF program using a controlled ovarian stimulation protocol with either a short or a long protocol. A transvaginal ultrasound examination was performed before the start of the program. The short protocol began when the patient was on the 2nd day of menstruation, using recombinant FSH at a dose of 75\u0026ndash;450 IU/day. On the 6th or 7th day of stimulation, or when follicles\u0026thinsp;\u0026ge;\u0026thinsp;14 mm were found, a GnRH antagonist (Cetrorelix 0.25 mg) was administered. Triggers using recombinant human chorionic gonadotropin (hCG) or a GnRH agonist were performed when at least three follicles with a diameter of 18 mm were found. Oocyte picking was carried out 34\u0026ndash;36 hours after the triggering.\u003c/p\u003e \u003cp\u003eThe long protocol begins when the patient is on the 21st day of menstruation, down regulation was carried out using a 0.5 mg GnRH analog until the patient was menstruating. When the patient menstruated on the first day, endometrial thickness assessment and antral follicle counts were performed. If endometrial thickness was less than 5 mm, stimulation was performed using recombinant FSH at a dose of 75\u0026ndash;450 IU/day and a GnRH analog of 0.2 mg. Triggers using recombinant human chorionic gonadotropin were used when at least three follicles with a diameter of 18 mm were found. The ovum pick-up procedure was performed 34\u0026ndash;36 h after the triggering.\u003c/p\u003e \u003cp\u003eBefore the ovum pick-up procedure, the size of the follicles was examined by oocyte aspiration. Follicle size was measured using two diameter measurements and averaged. Oocytes were collected using a 17-gauge oocyte aspiration needle under transvaginal ultrasound guidance. The follicular fluid and oocytes were then aspirated without flushing and stored in a Falcon tube. In the present study, only one Falcon tube was used for each follicle. The tube containing follicular fluid was examined by an embryologist using a light microscope to identify oocytes and assess oocyte maturity. The follicular fluid in a tube containing oocytes was used as a research sample, and some codes were assigned to those tubes by the examiner. The tube containing the follicular fluid was stored at -85\u003csup\u003eo\u003c/sup\u003eC until the levels of 25-OH-vitamin D3, vitamin D-binding protein, albumin, and calcium were examined. Each patient had a maximum of eight follicular fluid samples.\u003c/p\u003e \u003cp\u003eExamination of 25-OH-vitamin D3, vitamin D-binding protein, albumin, and calcium levels was performed at the Clinical Pathology Laboratory of Dr. Hasan Sadikin General Hospital, Bandung. The tube containing follicular fluid would be centrifuged at 300 rpm, then they would be separated into 2 aliquots containing 0.5\u0026ndash;1 mL in each aliquot. Examination of 25(OH)D3, vitamin D-binding protein, albumin, and calcium levels in the follicles was performed using ELISA. Free 25(OH)D3 levels were calculated using a method Vermeulen formula:[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/p\u003e \u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1716472053.png\"\u003e\u003cbr\u003e\u003c/p\u003e\u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStudy Outcomes\u003c/h2\u003e \u003cp\u003eThe outcomes of this study were as follows: (1) Differences in 25(OH)D3 and calcium levels in ovarian follicular fluid between TQE and non-TQE normoresponder women who underwent IVF procedures. (2) Correlation between free 25(OH)D3 and calcium levels in ovarian follicular fluid and embryo quality in normoresponder patients undergoing IVF procedures.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eDuring the study period, 173 follicular fluid samples were obtained, with 86 and 87 samples from the TQE and non-TQE groups, respectively. Table\u0026nbsp;\u003cspan\u003e1\u003c/span\u003e presents the participants\u0026rsquo; characteristics. The two groups (TQE and non-TQE) were comparable in terms of age, BMI, infertility duration, total gonadotropin dose, estradiol level, progesterone level, basal AMH level, AFC, sperm analysis, and sperm counts. The mean age of patients in the TQE group was 30\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9 and in the non-TQE group 30.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9 (p\u0026thinsp;=\u0026thinsp;0.678). The mean value of estradiol levels on trigger day in the TQE group was 4645\u0026thinsp;\u0026plusmn;\u0026thinsp;386.7 and in the non-TQE group was 4727.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2386.9 (p\u0026thinsp;=\u0026thinsp;0.771), which means that statistically there was no difference (Table\u0026nbsp;\u003cspan\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eTable 1. Comparison of patient baseline demographics and clinical parameters between\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1716472711.png\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cdiv\u003e\n \u003cdiv align=\"left\"\u003e\u003cstrong\u003e*Notes:\u0026nbsp;\u003c/strong\u003e*) Chi-square test was performed on the Age, Type of Infertility, and Sperm Analysis variables; duration of infertility, total gonadotropin dose, and sperm count were analyzed with Mann-Whitney test; others were analyzed by t test.\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eComparison of various research variables of the two groups in Table\u0026nbsp;\u003cspan\u003e2\u003c/span\u003e, The two groups differed significantly in total follicular vitamin D levels (p\u0026thinsp;=\u0026thinsp;0.009), free follicular vitamin D levels (p\u0026thinsp;=\u0026thinsp;0.017), and follicle sizes (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The levels of 25-OH-vitamin D3 total follicles, 25-OH-vitamin D3 free follicles, and follicular calcium, which can be used as predictors of TQE, were analyzed using the ROC curve to determine the cut-off point, AUC (area under the curve (AUC), sensitivity, and specificity (Table\u0026nbsp;\u003cspan\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv\u003e\u003cbr\u003e\u003c/div\u003e\n\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1716472450.png\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cdiv\u003e\u003cbr\u003e\u003c/div\u003e\n\u003cp\u003eThe relationship between the cut-off value of total 25-OH-vitamin D3 levels and follicle-free 25-OH-vitamin D3 levels with embryo quality, which was divided into TQE and non-TQE groups, in normoresponder patients undergoing in vitro fertilization (FIV) procedures in Table\u0026nbsp;\u003cspan\u003e4\u003c/span\u003e. Patients with total follicular 25(OH)D3 levels of \u0026gt;\u0026thinsp;12.1 ng/mL had a 2.38 times tendency to develop TQE. Patients with follicular-free 25(OH)D3 levels\u0026thinsp;\u0026gt;\u0026thinsp;0.00175 ng/mL had a 3.05 times chance of developing TQE than those with 25(OH)D3 levels\u0026thinsp;\u0026le;\u0026thinsp;0.00175 ng/mL. The cutoff value for follicular calcium levels was \u0026gt;\u0026thinsp;4.88 mg/dL (p\u0026thinsp;=\u0026thinsp;0.144). There was no statistically significant correlation between the follicular calcium levels in the two study groups.\u003c/p\u003e\n\u003cp\u003eTable 4.\u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Correlation between Total 25(OH)D\u003csub\u003e3\u003c/sub\u003e and Free 25(OH)D\u003csub\u003e3\u003c/sub\u003e Levels in Follicular Fluid with Embryo Quality in Normoresponder Patients Underwent IVF Procedures\u003c/p\u003e\n\u003cdiv\u003e\n \u003cdiv align=\"left\"\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img171647244911.png\"\u003e\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003eThe follicle-free 25-25-OH-vitamin D3 cut-off value that can be used as a predictor of TQE was \u0026gt;\u0026thinsp;0.00175 ng/mL, and the follicle calcium cutoff value that can be used as a predictor of TQE was \u0026gt;\u0026thinsp;4.88 mg/dL. Here, we suggest cutoff values for predicting top-quality embryos. A follicular total 25(OH)D3 level\u0026thinsp;\u0026gt;\u0026thinsp;12.1 ng/mL (p\u0026thinsp;=\u0026thinsp;0.0075), free follicular 25(OH)D3 level\u0026thinsp;\u0026gt;\u0026thinsp;0.00175 (p\u0026thinsp;=\u0026thinsp;0.0146), and follicle size\u0026thinsp;\u0026gt;\u0026thinsp;19.5 mm (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) were all suggestive of top quality embryos (Fig. \u003cspan\u003e1\u003c/span\u003e).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003ePrincipal findings\u003c/h2\u003e \u003cp\u003eIn this study, the total follicle 25(OH)D3 and follicular free 25(OH)D3 levels were higher in the TQE group than in the non-TQE group. The results of this study were consistent with those reported by Alawad et al. Intrafollicular vitamin D levels are positively correlated with oocyte maturity, fertilization rate, and top-quality embryos.[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] Arnanz et al found that total follicle 25(OH)D3 levels and follicular free 25(OH)D3 levels were predictors of euploid embryos in the vitamin D-deficient group.[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] However, a study conducted by Ciepiela et al. showed different results; the TQE group had lower follicular 25(OH)D3 levels than the non-TQE group [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Rudick et al. revealed that race had an effect on the relationship between vitamin D status and post-IVF pregnancy rates.[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] The results of this study were in line with those of Zainab M. Alawad and Arnanz et al. due to the same sample (Asian racial group), while in the study of Ciepiela et al. the study subjects were the Hispanic race group.\u003c/p\u003e \u003cp\u003eA study conducted by Cai et al. showed that serum total 25(OH)D3 levels correlate with serum-free 25(OH)D3 levels.[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] In this study, total follicle 25(OH)D3 levels correlate with follicular free 25(OH)D3 levels. However, in this study, total follicular 25(OH)D3 levels were not correlated with total serum 25-(OH)-vitamin D3 levels, in contrast to the study by Liu et al., in which the levels of vitamin D in follicular fluid correlated with serum levels of vitamin D.[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] This is thought to be related to the pleiotropic effect of vitamin D, which is related to the vitamin D response element (VDRE) gene, which gives a different phenotype in each cell.\u003c/p\u003e \u003cp\u003eThe median follicular calcium level in the TQE group was higher than in the non-TQE group (6.30 (3.74\u0026ndash;18.49) mg/dL vs 6.23 (3.60\u0026ndash;17.82) mg/dL respectively), but not significantly different statistically (p\u0026thinsp;=\u0026thinsp;0.805). Follicular calcium levels were positively correlated with serum 25(OH)D3 levels in the TQE group (r\u0026thinsp;=\u0026thinsp;0.218, p\u0026thinsp;=\u0026thinsp;0.046) and all study samples (r\u0026thinsp;=\u0026thinsp;0.216, p\u0026thinsp;=\u0026thinsp;0.004). However, calcium levels did not correlate with serum 25(OH)D3 levels in the non-TQE group (r\u0026thinsp;=\u0026thinsp;0.051, p\u0026thinsp;=\u0026thinsp;0.216). Follicular calcium levels were positively correlated with serum calcium levels in the TQE group (r\u0026thinsp;=\u0026thinsp;0.222, p\u0026thinsp;=\u0026thinsp;0.040), the non-TQE (r\u0026thinsp;=\u0026thinsp;0.215, p\u0026thinsp;=\u0026thinsp;0.046), and all study samples (r\u0026thinsp;=\u0026thinsp;0.224, p\u0026thinsp;=\u0026thinsp;0.003). The results of this study are in line with those of Ozdamar et al., in which follicular calcium levels in the IVF group in pregnant vs. non-pregnant patients were not significantly different.[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] It was hypothesized that during follicular fluid sampling, a specific calcium oscillation process did not occur, so there was no activation of intracellular calcium ions.\u003c/p\u003e \u003cp\u003ePatients with total follicular 25(OH)D3 levels of \u0026gt;\u0026thinsp;12.1 ng/mL had a 2.38 times tendency to obtain high-quality embryos (TQE). Ciepiela et al. reported that the cut-off value of serum 25(OH)D3 as a predictor of top-quality embryos was \u0026gt;\u0026thinsp;12.7 ng/mL [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In a study by Liu et al., a cut-off value of 25(OH)D3 serum of \u0026gt;\u0026thinsp;14.1 ng/mL can predict a normal fertilization rate, pregnancy rate, and live birth rate.[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] The cut off value of follicular free 25(OH)D3, which can be used as a predictor of top quality embryo (TQE), is \u0026gt;\u0026thinsp;0.00175 ng/mL (95% CI 0.605 (0.528\u0026ndash;0.678), p\u0026thinsp;=\u0026thinsp;0.0146, sensitivity 34.88%, specificity 85.06%). The relationship between the cut off value of follicle-free 25(OH)D3 levels\u0026thinsp;\u0026gt;\u0026thinsp;0.00175 ng/mL between the TQE and non-TQE groups differed statistically significantly (p\u0026thinsp;=\u0026thinsp;0.002, OR (95% CI) 3.05(1.46\u0026ndash;6) ,38)). Patients with follicle-free 25(OH)D3 levels\u0026thinsp;\u0026gt;\u0026thinsp;0.00175 ng/mL showed a 3.05 times tendency to obtain top-quality embryos (TQE).\u003c/p\u003e \u003cp\u003eThe mechanism that could explain the results of this study is that follicular free 25(OH)D3 affects AMH signaling and steroidogenesis in granulosa cells; thus, good follicle development can occur. Modulation of steroidogenesis in granulosa cells through activation of the AMP-activated protein kinase (AMPK) pathway increases follicle maturation. Vitamin D also regulates oxidative stress at the cellular level, which plays an important role in follicular development. Mature oocytes can fertilize and form high-quality embryos.\u003c/p\u003e \u003c/div\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eFree 25(OH)D3 and calcium in follicular fluid act independently during steroidogenesis, folliculogenesis, and fertilization. Further studies regarding the correlation of free 25(OH)D3 levels in follicular fluid and serum with euploid embryo status, clinical pregnancy rate, and live birth rate are needed. Additional sequelae studies are needed to evaluate whether vitamin D3 supplementation improves oocyte and embryo quality, as well as the correlation of calcium ion levels in granulosa cells with embryo quality.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e25(OH)D3\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;: 25-hidroxy-vitamin D3\u003c/p\u003e\n\u003cp\u003eAFC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;:\u0026nbsp; \u0026nbsp;Antral Follicle Count\u003c/p\u003e\n\u003cp\u003eAMH\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;:\u0026nbsp; \u0026nbsp;Anti-Mullerian hormone\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIVF\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;:\u0026nbsp; \u0026nbsp;in vitro fertilization\u003c/p\u003e\n\u003cp\u003eIGF-1\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;: \u0026nbsp; insulin-like growth factor-1\u003c/p\u003e\n\u003cp\u003eIGFBP-1\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;:\u0026nbsp; \u0026nbsp;insulin-like growth factor-binding protein-1\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eVDBP\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;:\u0026nbsp; \u0026nbsp;vitamin D-binding protein\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eVDR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;: \u0026nbsp; vitamin D receptors\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dataset used and /or analysed during the current study available from the corresponding author on reasonable request\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Source\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe declare that we do not receive external funding for this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Sharing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) declare that they have no conflict of interest in this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatients Permisssion\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ch2\u003eEthics Approval and Institutional Review Board Statement\u003c/h2\u003e\n\u003cp\u003eThe study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board (or Ethics Committee) of Ethics Committee Review Board of Hasan Sadikin General Hospital \u0026ndash; Faculty of Medicine, Universitas Padjadjaran (protocol date LB.02.01/X.6.5/349/2022\u0026nbsp;and date of approval 06\u0026nbsp;October\u0026nbsp;2022).\u0026nbsp;All study participants provided written informed consent prior to engaging in any study-related procedures. All authors hereby declare that all patients were examined in accordance with the ethical standards specified in the 1964 Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the Department of Obstetrics and Gynecology, Faculty of Medicine, University of Padjadjaran/Dr. Hasan Sadikin General Hospital, Padjadjaran University, Ministry of Research, Technology, and Higher Education, as well as all patients who participated in our study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eVander Borght, M. and C. Wyns, \u003cem\u003eFertility and infertility: Definition and epidemiology.\u003c/em\u003e Clinical biochemistry, 2018. \u003cstrong\u003e62\u003c/strong\u003e: p. 2-10.\u003c/li\u003e\n\u003cli\u003eCarson, S.A. and A.N. Kallen, \u003cem\u003eDiagnosis and management of infertility: a review.\u003c/em\u003e Jama, 2021. \u003cstrong\u003e326\u003c/strong\u003e(1): p. 65-76.\u003c/li\u003e\n\u003cli\u003eBasile, S., et al., \u003cem\u003eVitamin D and infertility: a narrative review.\u003c/em\u003e\u003c/li\u003e\n\u003cli\u003eCiepiela, P., et al., \u003cem\u003eVitamin D as a follicular marker of human oocyte quality and a serum marker of in vitro fertilization outcome.\u003c/em\u003e Journal of assisted reproduction and genetics, 2018. \u003cstrong\u003e35\u003c/strong\u003e: p. 1265-1276.\u003c/li\u003e\n\u003cli\u003eMuscogiuri, G., et al., \u003cem\u003eShedding new light on female fertility: The role of vitamin D.\u003c/em\u003e Reviews in Endocrine and Metabolic Disorders, 2017. \u003cstrong\u003e18\u003c/strong\u003e: p. 273-283.\u003c/li\u003e\n\u003cli\u003eSkowrońska, P., et al., \u003cem\u003eThe role of vitamin D in reproductive dysfunction in women-a systematic review.\u003c/em\u003e Annals of Agricultural and Environmental Medicine, 2016. \u003cstrong\u003e23\u003c/strong\u003e(4).\u003c/li\u003e\n\u003cli\u003eHosseinisadat, R., et al., \u003cem\u003eAssessment of the effect of serum and follicular fluid vitamin D and glucose on assisted reproductive technique outcome: A cross-sectional study.\u003c/em\u003e International Journal of Reproductive BioMedicine, 2022. \u003cstrong\u003e20\u003c/strong\u003e(3): p. 221.\u003c/li\u003e\n\u003cli\u003eKo, J.K., et al., \u003cem\u003e100 YEARS OF VITAMIN D: Effect of serum vitamin D level before ovarian stimulation on the cumulative live birth rate of women undergoing in vitro fertilization: a retrospective analysis.\u003c/em\u003e Endocrine Connections, 2022. \u003cstrong\u003e11\u003c/strong\u003e(2).\u003c/li\u003e\n\u003cli\u003eThomson, R.L., S. Spedding, and J.D. Buckley, \u003cem\u003eVitamin D in the aetiology and management of polycystic ovary syndrome.\u003c/em\u003e Clinical endocrinology, 2012. \u003cstrong\u003e77\u003c/strong\u003e(3): p. 343-350.\u003c/li\u003e\n\u003cli\u003eJensen, M.B., \u003cem\u003eVitamin D and male reproduction.\u003c/em\u003e Nature Reviews Endocrinology, 2014. \u003cstrong\u003e10\u003c/strong\u003e(3): p. 175-186.\u003c/li\u003e\n\u003cli\u003eCozzolino, M., et al., \u003cem\u003eHow vitamin D level influences in vitro fertilization outcomes: results of a systematic review and meta-analysis.\u003c/em\u003e Fertility and Sterility, 2020. \u003cstrong\u003e114\u003c/strong\u003e(5): p. 1014-1025.\u003c/li\u003e\n\u003cli\u003eGrzeczka, A., et al., \u003cem\u003eRelevance of Vitamin D and Its Deficiency for the Ovarian Follicle and the Oocyte: An Update.\u003c/em\u003e Nutrients, 2022. \u003cstrong\u003e14\u003c/strong\u003e(18): p. 3712.\u003c/li\u003e\n\u003cli\u003eChun, R.F. and C.M. Nielson, \u003cem\u003eFree Vitamin D: concepts, assays, outcomes, and prospects.\u003c/em\u003e Vitamin D, 2018: p. 925-937.\u003c/li\u003e\n\u003cli\u003eAlawad, Z.M., \u003cem\u003eLevel of follicular fluid vitamin D and embryo quality in a sample of Iraqi women undergoing IVF.\u003c/em\u003e Journal of the Faculty of Medicine Baghdad, 2018. \u003cstrong\u003e60\u003c/strong\u003e(4): p. 215-221.\u003c/li\u003e\n\u003cli\u003eArnanz, A., et al., \u003cem\u003eVitamin D in follicular fluid correlates with the euploid status of blastocysts in a vitamin D deficient population.\u003c/em\u003e Frontiers in endocrinology, 2021. \u003cstrong\u003e11\u003c/strong\u003e: p. 609524.\u003c/li\u003e\n\u003cli\u003eMonastra, G., et al., \u003cem\u003eVitamin D: a steroid hormone with progesterone-like activity.\u003c/em\u003e European Review for Medical \u0026amp; Pharmacological Sciences, 2018. \u003cstrong\u003e22\u003c/strong\u003e(8).\u003c/li\u003e\n\u003cli\u003eRudick, B., et al., \u003cem\u003eCharacterizing the influence of vitamin D levels on IVF outcomes.\u003c/em\u003e Human reproduction, 2012. \u003cstrong\u003e27\u003c/strong\u003e(11): p. 3321-3327.\u003c/li\u003e\n\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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Vitamin D, Embryo, In Vitro Fertilization, Top-Quality Embryo","lastPublishedDoi":"10.21203/rs.3.rs-4375279/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4375279/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eIntroduction:\u003c/h2\u003e \u003cp\u003eVitamin D and calcium in follicular fluid play essential roles in modulating steroidogenesis, folliculogenesis, and oocyte quality determination. Both collaborate to produce high-quality embryos (TQE) during in vitro fertilization. This study aimed to compare free 25(OH)D3 and calcium levels in follicular fluid between TQE and non-TQE groups (non-TQE).\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis cross-sectional study included women who underwent IVF (In Vitro Fertilization) procedures at tertiary hospitals in Bandung, Indonesia. Ovarian follicular fluid was collected during the ovum pick-up procedure. Examination of 25(OH)D3 levels, vitamin D-binding protein and calcium in the follicles was performed using an enzyme-linked immunosorbent assay (ELISA). Free 25(OH)D3 levels were calculated using a method of Vermeulen formula.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eA total of 173 samples, consisting of 86 subjects in the TQE group and 87 subjects in the non-TQE group, met the study criteria. There was a significant difference in free 25(OH)D3 follicular fluid levels between the TQE and non-TQE groups (p\u0026thinsp;=\u0026thinsp;0,017), there was no significant difference in calcium levels between the two groups (p\u0026thinsp;=\u0026thinsp;0,805). This study also revealed that there was a significant association between free 25(OH)D3 follicular fluid level and embryo quality (OR 3.05, 95% CI, 1.46\u0026ndash;6.38; p value\u0026thinsp;=\u0026thinsp;0.002), but there was no significant association between follicular fluid calcium and embryo quality (p\u0026thinsp;=\u0026thinsp;0.144 and OR, 1.74 (95% CI 0.82\u0026ndash;3.68)).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThese circumstances allow free 25(OH)D3 and calcium in the follicular fluid to act independently in the processes of steroidogenesis, folliculogenesis, and fertilization.\u003c/p\u003e","manuscriptTitle":"Free 25(Oh)d3 Levels in Follicular Ovarian Fluid Top Quality Embryo is Higher Than Non- Top Quality Embryo in Normoresponders Group","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-23 13:59:16","doi":"10.21203/rs.3.rs-4375279/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-28T02:33:52+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-23T22:53:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"11739260500333005455108336104828232673","date":"2024-06-12T21:10:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-10T15:52:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"98350900764716658866418756545670590298","date":"2024-05-31T09:51:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-30T09:14:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-30T09:12:31+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-05-16T09:08:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-15T04:30:29+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-05-06T08:26:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6042879a-2fa1-4e62-9fce-c72121c853ec","owner":[],"postedDate":"May 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-11-25T16:07:07+00:00","versionOfRecord":{"articleIdentity":"rs-4375279","link":"https://doi.org/10.1038/s41598-024-71769-6","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-11-22 15:57:39","publishedOnDateReadable":"November 22nd, 2024"},"versionCreatedAt":"2024-05-23 13:59:16","video":"","vorDoi":"10.1038/s41598-024-71769-6","vorDoiUrl":"https://doi.org/10.1038/s41598-024-71769-6","workflowStages":[]},"version":"v1","identity":"rs-4375279","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4375279","identity":"rs-4375279","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}