Melatonin treatment results in progression of endometrial thickness in infertile women in IUI cycle

Melatonin treatment results in progression of endometrial thickness in infertile women in IUI cycle | 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 Research Article Melatonin treatment results in progression of endometrial thickness in infertile women in IUI cycle Zohre Azizi, Jan Mohamad Malekzadeh, Parvin Alsadat Eslamnik, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6444765/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Infertility is a disease of the reproductive system, characterized by failure to establish a clinical pregnancy after 12 months of regular and unprotected sexual intercourse. This study was conducted with the aim of investigating the effect of melatonin on the thickness of the endometrium of infertile patients undergoing intrauterine insemination (IUI). Materials and methods This randomized, double-blind, placebo-controlled clinical trial was conducted in Shahid Jalil Infertility Clinic. 94 infertile patients 18–42 years of age undergoing IUI were randomly divided to a daily dose of 6 mg of melatonin or placebo for a 4-week period. Endometrial thickness was measured by transvaginal ultrasound on days 1, 11 and 14 for each patient. Also, the subjects' basic characteristics, including employment status, smoking, pregnancy rate, abortion rate, and live birth rate were recorded. Results The thickness of the endometrium in the group treated with melatonin was significantly different compared to the control group (P < 0.05). In addition, the clinical pregnancy rate in the intervention group receiving melatonin was about 40%, while it was 14% in the control group (P < 0.05). Therefore, it was concluded that the clinical pregnancy rate increases with the increase in endometrial thickness ratio. Conclusions In general, administration of melatonin for 4 weeks in infertile women undergoing IUI cycle had beneficial effects on endometrial thickness, which was associated with better pregnancy outcome. Trial registration EU CT Nr IRCT20221127056633N1 2023,04,03. Registered on 3 April 2023. Infertility melatonin endometrial thickness Figures Figure 1 Introduction Background and rationale {6a} Sterility or infertility is a disease of the reproductive system, which is defined as the absence of pregnancy after 12 months of sexual intercourse, without the use of contraceptive methods ( 1 ). Infertility is a condition with economic, psychological and medical consequences that currently 10–15% of couples suffer from this problem and the factors of men and women are almost equal ( 2 ). Several factors play a role in infertility, which cause defects in reproductive performance in men and women. These factors include genetic and hormonal abnormalities, physical problems, lifestyle factors, environmental hazards and psychological issues. ( 3 ). The most common causes of female infertility are aging of the mother, damage to the fallopian tubes caused by infection, ovulation disorders such as polycystic ovary syndrome, hypothalamus dysfunction, and endometriosis. In addition, it has been estimated that approximately 15–30% of infertility is due to unknown causes, which is defined as unexplained or idiopathic infertility.( 4 ). Many techniques for the treatment of infertility have been introduced, with the oldest one being intrauterine insemination (IUI) as well as artificial insemination; which includes: types of methods in which sperm are introduced into different parts of the female reproductive system ( 5 , 6 ). Intrauterine sperm insemination (IUI) is chosen as the first line of infertility treatment due to its low cost and minimal invasiveness. This method, along with controlled ovarian hyperstimulation (COH), is the most common treatment for couples with unexplained infertility, ovulation disorders, mild endometriosis, and mild male factor infertility.( 7 , 8 ). In general, two important factors in the pregnancy rate of patients that stimulate ovulation and then IUI include: the size of the follicle and the thickness of the endometrium ( 9 ). The endometrium is the innermost layer of the uterus, which is made of two basal and functional layers. The basal layer has a constant thickness during the monthly cycle, but the thickness of the functional layer is constantly changing during the cycle ( 10 , 11 ). After the end of menstruation and the increase of estrogen hormone again, due to the growth and proliferation of cells, the thickness of this layer increases again until it reaches the desired value during implantation; Because at the time of implantation, the fertilized egg places itself in the cells of the functional layer of the endometrium. If this layer is not thick enough, implantation will not take place and the fertilized egg will be discarded ( 12 , 13 ). Among the causes of low endometrial thickness, we can point out reasons such as: poor blood circulation in the body, inappropriate growth rate of the endometrium, decreased estrogen levels, decreased progesterone function, complications due to the use of pregnancy drugs, high stress levels ( 14 – 16 ). Melatonin is an indoleamine and the final secretory product of the pineal gland that is produced by humans and other animals during the night and is regulated by the suprachiasmatic nuclei of the hypothalamus (SCN) and inhibited by light. ( 17 , 18 ). The main producer of this hormone is the pineal gland, and it has also been reported that it is produced in extra-pineal organs that are not regulated by circadian cycles in the digestive system, skin, retina, ovary, and bone marrow. ( 19 , 20 ). As a hormone of darkness, melatonin is considered one of the important components of the body's biological clock regulation system and is responsible for other important functions such as regulation of the immune system, the body's circadian rhythm, oncostatic effects, cardiovascular functions, reproduction and fetal growth. It also has antioxidant, anti-inflammatory and anti-cancer properties ( 21 – 23 ). It is generally believed that the reproductive actions of melatonin are mediated by way of regulating gonadotropin release after effects on hypothalamic monoamine and GnRH and possibly on cAMP andCa2+ -dependent intracellular mechanisms in the hypophysis ( 24 ). Gonadotropin-releasing hormone (GnRH) causes the release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from the pituitary gland, which by controlling the blood level of these two hormones, sexual growth and maturation and sexual actions and characteristics in both sexes, straight or indirectly causes ( 25 ). Melatonin has a progonadotropic effect, i.e. increasing FSH concentration and LH pulses, possibly by inhibiting the inhibitory effects of sex steroids on ovulation. The endometrium or the mucous layer of the uterus is more complex than other parts of the reproductive tract and has simple glands. Estrogen increases the vessels and thickness of the endometrium and stimulates the growth of endometrial glands. FSH stimulates follicle growth and estrogen production by the granulosa cells of the ovarian follicle. Stimulation of the internal follicle cells by LH causes the production of androgen, which can be released through the basement membrane and there, under the effect of FSH, it is converted into estrogen by granulosa cells ( 21 , 26 , 27 ). Because of its antioxidant and anti-inflammatory properties, melatonin is used as a suitable alternative to reduce chronic pelvic pain in women with endometriosis ( 26 ). Therefore, considering the aforementioned effects regarding melatonin and also the lack of finding a study that measures the effects of melatonin during infertility treatments and assisted reproduction, in this research, the effect of this hormone on the thickness of the uterine wall in infertile women with unknown cause was investigated. Material and Methods Trial design {8} This study was registered as a randomized, double-blind, placebo-controlled clinical trial. It was also approved by the ethics committee of Yasuj University of Medical Sciences (code: IR.YUMS.REC.1401.094). Written informed consent was obtained from all participants prior to the intervention. Study setting {9} Ninety-four infertile patients (women aged 42 − 18 years) were assessed for eligibility for infertility treatment by doctors undergoing IUI treatment from Shahid Jalil Infertility Clinic, Yasuj, Iran. The inclusion criteria for patients were: full consent to participate in the study, BMI less than 35, women with endometrial thickness less or equal 5 mm, no history of hospitalization and underlying diseases. The non-inclusion criteria for patients were: severe male factor, simultaneous uterine abnormalities, fallopian tube obstruction, and history of endometriosis, AFC (Antral follicles) less than 7. The exclusion criteria were: creating a complication during the study for the patient, unwillingness to continue the cooperation of the patient. A randomization list was created in Stata software to generate sequences of random numbers, in blocks of random size (2 or 4). This list was prepared by a third person who was not involved in the study design. Both investigators and patients were blinded to treatment or placebo groups. All the sealed and numbered envelopes were opened after completing the basic information and examining the participants, and then the participants were placed in two intervention or control groups. During the first visit and after signing the study consent form, patients’ baseline demographics, menstrual cycle length, last menstrual period and medications were recorded. In order to reach the diagnosis of the causes of infertility, the classic stages of infertility examination include: sperm analysis to diagnose male disorders (based on WHO criteria) and, if necessary, urological consultation, investigation of hypothalamus-pituitary-ovary axis function by tests, LH, FSH, estradiol, prolactin, TSH hormone (progesterone for ovulation factor) on the second or third day of the cycle and other signs of increased androgens and to rule out adrenal causes, the test along with other hormone tests was performed on 5-DHEA in the morning of the second or third day of the menstrual cycle. In order to examine the uterus and tubes, hysterosalpingography was performed in all cases. Intervention description {11a} Treatment procedure After initial evaluations by the physician, the vaginal ultrasound was performed to determine the thickness of the endometrium in the previous cycle, and if the thickness of the endometrium was less than 5 mm, estradiol 2 mg was prescribed to all subjects from the 8th day of menstruation. Also, to induce ovulation two clomiphene citrate 50-mg tablets and one tablet of letrozole were prescribed once a day from cycle days 3 to 7. Furthermore, on the 3rd day of menstruation, a 6-mg melatonin tablet or placebo (manufactured by Yasuj University of Medical Sciences) was given daily until the end of cycle, and then injectable gonadotropin was injected on days 8, 9, and 10. The placebo capsules were matched in color, shape, size, packaging, smell, and taste with the melatonin capsules. Then, 14 days after IUI, β-HCG blood test was performed using an antibody kit to determine the rate of chemical pregnancy ( 28 – 30 ). Statistical methods Statistical methods for primary and secondary outcomes {20a} SPSS version 21 software was used to analyze experimental data. Independent t-test was used to compare the mean of continuous variables following normal distribution before intervention between two groups, and Mann-Whitney's time was used to compare the score of continuous variables following non-normal distribution before and after in each group as mean + standard deviation (SD). Classification variables were presented as numbers and percentages and were analyzed with w2 or Fisher's exact test. A P-value < 0.05 was considered statistically significant. Results In the present study, information on clinical infertility treatments of 94 infertile patients from the age group of 18 to 42 years referring to Shahid Jalil Infertility Clinic was used. 47 patients in the control group receiving placebo and 47 patients in the intervention group receiving melatonin (treatment with 6 mg/day) were assessed. Baseline characteristics of subjects, including job status, smoking, pregnancy, abortion, and live birth rate are shown in Table 1 . We did not observe any significant changes in the baseline characteristics between the two groups, with the exception of pregnancy status which was increased significantly in the intervention subjects, and showed a remarkable change compared to the control group (P < 0.05). Table 1 Baseline characteristics of the control and intervention groups Characteristic Control group (n = 47) Intervention group (n = 47) P value Occupational status n (%) Employed Unemployed 4 (8.5%) 43 (91.5%) 7 (14.9%) 40 (85.1%) P = 0.26 Smoking status Yes No 7(58.3%) 40(48.8%) 5 (41.7%) 42 (51.2%) P = 0.38 Pregnancy Yes No 7 (14.9%) 40 (85.1%) 19 (40.4%) 28 (59.6%) P = 0.005 Abortion status Yes No 2 (28.6%) 5 (71.4%) 1 (5.3%) 18 (94.7%) P = 0.16 Live birth rate Yes No 5 (71.4%) 2 (28.6%) 18 (94.7%) 1 (5.3%) P = 0.17 In Fig. 1 , and Table 2 , the results of endometrial thickness at the beginning, day 11 and day 14 of the study are compared between the two groups. As shown in Table 2 , the comparison of the average thickness of the endometrium of the melatonin receiving group at the beginning and day 14 of the study shows a statistically significant difference (3.28 vs. 7.5 mm, p < 0.0001). Also, the average thickness of the endometrium in the placebo group showed a significant increase at the beginning and on the 14th day of the intervention (3.3 vs. 6.7 p < 0.0001). Nevertheless, the observed difference between the two groups indicates a greater increase in the thickness of the endometrium in the group receiving melatonin compared to the control group, which is statistically significant (p = 0.007). Table 2 Comparison of the average changes in endometrial thickness during the study period between the control and intervention groups Group Statistics P value Group Mean Std. Deviation Mean Rank age (years) Control group (n = 47) 30.77 6.938 49.64 0.44 Intervention group (n = 47) 30.26 5.231 45.36 History of infertility Control 2.872 2.0389 50.30 0.3 Intervention 2.340 1.3875 44.70 Primary endometrial thickness (mm) Control 3.32 1.065 47.91 0.87 Intervention 3.28 1.077 47.09 Endometrial thickness on day 11 (mm) Control 5.340 1.4637 43.27 0.12 Intervention 5.798 1.5834 51.73 Endometrial thickness on day 14 (mm) Control 6.7447 1.55645 40.54 0.012 Intervention 7.5043 1.53112 54.46 Endometrial thickness change (mm) Control 3.4255 1.70032 40.09 0.007 Intervention 4.2277 1.67951 54.91 Discussion Any abnormality of the reproductive system that leads to the failure of pregnancy after one year of unprotected sex is defined as infertility. The etiology of female infertility is diverse, the most common causes of which are ovulation disorders, menstrual disorders, hypothalamus dysfunction, endometriosis, tubal infertility, uterine and cervical causes ( 4 , 31 ). Intrauterine insemination (IUI) is an assisted reproductive technique for couples suffering from infertility ( 6 ). Despite significant progress in infertility treatment techniques, few studies have been made in the field of IUI to explore improvement in endometrial thickness as the mainly used marker as part of standard cycle monitoring of pregnancy rates. In the present study, for the first time, the effect of oral melatonin on endometrial thickness of infertile women undergoing IUI cycle was evaluated. It was observed that the treatment of infertile patients undergoing IUI with melatonin for four weeks led to a significant increase in the thickness of the endometrium and the pregnancy rate. This was consistent with Mokhtari et al.'s study, which concluded that adding melatonin to the treatment cycles of PCOS subjects significantly improved follicle size, pregnancy rate, and mean endometrial thickness during IUI ( 29 ). The endometrium undergoes morphological and functional changes during the menstrual cycle under the concentration of ovarian hormones (estrogen and progesterone), the density of endometrial hormone receptors, and blood flow. In natural menstrual cycle, the functional layer of the endometrium (stromal cells) from the proliferative phase continues to grow with the action of estrogen and progesterone secreted by the corpus luteum to prepare for embryo implantation ( 30 ). According to the results of the study conducted by ke et al., the minimum thickness of the endometrium at the end of the follicular phase for implantation is 7–8 mm ( 32 ). Based on this fact, the thickness of the endometrium less than 7 mm has a negative effect on the pregnancy rate. At present, various treatments have been suggested to maintain optimal endometrial lining such as hormone therapy, drugs to improve blood flow, aspirin, sildenafil, vitamin E, estradiol valerate, surgical treatment, intrauterine perfusions, and stem cell therapy ( 33 , 34 ). But to recommend these treatments, there are still many disagreements about their safety and standardization. Recently, some reports have discussed possible beneficial effects of melatonin on endometrial thickness ( 29 , 35 ). Melatonin is a circadian regulatory hormone that plays an important role in the secretion of reproductive hormones, luteal function, egg quality, ovulation and early embryo development through the regulation of pituitary-hypothalamus-ovary pathways ( 36 , 37 ). It seems that melatonin exerts its actions directly or indirectly through a receptor-mediated event (melatonin receptor type 1 and 2: MT1 and MT2) at the level of the ovary, to modulate steroidogenesis ( 24 ). It has been shown that melatonin, by directly affecting gonadotropes and GnRH of the hypothalamus, causes the secretion of FSH and LH from the pituitary gland. Secretion of LH before ovulation can result in high expression of MTI in granulosa/luteal cells and endometrial stromal cells ( 30 , 38 ). Probably, melatonin could promote the proliferation activity of stromal cells through interaction with MTI receptor and inhibits their apoptosis through signaling pathways. In addition, it is known that FSH stimulates the secretion of progesterone and estrogen from granulosa cells and increases follicle growth. Subsequently, estrogen and progesterone increase the proliferation of stromal cells and thus increase the thickness of the endometrium ( 27 , 39 , 40 ). Conclusion In summary, we report that four weeks of melatonin treatment in infertile women undergoing IUI not only had beneficial effects on endometrial thickness but also significantly improved clinical pregnancy rates in patients whose endometrium was unable to achieve optimal lining. Based on these data, we demonstrated that oral melatonin is a promising and inexpensive treatment option in endometrial optimization, especially in infertile patients undergoing IUI cycles. However, further research with a larger sample size is needed to better evaluate the effects of melatonin on endometrial thickness and pregnancy outcomes in the IUI process. Declarations Acknowledgements: We would like to thank the Yasuj University of Medical Sciences for the supporting of the study. Code Availability IBM SPSS version 25 was used for statistical analyses. Author contributions ZA, JMM, PAE, RV, PG, and SA wrote the manuscript. RV, PG, and SA devised the study concept and design. SA was responsible for overseeing the statistical section. ZA, JMM, PAE, RV, PG, and SA contributed to the study protocol, read and approved the final manuscript. Each author has been sufficiently involved in the work to take public responsibility for appropriate portions of the content. Figures and illustrations were designed and created by ZA and JMM. Funding was obtained by SA. Funding This work is supported by Yasuj University of Medical Sciences. Data availability Data was available. Ethics approval and consent to participate This study has received ethical approval from the Research Ethics Committees of Yasuj University of Medical Sciences (IR.YUMS.REC.1401.094) on 2022-09-07. The clinical trial has been registered at ClinicalTrials.gov with the identifier IRCT20221127056633N1- registered on April 3th, 2023. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Author details 1 Student Research Committee, Yasuj University of Medical Sciences, Yasuj, Iran 2 Department of Nutrition, School of Health, Yasuj University of Medical Sciences, Yasuj, Iran. 3 Department of Gynecology and Obstetrics, Yasuj University of Medical Sciences, Yasuj, Iran. References Obeagu EI, Njar VE, Obeagu GU. Infertility: Prevalence and consequences. Int J Curr Res Chem Pharm Sci. 2023;10(7):43–50. Babakhanzadeh E, Nazari M, Ghasemifar S, Khodadadian A. Some of the factors involved in male infertility: a prospective review. International journal of general medicine. 2020:29–41. Vander Borght M, Wyns C. Fertility and infertility: Definition and epidemiology. Clinical biochemistry. 2018;62:2–10. Bala R, Singh V, Rajender S, Singh K. Environment, lifestyle, and female infertility. Reproductive sciences. 2021;28:617–38. Marschalek J, Egarter C, Vytiska-Binsdorfer E, Obruca A, Campbell J, Harris P, et al. 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Melatonin reduces LH, 17 beta-estradiol and induces differential regulation of sex steroid receptors in reproductive tissues during rat ovulation. Reproductive Biology and Endocrinology. 2011;9:1–9. Yong W, Ma H, Na M, Gao T, Zhang Y, Hao L, et al. Roles of melatonin in the field of reproductive medicine. Biomedicine & Pharmacotherapy. 2021;144:112001. Woo MM, Tai C-J, Kang SK, Nathwani PS, Pang SF, Leung PC. Direct action of melatonin in human granulosa-luteal cells. The Journal of Clinical Endocrinology & Metabolism. 2001;86(10):4789–97. Seymen CM, Sağlam ASY, Elmazoğlu Z, Arık GN, Kaplanoğlu GT. Involvement of endometrial IGF-1R/IGF-1/Bcl-2 pathways in experimental polycystic ovary syndrome: Identification of the regulatory effect of melatonin. Tissue and Cell. 2021;73:101585. Additional Declarations No competing interests reported. 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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-6444765","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":451210752,"identity":"9889550a-6bf6-416e-b862-972e432ae105","order_by":0,"name":"Zohre Azizi","email":"","orcid":"","institution":"Yasuj University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Zohre","middleName":"","lastName":"Azizi","suffix":""},{"id":451210753,"identity":"6eb49658-dc7a-4ba2-8e5a-9c6ae50d885b","order_by":1,"name":"Jan Mohamad Malekzadeh","email":"","orcid":"","institution":"Yasuj University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jan","middleName":"Mohamad","lastName":"Malekzadeh","suffix":""},{"id":451210754,"identity":"1a0e6f21-4cbf-4964-b205-586fbeaf2bfb","order_by":2,"name":"Parvin Alsadat Eslamnik","email":"","orcid":"","institution":"Yasuj University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Parvin","middleName":"Alsadat","lastName":"Eslamnik","suffix":""},{"id":451210755,"identity":"fc78e8d0-4c33-41e5-ac0b-af8de30aab1e","order_by":3,"name":"Razieh Vanda","email":"","orcid":"","institution":"Yasuj University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Razieh","middleName":"","lastName":"Vanda","suffix":""},{"id":451210756,"identity":"f8c28644-5ebb-4c1b-8bf6-670b9f2ebdfe","order_by":4,"name":"Parvin Ghaffari","email":"","orcid":"","institution":"Yasuj University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Parvin","middleName":"","lastName":"Ghaffari","suffix":""},{"id":451210757,"identity":"df2159c9-e685-43d5-a028-8b14992842b5","order_by":5,"name":"Shahintaj Aramesh","email":"data:image/png;base64,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","orcid":"","institution":"Yasuj University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Shahintaj","middleName":"","lastName":"Aramesh","suffix":""}],"badges":[],"createdAt":"2025-04-14 09:53:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6444765/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6444765/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82177189,"identity":"a8ad5767-18a9-49c6-82a4-d78ea33c173c","added_by":"auto","created_at":"2025-05-07 11:19:52","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":37374,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the endometrium thickness in the first, eleventh and fourteenth days in two groups.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6444765/v1/609921ef2d55ecd93428da28.jpeg"},{"id":88545611,"identity":"53e3f44b-4cf0-4e60-8efc-f41266b11b12","added_by":"auto","created_at":"2025-08-07 14:31:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":780849,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6444765/v1/52c809d8-f9e1-4272-abee-d4e61eb8a08a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Melatonin treatment results in progression of endometrial thickness in infertile women in IUI cycle","fulltext":[{"header":"Introduction","content":"\n\u003ch3\u003eBackground and rationale {6a}\u003c/h3\u003e\n\u003cp\u003eSterility or infertility is a disease of the reproductive system, which is defined as the absence of pregnancy after 12 months of sexual intercourse, without the use of contraceptive methods (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Infertility is a condition with economic, psychological and medical consequences that currently 10\u0026ndash;15% of couples suffer from this problem and the factors of men and women are almost equal (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Several factors play a role in infertility, which cause defects in reproductive performance in men and women. These factors include genetic and hormonal abnormalities, physical problems, lifestyle factors, environmental hazards and psychological issues. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). The most common causes of female infertility are aging of the mother, damage to the fallopian tubes caused by infection, ovulation disorders such as polycystic ovary syndrome, hypothalamus dysfunction, and endometriosis. In addition, it has been estimated that approximately 15\u0026ndash;30% of infertility is due to unknown causes, which is defined as unexplained or idiopathic infertility.(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMany techniques for the treatment of infertility have been introduced, with the oldest one being intrauterine insemination (IUI) as well as artificial insemination; which includes: types of methods in which sperm are introduced into different parts of the female reproductive system (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Intrauterine sperm insemination (IUI) is chosen as the first line of infertility treatment due to its low cost and minimal invasiveness. This method, along with controlled ovarian hyperstimulation (COH), is the most common treatment for couples with unexplained infertility, ovulation disorders, mild endometriosis, and mild male factor infertility.(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). In general, two important factors in the pregnancy rate of patients that stimulate ovulation and then IUI include: the size of the follicle and the thickness of the endometrium (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). The endometrium is the innermost layer of the uterus, which is made of two basal and functional layers. The basal layer has a constant thickness during the monthly cycle, but the thickness of the functional layer is constantly changing during the cycle (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). After the end of menstruation and the increase of estrogen hormone again, due to the growth and proliferation of cells, the thickness of this layer increases again until it reaches the desired value during implantation; Because at the time of implantation, the fertilized egg places itself in the cells of the functional layer of the endometrium. If this layer is not thick enough, implantation will not take place and the fertilized egg will be discarded (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Among the causes of low endometrial thickness, we can point out reasons such as: poor blood circulation in the body, inappropriate growth rate of the endometrium, decreased estrogen levels, decreased progesterone function, complications due to the use of pregnancy drugs, high stress levels (\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMelatonin is an indoleamine and the final secretory product of the pineal gland that is produced by humans and other animals during the night and is regulated by the suprachiasmatic nuclei of the hypothalamus (SCN) and inhibited by light. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). The main producer of this hormone is the pineal gland, and it has also been reported that it is produced in extra-pineal organs that are not regulated by circadian cycles in the digestive system, skin, retina, ovary, and bone marrow. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). As a hormone of darkness, melatonin is considered one of the important components of the body's biological clock regulation system and is responsible for other important functions such as regulation of the immune system, the body's circadian rhythm, oncostatic effects, cardiovascular functions, reproduction and fetal growth. It also has antioxidant, anti-inflammatory and anti-cancer properties (\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). It is generally believed that the reproductive actions of melatonin are mediated by way of regulating gonadotropin release after effects on hypothalamic monoamine and GnRH and possibly on cAMP andCa2+ -dependent intracellular mechanisms in the hypophysis (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Gonadotropin-releasing hormone (GnRH) causes the release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from the pituitary gland, which by controlling the blood level of these two hormones, sexual growth and maturation and sexual actions and characteristics in both sexes, straight or indirectly causes (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Melatonin has a progonadotropic effect, i.e. increasing FSH concentration and LH pulses, possibly by inhibiting the inhibitory effects of sex steroids on ovulation. The endometrium or the mucous layer of the uterus is more complex than other parts of the reproductive tract and has simple glands. Estrogen increases the vessels and thickness of the endometrium and stimulates the growth of endometrial glands. FSH stimulates follicle growth and estrogen production by the granulosa cells of the ovarian follicle. Stimulation of the internal follicle cells by LH causes the production of androgen, which can be released through the basement membrane and there, under the effect of FSH, it is converted into estrogen by granulosa cells (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Because of its antioxidant and anti-inflammatory properties, melatonin is used as a suitable alternative to reduce chronic pelvic pain in women with endometriosis (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Therefore, considering the aforementioned effects regarding melatonin and also the lack of finding a study that measures the effects of melatonin during infertility treatments and assisted reproduction, in this research, the effect of this hormone on the thickness of the uterine wall in infertile women with unknown cause was investigated.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eTrial design {8}\u003c/h2\u003e \u003cp\u003eThis study was registered as a randomized, double-blind, placebo-controlled clinical trial. It was also approved by the ethics committee of Yasuj University of Medical Sciences (code: IR.YUMS.REC.1401.094). Written informed consent was obtained from all participants prior to the intervention.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStudy setting {9}\u003c/h3\u003e\n\u003cp\u003eNinety-four infertile patients (women aged 42\u0026thinsp;\u0026minus;\u0026thinsp;18 years) were assessed for eligibility for infertility treatment by doctors undergoing IUI treatment from Shahid Jalil Infertility Clinic, Yasuj, Iran. The inclusion criteria for patients were: full consent to participate in the study, BMI less than 35, women with endometrial thickness less or equal 5 mm, no history of hospitalization and underlying diseases. The non-inclusion criteria for patients were: severe male factor, simultaneous uterine abnormalities, fallopian tube obstruction, and history of endometriosis, AFC (Antral follicles) less than 7. The exclusion criteria were: creating a complication during the study for the patient, unwillingness to continue the cooperation of the patient.\u003c/p\u003e \u003cp\u003eA randomization list was created in Stata software to generate sequences of random numbers, in blocks of random size (2 or 4). This list was prepared by a third person who was not involved in the study design. Both investigators and patients were blinded to treatment or placebo groups. All the sealed and numbered envelopes were opened after completing the basic information and examining the participants, and then the participants were placed in two intervention or control groups.\u003c/p\u003e \u003cp\u003eDuring the first visit and after signing the study consent form, patients\u0026rsquo; baseline demographics, menstrual cycle length, last menstrual period and medications were recorded. In order to reach the diagnosis of the causes of infertility, the classic stages of infertility examination include: sperm analysis to diagnose male disorders (based on WHO criteria) and, if necessary, urological consultation, investigation of hypothalamus-pituitary-ovary axis function by tests, LH, FSH, estradiol, prolactin, TSH hormone (progesterone for ovulation factor) on the second or third day of the cycle and other signs of increased androgens and to rule out adrenal causes, the test along with other hormone tests was performed on 5-DHEA in the morning of the second or third day of the menstrual cycle. In order to examine the uterus and tubes, hysterosalpingography was performed in all cases.\u003c/p\u003e\n\u003ch3\u003eIntervention description {11a}\u003c/h3\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eTreatment procedure\u003c/h2\u003e \u003cp\u003eAfter initial evaluations by the physician, the vaginal ultrasound was performed to determine the thickness of the endometrium in the previous cycle, and if the thickness of the endometrium was less than 5 mm, estradiol 2 mg was prescribed to all subjects from the 8th day of menstruation. Also, to induce ovulation two clomiphene citrate 50-mg tablets and one tablet of letrozole were prescribed once a day from cycle days 3 to 7. Furthermore, on the 3rd day of menstruation, a 6-mg melatonin tablet or placebo (manufactured by Yasuj University of Medical Sciences) was given daily until the end of cycle, and then injectable gonadotropin was injected on days 8, 9, and 10. The placebo capsules were matched in color, shape, size, packaging, smell, and taste with the melatonin capsules. Then, 14 days after IUI, β-HCG blood test was performed using an antibody kit to determine the rate of chemical pregnancy (\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStatistical methods Statistical methods for primary and secondary outcomes {20a}\u003c/h3\u003e\n\u003cp\u003eSPSS version 21 software was used to analyze experimental data. Independent t-test was used to compare the mean of continuous variables following normal distribution before intervention between two groups, and Mann-Whitney's time was used to compare the score of continuous variables following non-normal distribution before and after in each group as mean\u0026thinsp;+\u0026thinsp;standard deviation (SD). Classification variables were presented as numbers and percentages and were analyzed with w2 or Fisher's exact test. A P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIn the present study, information on clinical infertility treatments of 94 infertile patients from the age group of 18 to 42 years referring to Shahid Jalil Infertility Clinic was used. 47 patients in the control group receiving placebo and 47 patients in the intervention group receiving melatonin (treatment with 6 mg/day) were assessed.\u003c/p\u003e \u003cp\u003eBaseline characteristics of subjects, including job status, smoking, pregnancy, abortion, and live birth rate are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. We did not observe any significant changes in the baseline characteristics between the two groups, with the exception of pregnancy status which was increased significantly in the intervention subjects, and showed a remarkable change compared to the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\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\u003eBaseline characteristics of the control and intervention groups\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=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl group (n\u0026thinsp;=\u0026thinsp;47)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIntervention group (n\u0026thinsp;=\u0026thinsp;47)\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\u003e\u003cb\u003eOccupational status n (%)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eEmployed\u003c/p\u003e \u003cp\u003eUnemployed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 (8.5%)\u003c/p\u003e \u003cp\u003e43 (91.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7 (14.9%)\u003c/p\u003e \u003cp\u003e40 (85.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eP\u0026thinsp;=\u0026thinsp;0.26\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSmoking status\u003c/b\u003e\u003c/p\u003e \u003cp\u003eYes\u003c/p\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7(58.3%)\u003c/p\u003e \u003cp\u003e40(48.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 (41.7%)\u003c/p\u003e \u003cp\u003e42 (51.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eP\u0026thinsp;=\u0026thinsp;0.38\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePregnancy\u003c/b\u003e\u003c/p\u003e \u003cp\u003eYes\u003c/p\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7 (14.9%)\u003c/p\u003e \u003cp\u003e40 (85.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19 (40.4%)\u003c/p\u003e \u003cp\u003e28 (59.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eP\u0026thinsp;=\u0026thinsp;0.005\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAbortion status\u003c/b\u003e\u003c/p\u003e \u003cp\u003eYes\u003c/p\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 (28.6%)\u003c/p\u003e \u003cp\u003e5 (71.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (5.3%)\u003c/p\u003e \u003cp\u003e18 (94.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eP\u0026thinsp;=\u0026thinsp;0.16\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLive birth rate\u003c/b\u003e\u003c/p\u003e \u003cp\u003eYes\u003c/p\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 (71.4%)\u003c/p\u003e \u003cp\u003e2 (28.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18 (94.7%)\u003c/p\u003e \u003cp\u003e1 (5.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eP\u0026thinsp;=\u0026thinsp;0.17\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the results of endometrial thickness at the beginning, day 11 and day 14 of the study are compared between the two groups. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the comparison of the average thickness of the endometrium of the melatonin receiving group at the beginning and day 14 of the study shows a statistically significant difference (3.28 vs. 7.5 mm, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Also, the average thickness of the endometrium in the placebo group showed a significant increase at the beginning and on the 14th day of the intervention (3.3 vs. 6.7 p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Nevertheless, the observed difference between the two groups indicates a greater increase in the thickness of the endometrium in the group receiving melatonin compared to the control group, which is statistically significant (p\u0026thinsp;=\u0026thinsp;0.007).\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\u003eComparison of the average changes in endometrial thickness during the study period between the control and intervention groups\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eGroup Statistics\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eStd. Deviation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMean Rank\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eage (years)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl group (n\u0026thinsp;=\u0026thinsp;47)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.938\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e49.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.44\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIntervention group (n\u0026thinsp;=\u0026thinsp;47)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.231\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e45.36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eHistory of infertility\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.872\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.0389\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e50.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIntervention\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.340\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.3875\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e44.70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003ePrimary endometrial thickness (mm)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.065\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e47.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIntervention\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.077\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e47.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eEndometrial thickness on day 11 (mm)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.340\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.4637\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e43.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIntervention\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.798\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.5834\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e51.73\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eEndometrial thickness on day 14 (mm)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.7447\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.55645\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e40.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.012\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIntervention\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.5043\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.53112\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e54.46\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eEndometrial thickness change (mm)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.4255\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.70032\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e40.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.007\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIntervention\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.2277\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.67951\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e54.91\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAny abnormality of the reproductive system that leads to the failure of pregnancy after one year of unprotected sex is defined as infertility. The etiology of female infertility is diverse, the most common causes of which are ovulation disorders, menstrual disorders, hypothalamus dysfunction, endometriosis, tubal infertility, uterine and cervical causes (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Intrauterine insemination (IUI) is an assisted reproductive technique for couples suffering from infertility (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Despite significant progress in infertility treatment techniques, few studies have been made in the field of IUI to explore improvement in endometrial thickness as the mainly used marker as part of standard cycle monitoring of pregnancy rates. In the present study, for the first time, the effect of oral melatonin on endometrial thickness of infertile women undergoing IUI cycle was evaluated. It was observed that the treatment of infertile patients undergoing IUI with melatonin for four weeks led to a significant increase in the thickness of the endometrium and the pregnancy rate. This was consistent with Mokhtari et al.'s study, which concluded that adding melatonin to the treatment cycles of PCOS subjects significantly improved follicle size, pregnancy rate, and mean endometrial thickness during IUI (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe endometrium undergoes morphological and functional changes during the menstrual cycle under the concentration of ovarian hormones (estrogen and progesterone), the density of endometrial hormone receptors, and blood flow. In natural menstrual cycle, the functional layer of the endometrium (stromal cells) from the proliferative phase continues to grow with the action of estrogen and progesterone secreted by the corpus luteum to prepare for embryo implantation (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). According to the results of the study conducted by ke et al., the minimum thickness of the endometrium at the end of the follicular phase for implantation is 7\u0026ndash;8 mm (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Based on this fact, the thickness of the endometrium less than 7 mm has a negative effect on the pregnancy rate. At present, various treatments have been suggested to maintain optimal endometrial lining such as hormone therapy, drugs to improve blood flow, aspirin, sildenafil, vitamin E, estradiol valerate, surgical treatment, intrauterine perfusions, and stem cell therapy (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). But to recommend these treatments, there are still many disagreements about their safety and standardization. Recently, some reports have discussed possible beneficial effects of melatonin on endometrial thickness (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Melatonin is a circadian regulatory hormone that plays an important role in the secretion of reproductive hormones, luteal function, egg quality, ovulation and early embryo development through the regulation of pituitary-hypothalamus-ovary pathways (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). It seems that melatonin exerts its actions directly or indirectly through a receptor-mediated event (melatonin receptor type 1 and 2: MT1 and MT2) at the level of the ovary, to modulate steroidogenesis (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). It has been shown that melatonin, by directly affecting gonadotropes and GnRH of the hypothalamus, causes the secretion of FSH and LH from the pituitary gland. Secretion of LH before ovulation can result in high expression of MTI in granulosa/luteal cells and endometrial stromal cells (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Probably, melatonin could promote the proliferation activity of stromal cells through interaction with MTI receptor and inhibits their apoptosis through signaling pathways. In addition, it is known that FSH stimulates the secretion of progesterone and estrogen from granulosa cells and increases follicle growth. Subsequently, estrogen and progesterone increase the proliferation of stromal cells and thus increase the thickness of the endometrium (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, we report that four weeks of melatonin treatment in infertile women undergoing IUI not only had beneficial effects on endometrial thickness but also significantly improved clinical pregnancy rates in patients whose endometrium was unable to achieve optimal lining. Based on these data, we demonstrated that oral melatonin is a promising and inexpensive treatment option in endometrial optimization, especially in infertile patients undergoing IUI cycles. However, further research with a larger sample size is needed to better evaluate the effects of melatonin on endometrial thickness and pregnancy outcomes in the IUI process.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the Yasuj University of Medical Sciences for the supporting of the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIBM SPSS version 25 was used for statistical analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZA, JMM, PAE, RV, PG, and SA wrote the manuscript. RV, PG, and SA devised the study concept and design. SA was responsible for overseeing the statistical section. ZA, JMM, PAE, RV, PG, and SA contributed to the study protocol, read and approved the final manuscript. Each author has been sufficiently involved in the work to take public responsibility for appropriate portions of the content. Figures and illustrations were designed and created by ZA and JMM. Funding was obtained by SA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is supported by Yasuj University of Medical Sciences.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData was available.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study has received ethical approval from the Research Ethics Committees of Yasuj University of Medical Sciences (IR.YUMS.REC.1401.094) on 2022-09-07. \u0026nbsp;The clinical trial has been registered at ClinicalTrials.gov with the identifier IRCT20221127056633N1- registered on April 3th, 2023.\u0026nbsp;\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\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eStudent Research Committee, Yasuj University of Medical Sciences, Yasuj, Iran\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003eDepartment of Nutrition, School of Health, Yasuj University of Medical Sciences, Yasuj, Iran.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e3\u003c/sup\u003eDepartment of Gynecology and Obstetrics, Yasuj University of Medical Sciences, Yasuj, Iran.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eObeagu EI, Njar VE, Obeagu GU. 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Tissue and Cell. 2021;73:101585.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Infertility, melatonin, endometrial thickness","lastPublishedDoi":"10.21203/rs.3.rs-6444765/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6444765/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eInfertility is a disease of the reproductive system, characterized by failure to establish a clinical pregnancy after 12 months of regular and unprotected sexual intercourse. This study was conducted with the aim of investigating the effect of melatonin on the thickness of the endometrium of infertile patients undergoing intrauterine insemination (IUI).\u003c/p\u003e\u003ch2\u003eMaterials and methods\u003c/h2\u003e \u003cp\u003eThis randomized, double-blind, placebo-controlled clinical trial was conducted in Shahid Jalil Infertility Clinic. 94 infertile patients 18\u0026ndash;42 years of age undergoing IUI were randomly divided to a daily dose of 6 mg of melatonin or placebo for a 4-week period. Endometrial thickness was measured by transvaginal ultrasound on days 1, 11 and 14 for each patient. Also, the subjects' basic characteristics, including employment status, smoking, pregnancy rate, abortion rate, and live birth rate were recorded.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe thickness of the endometrium in the group treated with melatonin was significantly different compared to the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, the clinical pregnancy rate in the intervention group receiving melatonin was about 40%, while it was 14% in the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Therefore, it was concluded that the clinical pregnancy rate increases with the increase in endometrial thickness ratio.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eIn general, administration of melatonin for 4 weeks in infertile women undergoing IUI cycle had beneficial effects on endometrial thickness, which was associated with better pregnancy outcome.\u003c/p\u003e\u003ch2\u003eTrial registration\u003c/h2\u003e \u003cp\u003eEU CT Nr IRCT20221127056633N1 2023,04,03. Registered on 3 April 2023.\u003c/p\u003e","manuscriptTitle":"Melatonin treatment results in progression of endometrial thickness in infertile women in IUI cycle","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-07 11:19:48","doi":"10.21203/rs.3.rs-6444765/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d3fea0a8-f300-4ab7-a0fe-eb1ceb860766","owner":[],"postedDate":"May 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-07T14:23:45+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-07 11:19:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6444765","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6444765","identity":"rs-6444765","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}