Assessment of Gonadal Hormones levels in Iron Deficient Females of Reproductive Age

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Bani-Ahmad, Jood M. Hashim, Alia S. Khawaldeh, Ibrahim A. Al-Odat, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5822617/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 Iron deficiency is a common malnutritional disorder with a wide variety of systemic clinical outcomes, including endocrine dysfunction. This study is a randomized descriptive study that, primarily, aimed to investigate that impacts of iron deficiency on the serum levels of gonadal hormones levels among females on reproductive age. To achieve so, a total of one hundred and twenty-three Female were included and were categorized by the menstrual phase based on obtained information form included subjects. Furthermore, based on their serum iron levels, subjects were subcategorized into iron sufficient (IS) and iron deficient (ID) females. Serum levels of iron, ferritin, Follicle-stimulating factor (FSH), Luteinizing hormone (LH), Progesterone, and estrogen were measured. Results No remarkable change in serum hormones levels during the follicular phase. During midcycle phase, ID subjects had significantly lower LH (17.49 ± 1.14 vs. 45.22 ± 6.31 IU/L, P < 0.001) and higher progesterone (11.2 ± 3.5 nmol/L vs. 1.95 ± 0.4 nmol/L, P < 0.05). Though it was statistically insignificant, the average serum estrogen among ID subjects was 128.4 ± 14.9 IU/L as compared to 202.4 ± 52.5 IU/L among IS females, P = 0.212. Serum LH and progesterone were significantly associated with serum iron levels (0.581, P < 0.005 and − 0.481, P < 0.05, respectively). Among ID females on luteal phase, significantly higher serum levels of estrogen (170.6 ± 9.5 pg/mL vs. 121.1 ± 13.1 pg/mL, P < 0.005) and progesterone (43.9 ± 3.3 nmol/L vs. 28.0 ± 4.1 nmol/L, P < 0.01). Furthermore, they were significantly associated with serum iron (-0.421, P < 0.05 and − 0.400, P < 0.05, respectively). Conclusion our data offers initial insights into the dysregulatory impacts of iron deficiency on the dynamic balance of gonadal hormones via the disruption of the hypothalamic-pituitary-gonadal-axis. Considering the positive feedback interaction, the main findings are the reduced levels of LH and estrogen below the optimal levels, during the luteal phase, in the context of iron deficiency. The reported impaired or delayed LH surge during the midcycle may reveal clinical impacts of iron deficiency on fertility and ability to conceive among females on reproductive age. Further investigation is required to validate our findings on a larger sample size and multiple time-points measurements throughout the menstrual phases of study subjects. Iron Deficiency Follicle-Stimulating Hormone Luteinizing Hormone Progesterone Estrogen Figures Figure 1 Figure 2 Introduction Iron is an vital trace element that is essential for the physiological integrity of various cellular and systemic constituents [ 1 ]. Iron deficiency (ID) is a wide spread health concern that can lead multiple unfavorable clinical outcomes such as anemia, the earliest and the most common clinical presentation of ID [ 13 ]. It is characterized by the lack of mobilizable iron which leads to insufficient availability to satisfy the physiological demands by tissues [ 1 ]. Iron homeostasis significantly contributes to hormonal homeostasis and subsequently normal endocrine functions [ 23 ]. Endocrine dysfunction is linked to iron homeostasis where iron overload is associated with its harmful deposition within endocrine tissues. In the extent of iron overload, adult β-thalassemic females demonstrates gonadal hormones deficiency [ 14 ]. β-thalassemic patients have hypogonadotropic hypogonadism in consequence to defective luteinizing hormone (LH) synthesis in consequence to iron accumulation and deposition in the pituitary gland where follicle-stimulating hormone (FSH) is less vulnerable to such a defect [ 5 , 21 ]. A recent study has demonstrated that iron play a role in the dysregulation of hypothalamic-pituitary-gonadal signaling with deleterious clinical impacts on spermatogenesis and male fertility [ 23 ]. Authors defined an inverse association of serum levels of LH and gonadotropins with serum levels of iron and ferritin, respectively [ 23 ]. In females, 17β-estradiol (E2), the most abundant form of estrogen, is a potential physiological indicator of body iron demands [ 2 , 15 ]. Elevated levels of estrogen have been correlated with an interrupted iron hemostasis by inducing higher iron demand and reduced ferritin levels [ 15 ]. The exact mechanism through which estrogen interferes with iron hemostasis is still not been understood [ 2 ]. To the best of our knowledge, few studies have investigated the influential impact of menstrual cycles and regulatory hormones on iron homeostasis. Most studies focused on males or mixed populations, emphasizing iron overload. Conversely, less studies have investigated the impact of iron bioavailability on the menstrual hormones levels which highlighted iron overload, specifically among thalassemic patients. Considering that iron deficiency is a common malnutrition among females, this study aimed to investigate the impact of reduced iron availability on the serum levels of the menstrual cycle’s regulatory hormones in females of reproductive age. Materials and Methods Subjects: One hundred and twenty-three (N = 123) randomly-selected female of reproductive age (18–40 years old) were recruited and included in the study population. The inclusion criteria included females with no clinical evidence of having chronic inflammatory disease or any a personal or family history of hereditary hematological disorders. Prior to their inclusion, the study was thoroughly explained to all participants after which they approved their inclusion, and an informed written consent was obtained. This study was reviewed for ethical considerations in accordance with the declaration of Helsinki and approved by an institutional review board (Approval number 57/149/2022) Under aseptic conditions, five milliliters of plain blood samples were withdrawn from participants by venipuncture. Blood samples were allowed to clot after which serum samples were obtained by centrifugation at 4500 rpm for five minutes. Serum iron levels were measured in which 60 µg/dL was defined as a cut-off value. Accordingly, study subjects were categorized into iron-sufficient (IS) and iron-deficient (ID) subjects. Methods Serum iron and ferritin levels were measured using automated instruments (Beckman coulter, Europe). Results were presented in microgram per deciliter (µg/dL). Serum levels of FSH, LH, progesterone, and estrogen were measured using automated Cobas 8000 (Roche Diagnostics GmbH, Germany). Statistical Analysis: Statistical analysis was conducted using version 23 of the statistical package for the social sciences (SPSS) (SPSS Inc. Headquarter, Chicago, USA). Descriptive findings were conducted, and results were presented as mean ± standard error mean (SEM). The independent two-tailed student t-test was used for comparative analysis and results were considered significant when the p-value was less than (0.05). Correlation analysis was conducted using parametric Pearson’s (r) correlation analysis. Non-parametric Mann-Whitney U analysis was performed to evaluate distributional differences between study groups. Box and Whiskers analysis and graphics were prepared using GraphPad Prism 9 software (Boston, MA, USA). Results The study included a total of one hundred and twenty-three (N = 123) adult female of active reproductive age, with an average age of 27.0 ± 6.3 (range 18 to 40) years old. Based on the normal range of serum iron levels in healthy adult, which is 60.0–158.0 µg/dL (10.6–28.3 µmol/L), the participants were categorized into iron-sufficient (IS) and iron-deficient (ID). Included females were further subcategorized based on their phase of the menstrual cycle at the time of their inclusion. A total of seventy females were ID among whom 40.0% (n = 28) were in the follicular phase, 25.7% (n = 18) were in the midcycle (ovulation) phase and 34.3% (n = 24) were in the luteal phase. In contrast, fifty-three females were IS among whom 43.4% (n = 23) were in their follicular phase, 28.3% (n = 15) were in the midcycle phase and 28.3% (n = 15) were in the luteal phase. As demonstrated in Table 1 , there were significant differences in the serum iron levels between ID and IS subjects across three phases of menstrual cycle (P < 0.001). However, no significant differences in the serum levels of ferritin between ID and IS subjects across the three phases. Additionally, a significant correlation was found between the serum levels of iron and ferritin levels only among subjects who were in the follicular phase. Yet, no association was evident among subjects who were in the midcycle or luteal phases. Table 1 Demographic features and the average serum concentration of iron, ferritin and gonadal hormones among iron deficient (ID) and iron sufficient (IS) subjects of the three phases of their menstrual cycle : results are presented as mean ± standard error of means and comparison is considered significant when the corresponding P value is less than 0.05. Follicular (n = 51) Midcycle (n = 33) Luteal (n = 39) IS (n = 23) ID (n = 28) P IS (n = 15) ID (n = 18) P IS (n = 15) ID (n = 24) P Age (Years) 25.1 ± 1.0 26.4 ± 1.1 0.385 27.6 ± 1.8 29.2 ± 2.2 0.590 30.2 ± 1.7 26.1 ± 1.2 0.054 Iron ( µg/dL) 79.3 ± 4.9 28.0 ± 2.1 < 0.001 84.5 ± 4.8 29.0 ± 3.3 < 0.001 100.1 ± 12.2 31.7 ± 2.1 < 0.001 Ferritin (ng/mL) 27.2 ± 2.9 20.2 ± 3.6 0.148 39.15 ± 7.5 24.2 ± 7.6 0.174 27.5 ± 4.4 20.5 ± 5.7 0.406 FSH (IU/L) 5.56 ± 0.29 6.31 ± 0.37 0.130 16.09 ± 5.38 8.43 ± 1.14 0.212 3.54 ± 0.65 3.25 ± 0.30 0.645 LH (IU/L) 6.49 ± 0.65 6.24 ± 0.61 0.777 45.22 ± 6.31 17.49 ± 1.14 < 0.001 5.25 ± 1.39 5.53 ± 0.68 0.843 Prog (nmol/L) 0.94 ± 0.15 0.60 ± 0.06 0.04 1.95 ± 0.36 11.21 ± 3.48 0.024 27.98 ± 4.12 43.89 ± 3.33 0.004 17β-E (pg/mL) 49.2 ± 3.4 63.4 ± 7.0 0.909 202.4 ± 52.5 128.4 ± 14.9 0.212 121.1 ± 13.1 170.6 ± 9.5 0.49 n: number of subjects, IS: Iron sufficient, ID: Iron Deficient. FSH: Follicle-Stimulating Hormone, LH: Luteinizing Hormone, Prog: Progesterone, 17β-E: 17β-Estradiol. Regarding the alteration in gonadal hormones in the context of iron deficiency, comparative analysis was conducted, and results are illustrated in Table 1 . Regarding pituitary hormones (FSH and LH), no significant differences were observed between ID and IS subjects who were in their follicular and luteal phase (P > 0.05). However, in the midcycle phase, ID subjects had significantly reduced average serum LH levels ( p 0.05). As far as ovarian hormones are concerned, ID females, in the follicular phase, had significant slight reduction in the average serum progesterone level in (p < 0.05) as compared to significant higher average level during the midcycle phase (p < 0.05) and luteal phases (P 0.05) when compared to corresponding average levels in IS subjects. To further investigate the impact of serum iron levels to the dysregulation of gonadal hormones among the three phases of menstrual cycles, correlation analysis was conducted. The data depicted in Table 2 . Even though serum ferritin is an iron biomarker, significantly moderate association between serum iron level and serum ferritin level was evident only among subjects who were in their follicular phase (p < 0.05). To elucidate the relationship between serum iron levels and gonadal hormones levels, correlation analysis indicated that serum progesterone levels were significantly correlated with serum iron levels during the three phases of menstrual cycles. Furthermore, there was significant direct association between serum iron and LH levels among subjects in their midcycle phase (p < 0.005) and a significant inverse association with serum estrogen levels among subjects in their luteal phase (p < 0.05). Table 2 Parametric Pearson’s correlation coefficient (ρ) analysis of serum iron association with serum ferritin and serum levels of measured gonadal hormones : Correlation is considered significant when P value is less than 0.05. Menstrual phase Correlation coefficient Ferritin FSH LH Progesterone Estrogen Follicular ρ 0.317 -0.186 0.106 0.409 -0.237 P value 0.021 * 0.210 0.455 0.003 ** 0.101 Midcycle ρ 0.192 0.167 0.581 -0.456 0.130 P value 0.359 0.436 0.002 ** 0.029 * 0.585 Luteal ρ 0.072 0.133 0.131 -0.421 -0.400 P value 0.672 0.460 0.467 0.013 * 0.032 * FSH: Follicle-stimulating Hormones, LH: Luteinizing hormones To show the distribution of serum gonadal hormones levels through the quartiles among study subjects, box plot analysis was conducted, and results are depicted in Fig. 1 and Fig. 2 . The most stupendous observation in Fig. 1 is the narrower interquartile range of LH levels during the midcycle phase of ID subjects, compared to wider interquartile range with negatively skewed distribution in the corresponding IS subjects. No observed differences in LH distribution between ID and IS during the follicular and luteal phases. Regarding FSH, no significant distributional differences was observed between ID and IS subjects across the three menstrual phases. As far as ovarian hormones are concerned, Fig. 2 shows that ID subjects exhibited different interquartile ranges of progesterone during the midcycle and luteal phase, with medians falling outside the corresponding interquartile ranges of IS subjects. Regarding estrogen distribution, the interquartile range of estrogen levels was noticeably narrower among ID subjects compared to IS subjects. However, the median estrogen concentration in ID subjects falls within the interquartile range of the corresponding concentrations in IS subjects, suggesting that the differences are less likely to be significant. To further assess the statistical significance of differences in the distribution of serum gonadal hormones levels between ID and IS subjects across the menstrual phases, a nonparametric Mann-Whitney U test was conducted, and results are presented in Table 3 . ID subjects, in the midcycle phase, had significantly different distribution of serum LH levels when compared to IS subjects (P < 0.05). Additionally, Additionally, there were significant differences in the distribution of serum progesterone and estrogen levels between ID and IS subjects in the luteal phase with p values of < 0.05 and < 0.01, respectively. Table 3 Non-Parametric Matt-Whitney U analysis of the serum levels of gonadal hormones throughout the three phases of menstrual cycle among iron deficient (ID) and iron sufficient (IS) subjects. Analysis is considered significant when P value is less than 0.05. Menstrual phase Mann-Whitney Gonadal Hormone FSH LH Prog. Estrogen Follicular Mean Rank IS 21.4 27.2 30.0 21.59 ID 26.1 25.9 22.8 27.78 Mann-Whitney U 218.0 320.5 231.0 222.0 Z -1.178 -0.311 -1.723 -1.508 P value 0.239 0.756 0.085 0.132 Midcycle Mean Rank IS 14.5 16.1 10.1 12.00 ID 10.1 9.7 14.1 9.5 Mann-Whitney U 45.0 38.0 43.0 36.0 Z -1.536 -2.176 -1.461 -0.926 P value 0.134 0.03 * 0.169 0.355 Luteal Mean Rank IS 16.7 15.3 11.8 9.3 ID 17.2 18.0 20.6 18.5 Mann-Whitney U 122.5 105.0 63.0 36.0 Z -0.131 -0.786 -2.487 -2.832 P value 0.897 0.432 0.013 * 0.005 * FSH: Follicle-stimulating Hormones, LH: Luteinizing hormones, Prog.: Progesterone Discussion The regulation of the menstrual cycle relies on a series of complex interactions between hypothalamic–pituitary-ovarian axis hormones [ 3 , 16 ]. The dynamic balance of gonadal hormones drives the rhythmicity of the three menstrual phases [ 10 ]. Therefore, assessing the levels of these hormones is essential to understand their dynamic equilibrium and their impact on folliculogenesis and female fertility. In the current study, we evaluated the amplitude and fluctuation pattern of gonadal hormones across the three phases of menstrual cycle in the context of iron deficiency. Inflammation is a prominent feature of the ovulatory phase of menstruation that aids in tissue breakdown and bleeding characteristic of menstruation [ 11 ]. Ferritin is an acute phase reactant and a key mediator of inflammation [ 18 ]. This may explain the insignificant correlation between serum iron and serum ferritin levels among study subjects in their midcycle and luteal phases. Therefore, in this study, serum iron level was used as a biomarker to define iron bioavailability and to categorize study subjects as IS and ID. The insignificant difference in the serum ferritin levels between ID and IS subjects, supports the appropriateness of the utilization of serum iron levels as a determinant of iron bioavailability among study subjects. Compared with IS subjects, ID subjects have shown similar patterns of fluctuation of gonadal hormones secretions across the three phases that coincide with dysregulation of their circulatory levels at the midcycle and luteal phases. The rhythmicity is consistent among the two study groups and the amplitudes of their average serum levels were within the normal physiological reference range. During the follicular phase, gradual and slowly elevated levels of FSH and LH is responsible for follicular growth within the ovaries [ 3 ]. At this stage, LH and FSH levels also promote ovarian thecal and granulosa cells to synthesize progesterone and estrogen hormones, respectively which lead to follicular growth and development [ 3 ]. Except for progesterone, the lack of insignificant differences in the average serum levels of FSH, LH and estrogens between ID and IS subjects are indicatives that iron deficiency does not affect the basal levels of pituitary and ovarian gonadal hormones during the follicular phase of menstrual cycle. The negative feedback associated with the release of ovarian progesterone and estrogen hormones is essential to maintain low levels of these four gonadal hormones [ 17 ]. However, as the follicles develop, estrogen level rises due to positive feedback from induced estrogen, reaching a critical level of 200 pg/dL that is required to promote the LH surge [ 26 ]. in turn, LH surge is the key determinant of ovulation within hours [ 26 , 29 ]. Herein, our results demonstrate that the majority of IS subjects have reached the required critical estrogen level. This is compared to ID subjects who demonstrated markedly reduced levels where the majority have failed to achieve the required level to initiate ovulation. . Serum iron level was significantly associated with both LH and progesterone levels but not with estrogen level. In that regard, it is noteworthy that ovarian estrogen synthesis is controlled by the two-cell two-gonadotropin hypothesis [ 17 ]. According to this hypothesis, in addition to ovarian gransulosa cells and FSH, theca cells and LH are also indirectly responsible for the regulation of estrogen synthesis through producing androstenedione that is up taken by granulosa cells to be converted into estrogen under the enzymatic activity of Aromatase enzyme [ 29 ]. Aromatase is a cytochrome P450 enzyme that contains a heme group, and its catalytic activity requires an interaction with the electron transport system through a mechanism that is not fully understood yet [ 4 ]. It has been demonstrated that decreased aromatase activity is associated with decreased estradiol synthesis in addition to insufficient expression of LH receptors [ 6 ]. These findings indicate that iron is essential for the catalytic activity of aromatase and therefore may explain the significantly inverse association of serum iron with serum progesterone and LH levels but not estrogen levels. LH surge is the main event in the dynamics of ovulation [ 7 ]. Classically, LH surge is a single peak event of 2.5–14.8-folds induced LH levels over a duration of 3–5 days duration [ 22 ]. However, normal variability in the pattern, amplitude and duration are familiar manifestations even among heathy fertile women [ 9 ]. The dysregulated LH surge may reflect a clinical significance that can be represented by either delayed and/or failed ovulation [ 26 ]. While several studies have proposed different absolute cutoffs of LH surge, it has been demonstrated that a cutoff level of 40 IU/L is an optimal cutoff that is highly predictive of ovulation timing [ 19 ]. When comparing the average serum LH levels among ID and IS subjects, we have reported that most ID subjects had delayed or failed achievement of the optimal cutoff to initiate ovulation. Accordingly, it may be reasonable to claim that ID may contribute to impaired and defective ovulation and subsequently increase the risk of infertility issues among ID females. Following ovulation, mature follicular cells reprogramming results in its terminal differentiation into corpus luteum [ 28 ]. Primarily, corpus luteum is responsible for progesterone synthesis to induce endometrium and support gestation [ 20 ]. although LH surge induces corpus luteum and progesterone synthesis, progesterone augments its secretion [ 25 ]. Furthermore, it has been proposed that, even during normal menstrual cycle, progesterone secretion is complex and pulsatile where it’s levels may oscillate between 5 to 40 ng/ml over short period of time [ 12 , 27 ]. Therefore, it is difficult to interpret and compare progesterone levels during the luteal phase through single time-point measurement. However, the significant association of progesterone and estrogens as well as their significant association to serum iron levels may indicate that iron contributes to the amplitudes and possible pulsating progesterone and subsequently estrogen secretion following luteolysis (regression) of corpus luteum. In that regard, it has been postulated that high progesterone levels, that correlate to high estrogen levels, may not have any adverse clinical indications [ 24 ]. In conclusion, the data in this study emphasize the dysregulatory effect of iron deficiency on the hormonal balance of gonadal hormones and the subsequent disruption of the hypothalamic-pituitary-gonadal-axis. The primary finding is that ID is associated with a marked reduction in the magnitude of LH surge, which may reveal clinical consequences among females. Our study has limitations that mainly include the small sample size and the single-time points measurements of gonadal hormones. Considering that LH surge is variable between and within females [ 9 ], multiple-time points measurements should enable tracking the fluctuation in the pattern, duration and the amplitude of gonadal hormones throughout the menstrual cycle in ID and IS subjects. It is important to note that the timing of sampling is a limitation, as there is no established method for accurately estimating the timing of ovulation and monitoring the progression of the menstrual phases [ 8 , 19 ]. Declarations Ethics approval and consent to participate: This study was conducted in accordance with the ethical declaration of Helsinki of medical studies. The study was reviewed and approved by an institutional review board (approval number 57/149/2022) and the deanship of research (approval number 212/2022) at Jordan University of Science and Technology. Prior to their inclusion, the study was thoroughly explained to all participants verbally. Following their approval of inclusion, all participants provided informed written consent in that regard. Consent for publication: Not applicable Availability of data and material: All data generated or analyzed during the study are available from the corresponding author on reasonable request. Competing interests: The authors declare that they have no competing interests. Funding Sources: The authors disclosed receipt of financial support for this research by the Deanship of Scientific Research at Jadara University. Authors’ Contribution: M.B.A. is the principal investigator who contributed to the study of conceptualization, funding acquisition and statistical analysis. M.B.A., J.M.H., A.S.K., I.A.A., S.A.A., and A.A.A. have contributed to results interpretation and draft writing. H.A.N. has contributed to data curation and experimental analysis. M.B.A. drafted the original manuscript, and all co-authors read, edited and approved the final version of the manuscript. Acknowledgment The corresponding author is thankful to Jordan University of Science and Technology for their sponsorship of his sabbatical leave during which this study was conducted. Thanks are extended to the deanship of research at Jadara university, for their financial support of the study. References Abbaspour N, Hurrell R, Kelishadi R (2014) Review on iron and its importance for human health. J Res Med Sci 19:164-174. 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(1988) The corpus luteum: determinants of progesterone secretion in the normal menstrual cycle. Obstet Gynecol 71:659-666. Stocco C, Telleria C, Gibori G (2007) The molecular control of corpus luteum formation, function, and regression. Endocr Rev 28:117-149 DOI: 10.1210/er.2006-0022. Xu XL, Huang ZY, Yu K et al. (2022) Estrogen Biosynthesis and Signal Transduction in Ovarian Disease. Front Endocrinol (Lausanne) 13:827032 DOI: 10.3389/fendo.2022.827032. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-5822617","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":401990852,"identity":"89b7afa1-1445-41a7-b7c3-088f03eb84a0","order_by":0,"name":"Mohammad A. Bani-Ahmad","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIiWNgGAWjYFACHiA2YEgAkoYPJCBCBhBRIrQYGwC1SBCphQGsxQykHKoFD9BtP3vwc0GBXZ5u++FtFZZtdXUM7M3bJBhkbHBqMTuTlyw9wyC52OxMWtkNybbDEgw8x8qARBpuLQdyDKR5DJgTtx3IMQNqOSDBIJEDdCHPYdxazr8x/s1jUJ+47fwbswLJtjoJBvk3BLTcyDED2nI4cRuQwSDZxgy0hYeQlndp1jwGx4FanhVLSJw7LNnGk1ZskYDPL+dzD9/m+VMNdFjyxs8SZXX8/OyHN9742IM7xFAAMyhS2ECsxB7idDAwfoAzfxCpZRSMglEwCkYCAADME08MZMnzKwAAAABJRU5ErkJggg==","orcid":"","institution":"Jordan University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Mohammad","middleName":"A.","lastName":"Bani-Ahmad","suffix":""},{"id":401990853,"identity":"89de0403-9950-4640-912b-307ac0663361","order_by":1,"name":"Jood M. Hashim","email":"","orcid":"","institution":"Jordan University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Jood","middleName":"M.","lastName":"Hashim","suffix":""},{"id":401990854,"identity":"4b2be08d-3651-4563-ab30-94c2ad4fe04b","order_by":2,"name":"Alia S. Khawaldeh","email":"","orcid":"","institution":"Jadara University","correspondingAuthor":false,"prefix":"","firstName":"Alia","middleName":"S.","lastName":"Khawaldeh","suffix":""},{"id":401990855,"identity":"05395a6d-69db-44ff-8845-9e8bed6fb751","order_by":3,"name":"Ibrahim A. Al-Odat","email":"","orcid":"","institution":"Jadara University","correspondingAuthor":false,"prefix":"","firstName":"Ibrahim","middleName":"A.","lastName":"Al-Odat","suffix":""},{"id":401990856,"identity":"68c18bff-e1e4-45fd-94eb-547c2e9efd44","order_by":4,"name":"Saad A. Al-Fawaeir","email":"","orcid":"","institution":"Jadara University","correspondingAuthor":false,"prefix":"","firstName":"Saad","middleName":"A.","lastName":"Al-Fawaeir","suffix":""},{"id":401990857,"identity":"1d883998-e402-4196-82a8-b965573c2a04","order_by":5,"name":"Abdelraheem A. Bani Ahmad","email":"","orcid":"","institution":"Al Al-bayt University","correspondingAuthor":false,"prefix":"","firstName":"Abdelraheem","middleName":"A. Bani","lastName":"Ahmad","suffix":""},{"id":401990858,"identity":"5a1c00fe-0ded-4bf9-952a-2e9eb3dd8a1a","order_by":6,"name":"Hiba A. Nazazleh","email":"","orcid":"","institution":"Medlab","correspondingAuthor":false,"prefix":"","firstName":"Hiba","middleName":"A.","lastName":"Nazazleh","suffix":""}],"badges":[],"createdAt":"2025-01-13 21:08:11","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-5822617/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5822617/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":73933335,"identity":"705c5a4b-6334-47bc-98eb-1a12f6e90ffd","added_by":"auto","created_at":"2025-01-16 06:28:04","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1107466,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBox plot analysis of the distribution of the serum levels of pituitary gonadal hormones (A) FSH and (B) LH among iron-sufficient (white box) and iron-deficient (gray box) subjects on their follicular, midcycle and luteal phase of the menstrual cycle.\u003c/strong\u003e The boxes define the interquartile range while the lower and the upper whiskers define the minimum and maximum concentration per corresponding group/phase and hormone. Within the box, the middle line defines the median and the plus sign (+) defines the means of the corresponding hormone concentrations within group. The upper and lower dotted horizontal lines represent the normal range of corresponding hormones concentration during the specified phase of menstrual cycle.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5822617/v1/0baf3e55c9efaa348cbc3702.jpg"},{"id":73933610,"identity":"df3e0dc3-7cde-42c7-9c64-ff3895ca696a","added_by":"auto","created_at":"2025-01-16 06:36:04","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1214139,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBox plot analysis of the distribution of the serum levels of ovarian gonadal hormones (A) Progesterone and (B) 17-β Estradiol among iron-sufficient (white box) and iron-deficient (gray box) subjects on their follicular, midcycle and luteal phase of the menstrual cycle. \u003c/strong\u003eThe boxes define the interquartile range while the lower and the upper whiskers define the minimum and maximum concentration per corresponding group/phase and hormone. Within the box, the middle line defines the median and the plus sign (+) defines the means of the corresponding hormone concentrations within group. The upper and lower dotted horizontal lines represent the normal range of corresponding hormones concentration during the specified phase of menstrual cycle.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5822617/v1/e576d510c630de7209400e9b.jpg"},{"id":80081597,"identity":"05f282eb-30f6-4e0b-a296-acbcd4b30765","added_by":"auto","created_at":"2025-04-07 15:53:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3459063,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5822617/v1/c0a0d306-8e12-4004-900e-d062cb115a02.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Assessment of Gonadal Hormones levels in Iron Deficient Females of Reproductive Age","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIron is an vital trace element that is essential for the physiological integrity of various cellular and systemic constituents [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Iron deficiency (ID) is a wide spread health concern that can lead multiple unfavorable clinical outcomes such as anemia, the earliest and the most common clinical presentation of ID [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. It is characterized by the lack of mobilizable iron which leads to insufficient availability to satisfy the physiological demands by tissues [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Iron homeostasis significantly contributes to hormonal homeostasis and subsequently normal endocrine functions [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEndocrine dysfunction is linked to iron homeostasis where iron overload is associated with its harmful deposition within endocrine tissues. In the extent of iron overload, adult β-thalassemic females demonstrates gonadal hormones deficiency [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. β-thalassemic patients have hypogonadotropic hypogonadism in consequence to defective luteinizing hormone (LH) synthesis in consequence to iron accumulation and deposition in the pituitary gland where follicle-stimulating hormone (FSH) is less vulnerable to such a defect [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. A recent study has demonstrated that iron play a role in the dysregulation of hypothalamic-pituitary-gonadal signaling with deleterious clinical impacts on spermatogenesis and male fertility [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Authors defined an inverse association of serum levels of LH and gonadotropins with serum levels of iron and ferritin, respectively [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In females, 17β-estradiol (E2), the most abundant form of estrogen, is a potential physiological indicator of body iron demands [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Elevated levels of estrogen have been correlated with an interrupted iron hemostasis by inducing higher iron demand and reduced ferritin levels [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The exact mechanism through which estrogen interferes with iron hemostasis is still not been understood [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo the best of our knowledge, few studies have investigated the influential impact of menstrual cycles and regulatory hormones on iron homeostasis. Most studies focused on males or mixed populations, emphasizing iron overload. Conversely, less studies have investigated the impact of iron bioavailability on the menstrual hormones levels which highlighted iron overload, specifically among thalassemic patients. Considering that iron deficiency is a common malnutrition among females, this study aimed to investigate the impact of reduced iron availability on the serum levels of the menstrual cycle\u0026rsquo;s regulatory hormones in females of reproductive age.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSubjects:\u003c/h2\u003e \u003cp\u003eOne hundred and twenty-three (N\u0026thinsp;=\u0026thinsp;123) randomly-selected female of reproductive age (18\u0026ndash;40 years old) were recruited and included in the study population. The inclusion criteria included females with no clinical evidence of having chronic inflammatory disease or any a personal or family history of hereditary hematological disorders. Prior to their inclusion, the study was thoroughly explained to all participants after which they approved their inclusion, and an informed written consent was obtained. This study was reviewed for ethical considerations in accordance with the declaration of Helsinki and approved by an institutional review board (Approval number 57/149/2022)\u003c/p\u003e \u003cp\u003eUnder aseptic conditions, five milliliters of plain blood samples were withdrawn from participants by venipuncture. Blood samples were allowed to clot after which serum samples were obtained by centrifugation at 4500 rpm for five minutes. Serum iron levels were measured in which 60 \u0026micro;g/dL was defined as a cut-off value. Accordingly, study subjects were categorized into iron-sufficient (IS) and iron-deficient (ID) subjects.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMethods\u003c/h3\u003e\n\u003cp\u003eSerum iron and ferritin levels were measured using automated instruments (Beckman coulter, Europe). Results were presented in microgram per deciliter (\u0026micro;g/dL).\u003c/p\u003e \u003cp\u003eSerum levels of FSH, LH, progesterone, and estrogen were measured using automated Cobas 8000 (Roche Diagnostics GmbH, Germany).\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis:\u003c/h2\u003e \u003cp\u003eStatistical analysis was conducted using version 23 of the statistical package for the social sciences (SPSS) (SPSS Inc. Headquarter, Chicago, USA). Descriptive findings were conducted, and results were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error mean (SEM). The independent two-tailed student t-test was used for comparative analysis and results were considered significant when the p-value was less than (0.05). Correlation analysis was conducted using parametric Pearson\u0026rsquo;s (r) correlation analysis. Non-parametric Mann-Whitney U analysis was performed to evaluate distributional differences between study groups. Box and Whiskers analysis and graphics were prepared using GraphPad Prism 9 software (Boston, MA, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe study included a total of one hundred and twenty-three (N\u0026thinsp;=\u0026thinsp;123) adult female of active reproductive age, with an average age of 27.0\u0026thinsp;\u0026plusmn;\u0026thinsp;6.3 (range 18 to 40) years old. Based on the normal range of serum iron levels in healthy adult, which is 60.0\u0026ndash;158.0 \u0026micro;g/dL (10.6\u0026ndash;28.3 \u0026micro;mol/L), the participants were categorized into iron-sufficient (IS) and iron-deficient (ID). Included females were further subcategorized based on their phase of the menstrual cycle at the time of their inclusion. A total of seventy females were ID among whom 40.0% (n\u0026thinsp;=\u0026thinsp;28) were in the follicular phase, 25.7% (n\u0026thinsp;=\u0026thinsp;18) were in the midcycle (ovulation) phase and 34.3% (n\u0026thinsp;=\u0026thinsp;24) were in the luteal phase. In contrast, fifty-three females were IS among whom 43.4% (n\u0026thinsp;=\u0026thinsp;23) were in their follicular phase, 28.3% (n\u0026thinsp;=\u0026thinsp;15) were in the midcycle phase and 28.3% (n\u0026thinsp;=\u0026thinsp;15) were in the luteal phase.\u003c/p\u003e \u003cp\u003eAs demonstrated in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, there were significant differences in the serum iron levels between ID and IS subjects across three phases of menstrual cycle (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). However, no significant differences in the serum levels of ferritin between ID and IS subjects across the three phases. Additionally, a significant correlation was found between the serum levels of iron and ferritin levels only among subjects who were in the follicular phase. Yet, no association was evident among subjects who were in the midcycle or luteal phases.\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\u003e\u003cb\u003eDemographic features and the average serum concentration of iron, ferritin and gonadal hormones among iron deficient (ID) and iron sufficient (IS) subjects of the three phases of their menstrual cycle\u003c/b\u003e: results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of means and comparison is considered significant when the corresponding P value is less than 0.05.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eFollicular (n\u0026thinsp;=\u0026thinsp;51)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eMidcycle (n\u0026thinsp;=\u0026thinsp;33)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003eLuteal (n\u0026thinsp;=\u0026thinsp;39)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIS\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;23)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eID\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;28)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIS\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;15)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eID\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;18)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIS\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;15)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eID\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;24)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eP\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\u003eAge\u003c/b\u003e\u003c/p\u003e \u003cp\u003e(Years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e25.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e26.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.385\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e27.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e29.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.590\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e30.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e26.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.054\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIron\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(\u003c/b\u003e\u0026micro;g/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e79.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e28.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e84.5\u0026thinsp;\u0026plusmn;\u0026thinsp;4.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e29.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e100.1\u0026thinsp;\u0026plusmn;\u0026thinsp;12.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e31.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFerritin\u003c/b\u003e\u003c/p\u003e \u003cp\u003e(ng/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e27.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e20.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.148\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e39.15\u0026thinsp;\u0026plusmn;\u0026thinsp;7.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e24.2\u0026thinsp;\u0026plusmn;\u0026thinsp;7.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.174\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e27.5\u0026thinsp;\u0026plusmn;\u0026thinsp;4.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e20.5\u0026thinsp;\u0026plusmn;\u0026thinsp;5.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.406\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFSH\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(IU/L)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e5.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e6.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.130\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e16.09\u0026thinsp;\u0026plusmn;\u0026thinsp;5.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e8.43\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.212\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e3.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e3.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.645\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLH\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(IU/L)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e6.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e6.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.777\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e45.22\u0026thinsp;\u0026plusmn;\u0026thinsp;6.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e17.49\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e5.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e5.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.843\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eProg\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(nmol/L)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.04\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e11.21\u0026thinsp;\u0026plusmn;\u0026thinsp;3.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e0.024\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e27.98\u0026thinsp;\u0026plusmn;\u0026thinsp;4.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e43.89\u0026thinsp;\u0026plusmn;\u0026thinsp;3.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e0.004\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e17β-E\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(pg/mL)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e49.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e63.4\u0026thinsp;\u0026plusmn;\u0026thinsp;7.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.909\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e202.4\u0026thinsp;\u0026plusmn;\u0026thinsp;52.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e128.4\u0026thinsp;\u0026plusmn;\u0026thinsp;14.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.212\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e121.1\u0026thinsp;\u0026plusmn;\u0026thinsp;13.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e170.6\u0026thinsp;\u0026plusmn;\u0026thinsp;9.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"10\"\u003en: number of subjects, IS: Iron sufficient, ID: Iron Deficient. FSH: Follicle-Stimulating Hormone, LH: Luteinizing Hormone, Prog: Progesterone, 17β-E: 17β-Estradiol.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eRegarding the alteration in gonadal hormones in the context of iron deficiency, comparative analysis was conducted, and results are illustrated in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Regarding pituitary hormones (FSH and LH), no significant differences were observed between ID and IS subjects who were in their follicular and luteal phase (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). However, in the midcycle phase, ID subjects had significantly reduced average serum LH levels (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and insignificant difference in the average serum levels of FSH (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). As far as ovarian hormones are concerned, ID females, in the follicular phase, had significant slight reduction in the average serum progesterone level in (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) as compared to significant higher average level during the midcycle phase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and luteal phases (P\u0026thinsp;\u0026lt;\u0026thinsp;0.005). Alternatively, ID females had higher average levels of 17β- estradiol during the three phases of menstrual cycle albeit statistically insignificant (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) when compared to corresponding average levels in IS subjects.\u003c/p\u003e \u003cp\u003eTo further investigate the impact of serum iron levels to the dysregulation of gonadal hormones among the three phases of menstrual cycles, correlation analysis was conducted. The data depicted in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Even though serum ferritin is an iron biomarker, significantly moderate association between serum iron level and serum ferritin level was evident only among subjects who were in their follicular phase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). To elucidate the relationship between serum iron levels and gonadal hormones levels, correlation analysis indicated that serum progesterone levels were significantly correlated with serum iron levels during the three phases of menstrual cycles. Furthermore, there was significant direct association between serum iron and LH levels among subjects in their midcycle phase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.005) and a significant inverse association with serum estrogen levels among subjects in their luteal phase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\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\u003e\u003cb\u003eParametric Pearson\u0026rsquo;s correlation coefficient (ρ) analysis of serum iron association with serum ferritin and serum levels of measured gonadal hormones\u003c/b\u003e: Correlation is considered significant when P value is less than 0.05.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\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=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMenstrual phase\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCorrelation coefficient\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFerritin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFSH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eProgesterone\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eEstrogen\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eFollicular\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eρ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.317\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.186\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.409\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.237\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.021\u003c/b\u003e\u003csup\u003e\u003cb\u003e*\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.210\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.455\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.003\u003c/b\u003e\u003csup\u003e\u003cb\u003e**\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.101\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eMidcycle\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eρ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.192\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.167\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.581\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e-0.456\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.130\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.359\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.436\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.002\u003c/b\u003e\u003csup\u003e\u003cb\u003e**\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.029\u003c/b\u003e\u003csup\u003e\u003cb\u003e*\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.585\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eLuteal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eρ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.072\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.133\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e-0.421\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e-0.400\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.672\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.460\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.467\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.013\u003c/b\u003e\u003csup\u003e\u003cb\u003e*\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e0.032\u003c/b\u003e\u003csup\u003e\u003cb\u003e*\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eFSH: Follicle-stimulating Hormones, LH: Luteinizing hormones\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo show the distribution of serum gonadal hormones levels through the quartiles among study subjects, box plot analysis was conducted, and results are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The most stupendous observation in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e is the narrower interquartile range of LH levels during the midcycle phase of ID subjects, compared to wider interquartile range with negatively skewed distribution in the corresponding IS subjects. No observed differences in LH distribution between ID and IS during the follicular and luteal phases. Regarding FSH, no significant distributional differences was observed between ID and IS subjects across the three menstrual phases. As far as ovarian hormones are concerned, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows that ID subjects exhibited different interquartile ranges of progesterone during the midcycle and luteal phase, with medians falling outside the corresponding interquartile ranges of IS subjects. Regarding estrogen distribution, the interquartile range of estrogen levels was noticeably narrower among ID subjects compared to IS subjects. However, the median estrogen concentration in ID subjects falls within the interquartile range of the corresponding concentrations in IS subjects, suggesting that the differences are less likely to be significant.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further assess the statistical significance of differences in the distribution of serum gonadal hormones levels between ID and IS subjects across the menstrual phases, a nonparametric Mann-Whitney U test was conducted, and results are presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. ID subjects, in the midcycle phase, had significantly different distribution of serum LH levels when compared to IS subjects (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Additionally, Additionally, there were significant differences in the distribution of serum progesterone and estrogen levels between ID and IS subjects in the luteal phase with \u003cem\u003ep\u003c/em\u003e values of \u0026lt;\u0026thinsp;0.05 and \u0026lt;\u0026thinsp;0.01, respectively.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eNon-Parametric Matt-Whitney U analysis of the serum levels of gonadal hormones throughout the three phases of menstrual cycle among iron deficient (ID) and iron sufficient (IS) subjects.\u003c/b\u003e Analysis is considered significant when P value is less than 0.05.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMenstrual phase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c3\" namest=\"c2\" rowspan=\"2\"\u003e \u003cp\u003eMann-Whitney\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c7\" namest=\"c4\"\u003e \u003cp\u003eGonadal Hormone\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFSH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eProg.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eEstrogen\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e\u003cb\u003eFollicular\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMean Rank\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e27.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e30.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e21.59\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eID\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e22.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e27.78\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eMann-Whitney U\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e218.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e320.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e231.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e222.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eZ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-1.178\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-0.311\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-1.723\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-1.508\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.239\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.756\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.085\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.132\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e\u003cb\u003eMidcycle\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMean Rank\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eID\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eMann-Whitney U\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e45.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e38.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e43.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e36.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eZ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-1.536\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-2.176\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-1.461\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-0.926\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.134\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.03 \u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.169\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.355\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e\u003cb\u003eLuteal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMean Rank\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eID\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e20.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e18.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eMann-Whitney U\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e122.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e105.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e63.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e36.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eZ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-0.131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-0.786\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-2.487\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-2.832\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.897\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.432\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.013 \u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.005 \u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eFSH: Follicle-stimulating Hormones, LH: Luteinizing hormones, Prog.: Progesterone\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe regulation of the menstrual cycle relies on a series of complex interactions between hypothalamic\u0026ndash;pituitary-ovarian axis hormones [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The dynamic balance of gonadal hormones drives the rhythmicity of the three menstrual phases [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Therefore, assessing the levels of these hormones is essential to understand their dynamic equilibrium and their impact on folliculogenesis and female fertility. In the current study, we evaluated the amplitude and fluctuation pattern of gonadal hormones across the three phases of menstrual cycle in the context of iron deficiency.\u003c/p\u003e \u003cp\u003eInflammation is a prominent feature of the ovulatory phase of menstruation that aids in tissue breakdown and bleeding characteristic of menstruation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Ferritin is an acute phase reactant and a key mediator of inflammation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This may explain the insignificant correlation between serum iron and serum ferritin levels among study subjects in their midcycle and luteal phases. Therefore, in this study, serum iron level was used as a biomarker to define iron bioavailability and to categorize study subjects as IS and ID. The insignificant difference in the serum ferritin levels between ID and IS subjects, supports the appropriateness of the utilization of serum iron levels as a determinant of iron bioavailability among study subjects.\u003c/p\u003e \u003cp\u003eCompared with IS subjects, ID subjects have shown similar patterns of fluctuation of gonadal hormones secretions across the three phases that coincide with dysregulation of their circulatory levels at the midcycle and luteal phases. The rhythmicity is consistent among the two study groups and the amplitudes of their average serum levels were within the normal physiological reference range. During the follicular phase, gradual and slowly elevated levels of FSH and LH is responsible for follicular growth within the ovaries [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. At this stage, LH and FSH levels also promote ovarian thecal and granulosa cells to synthesize progesterone and estrogen hormones, respectively which lead to follicular growth and development [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Except for progesterone, the lack of insignificant differences in the average serum levels of FSH, LH and estrogens between ID and IS subjects are indicatives that iron deficiency does not affect the basal levels of pituitary and ovarian gonadal hormones during the follicular phase of menstrual cycle.\u003c/p\u003e \u003cp\u003eThe negative feedback associated with the release of ovarian progesterone and estrogen hormones is essential to maintain low levels of these four gonadal hormones [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, as the follicles develop, estrogen level rises due to positive feedback from induced estrogen, reaching a critical level of 200 pg/dL that is required to promote the LH surge [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. in turn, LH surge is the key determinant of ovulation within hours [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Herein, our results demonstrate that the majority of IS subjects have reached the required critical estrogen level. This is compared to ID subjects who demonstrated markedly reduced levels where the majority have failed to achieve the required level to initiate ovulation. .\u003c/p\u003e \u003cp\u003eSerum iron level was significantly associated with both LH and progesterone levels but not with estrogen level. In that regard, it is noteworthy that ovarian estrogen synthesis is controlled by the two-cell two-gonadotropin hypothesis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. According to this hypothesis, in addition to ovarian gransulosa cells and FSH, theca cells and LH are also indirectly responsible for the regulation of estrogen synthesis through producing androstenedione that is up taken by granulosa cells to be converted into estrogen under the enzymatic activity of Aromatase enzyme [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Aromatase is a cytochrome P450 enzyme that contains a heme group, and its catalytic activity requires an interaction with the electron transport system through a mechanism that is not fully understood yet [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. It has been demonstrated that decreased aromatase activity is associated with decreased estradiol synthesis in addition to insufficient expression of LH receptors [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. These findings indicate that iron is essential for the catalytic activity of aromatase and therefore may explain the significantly inverse association of serum iron with serum progesterone and LH levels but not estrogen levels.\u003c/p\u003e \u003cp\u003eLH surge is the main event in the dynamics of ovulation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Classically, LH surge is a single peak event of 2.5\u0026ndash;14.8-folds induced LH levels over a duration of 3\u0026ndash;5 days duration [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, normal variability in the pattern, amplitude and duration are familiar manifestations even among heathy fertile women [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The dysregulated LH surge may reflect a clinical significance that can be represented by either delayed and/or failed ovulation [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. While several studies have proposed different absolute cutoffs of LH surge, it has been demonstrated that a cutoff level of 40 IU/L is an optimal cutoff that is highly predictive of ovulation timing [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. When comparing the average serum LH levels among ID and IS subjects, we have reported that most ID subjects had delayed or failed achievement of the optimal cutoff to initiate ovulation. Accordingly, it may be reasonable to claim that ID may contribute to impaired and defective ovulation and subsequently increase the risk of infertility issues among ID females.\u003c/p\u003e \u003cp\u003eFollowing ovulation, mature follicular cells reprogramming results in its terminal differentiation into corpus luteum [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Primarily, corpus luteum is responsible for progesterone synthesis to induce endometrium and support gestation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. although LH surge induces corpus luteum and progesterone synthesis, progesterone augments its secretion [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Furthermore, it has been proposed that, even during normal menstrual cycle, progesterone secretion is complex and pulsatile where it\u0026rsquo;s levels may oscillate between 5 to 40 ng/ml over short period of time [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Therefore, it is difficult to interpret and compare progesterone levels during the luteal phase through single time-point measurement. However, the significant association of progesterone and estrogens as well as their significant association to serum iron levels may indicate that iron contributes to the amplitudes and possible pulsating progesterone and subsequently estrogen secretion following luteolysis (regression) of corpus luteum. In that regard, it has been postulated that high progesterone levels, that correlate to high estrogen levels, may not have any adverse clinical indications [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn conclusion, the data in this study emphasize the dysregulatory effect of iron deficiency on the hormonal balance of gonadal hormones and the subsequent disruption of the hypothalamic-pituitary-gonadal-axis. The primary finding is that ID is associated with a marked reduction in the magnitude of LH surge, which may reveal clinical consequences among females. Our study has limitations that mainly include the small sample size and the single-time points measurements of gonadal hormones. Considering that LH surge is variable between and within females [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], multiple-time points measurements should enable tracking the fluctuation in the pattern, duration and the amplitude of gonadal hormones throughout the menstrual cycle in ID and IS subjects. It is important to note that the timing of sampling is a limitation, as there is no established method for accurately estimating the timing of ovulation and monitoring the progression of the menstrual phases [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/strong\u003eThis study was conducted in accordance with the ethical declaration of Helsinki of medical studies. The study was reviewed and approved by an institutional review board (approval number 57/149/2022) and the deanship of research (approval number 212/2022) at Jordan University of Science and Technology. Prior to their inclusion, the study was thoroughly explained to all participants verbally. Following their approval of inclusion, all participants provided informed written consent in that regard.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eConsent for publication:\u003c/strong\u003e Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material:\u003c/strong\u003e All data generated or analyzed during the study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Sources:\u0026nbsp;\u003c/strong\u003eThe authors disclosed receipt of financial support for this research by the Deanship of Scientific Research at Jadara University. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contribution:\u0026nbsp;\u003c/strong\u003eM.B.A. is the principal investigator who contributed to the study of conceptualization, funding acquisition and statistical analysis. M.B.A., J.M.H., A.S.K., I.A.A., S.A.A., and A.A.A. have contributed to results interpretation and draft writing. H.A.N. has contributed to data curation and experimental analysis. M.B.A. drafted the original manuscript, and all co-authors read, edited and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe corresponding author is thankful to Jordan University of Science and Technology for their sponsorship of his sabbatical leave during which this study was conducted. Thanks are extended to the deanship of research at Jadara university, for their financial support of the study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbbaspour N, Hurrell R, Kelishadi R (2014) Review on iron and its importance for human health. J Res Med Sci 19:164-174.\u003c/li\u003e\n\u003cli\u003eBadenhorst CE, Forsyth AK, Govus AD (2022) A contemporary understanding of iron metabolism in active premenopausal females. Front Sports Act Living 4:903937 DOI: 10.3389/fspor.2022.903937.\u003c/li\u003e\n\u003cli\u003eChabbert Buffet N, Djakoure C, Maitre SC et al. (1998) Regulation of the human menstrual cycle. Front Neuroendocrinol 19:151-186 DOI: 10.1006/frne.1998.0167.\u003c/li\u003e\n\u003cli\u003eChan HJ, Petrossian K, Chen S (2016) Structural and functional characterization of aromatase, estrogen receptor, and their genes in endocrine-responsive and -resistant breast cancer cells. J Steroid Biochem Mol Biol 161:73-83 DOI: 10.1016/j.jsbmb.2015.07.018.\u003c/li\u003e\n\u003cli\u003eChatterjee R, Katz M (2000) Reversible hypogonadotrophic hypogonadism in sexually infantile male thalassaemic patients with transfusional iron overload. Clin Endocrinol (Oxf) 53:33-42 DOI: 10.1046/j.1365-2265.2000.00962.x.\u003c/li\u003e\n\u003cli\u003eCouse JF, Yates MM, Deroo BJ et al. (2005) Estrogen receptor-beta is critical to granulosa cell differentiation and the ovulatory response to gonadotropins. Endocrinology 146:3247-3262 DOI: 10.1210/en.2005-0213.\u003c/li\u003e\n\u003cli\u003eDemir A, Hero M, Alfthan H et al. (2022) Identification of the LH surge by measuring intact and total immunoreactivity in urine for prediction of ovulation time. Hormones (Athens) 21:413-420 DOI: 10.1007/s42000-022-00368-9.\u003c/li\u003e\n\u003cli\u003eDemir A, Hero M, Holopainen E et al. (2022) Quantification of urinary total luteinizing hormone immunoreactivity may improve the prediction of ovulation time. Front Endocrinol (Lausanne) 13:903831 DOI: 10.3389/fendo.2022.903831.\u003c/li\u003e\n\u003cli\u003eDireito A, Bailly S, Mariani A et al. (2013) Relationships between the luteinizing hormone surge and other characteristics of the menstrual cycle in normally ovulating women. Fertil Steril 99:279-285 e273 DOI: 10.1016/j.fertnstert.2012.08.047.\u003c/li\u003e\n\u003cli\u003eDraper CF, Duisters K, Weger B et al. (2018) Menstrual cycle rhythmicity: metabolic patterns in healthy women. Sci Rep 8:14568 DOI: 10.1038/s41598-018-32647-0.\u003c/li\u003e\n\u003cli\u003eEvans J, Salamonsen LA (2012) Inflammation, leukocytes and menstruation. Rev Endocr Metab Disord 13:277-288 DOI: 10.1007/s11154-012-9223-7.\u003c/li\u003e\n\u003cli\u003eFilicori M, Butler JP, Crowley WF, Jr. (1984) Neuroendocrine regulation of the corpus luteum in the human. Evidence for pulsatile progesterone secretion. J Clin Invest 73:1638-1647 DOI: 10.1172/JCI111370.\u003c/li\u003e\n\u003cli\u003eGanz T (2012) Macrophages and systemic iron homeostasis. J Innate Immun 4:446-453 DOI: 10.1159/000336423.\u003c/li\u003e\n\u003cli\u003eHagag AA, Badraia IM, Elfarargy MS et al. (2016) Gonadal Hormones in Adolescent Females with beta-Thalassemia in Relation to Iron Load. Endocr Metab Immune Disord Drug Targets 16:148-153 DOI: 10.2174/1871530316666160506150516.\u003c/li\u003e\n\u003cli\u003eHamad M, Bajbouj K, Taneera J (2020) The Case for an Estrogen-iron Axis in Health and Disease. Exp Clin Endocrinol Diabetes 128:270-277 DOI: 10.1055/a-0885-1677.\u003c/li\u003e\n\u003cli\u003eHerbison AE (2020) A simple model of estrous cycle negative and positive feedback regulation of GnRH secretion. Front Neuroendocrinol 57:100837 DOI: 10.1016/j.yfrne.2020.100837.\u003c/li\u003e\n\u003cli\u003eHillier SG, Whitelaw PF, Smyth CD (1994) Follicular oestrogen synthesis: the \u0026apos;two-cell, two-gonadotrophin\u0026apos; model revisited. Mol Cell Endocrinol 100:51-54 DOI: 10.1016/0303-7207(94)90278-x.\u003c/li\u003e\n\u003cli\u003eKernan KF, Carcillo JA (2017) Hyperferritinemia and inflammation. Int Immunol 29:401-409 DOI: 10.1093/intimm/dxx031.\u003c/li\u003e\n\u003cli\u003eMaman E, Adashi EY, Baum M et al. (2023) Prediction of ovulation: new insight into an old challenge. Sci Rep 13:20003 DOI: 10.1038/s41598-023-47241-2.\u003c/li\u003e\n\u003cli\u003eNiswender GD, Juengel JL, Silva PJ et al. (2000) Mechanisms controlling the function and life span of the corpus luteum. Physiol Rev 80:1-29 DOI: 10.1152/physrev.2000.80.1.1.\u003c/li\u003e\n\u003cli\u003ePapadimas J, Mandala E, Pados G et al. (1996) Pituitary-testicular axis in men with beta-thalassaemia major. Hum Reprod 11:1900-1904 DOI: 10.1093/oxfordjournals.humrep.a019515.\u003c/li\u003e\n\u003cli\u003ePark SJ, Goldsmith LT, Skurnick JH et al. (2007) Characteristics of the urinary luteinizing hormone surge in young ovulatory women. Fertil Steril 88:684-690 DOI: 10.1016/j.fertnstert.2007.01.045.\u003c/li\u003e\n\u003cli\u003eRastegar Panah M, Jarvi K, Lo K et al. (2024) Biomarkers of Iron Are Associated with Anterior-Pituitary-Produced Reproductive Hormones in Men with Infertility. Nutrients 16DOI: 10.3390/nu16020290.\u003c/li\u003e\n\u003cli\u003eRequena A, Cruz M, Bosch E et al. (2014) High progesterone levels in women with high ovarian response do not affect clinical outcomes: a retrospective cohort study. Reprod Biol Endocrinol 12:69 DOI: 10.1186/1477-7827-12-69.\u003c/li\u003e\n\u003cli\u003eRothchild I (1996) The corpus luteum revisited: are the paradoxical effects of RU486 a clue to how progesterone stimulates its own secretion? Biol Reprod 55:1-4 DOI: 10.1095/biolreprod55.1.1.\u003c/li\u003e\n\u003cli\u003eShoham Z, Schacter M, Loumaye E et al. (1995) The luteinizing hormone surge--the final stage in ovulation induction: modern aspects of ovulation triggering. Fertil Steril 64:237-251 DOI: 10.1016/s0015-0282(16)57717-6.\u003c/li\u003e\n\u003cli\u003eSoules MR, Clifton DK, Steiner RA et al. (1988) The corpus luteum: determinants of progesterone secretion in the normal menstrual cycle. Obstet Gynecol 71:659-666.\u003c/li\u003e\n\u003cli\u003eStocco C, Telleria C, Gibori G (2007) The molecular control of corpus luteum formation, function, and regression. Endocr Rev 28:117-149 DOI: 10.1210/er.2006-0022.\u003c/li\u003e\n\u003cli\u003eXu XL, Huang ZY, Yu K et al. (2022) Estrogen Biosynthesis and Signal Transduction in Ovarian Disease. Front Endocrinol (Lausanne) 13:827032 DOI: 10.3389/fendo.2022.827032.\u003c/li\u003e\n\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":"Iron Deficiency, Follicle-Stimulating Hormone, Luteinizing Hormone, Progesterone, Estrogen","lastPublishedDoi":"10.21203/rs.3.rs-5822617/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5822617/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eIron deficiency is a common malnutritional disorder with a wide variety of systemic clinical outcomes, including endocrine dysfunction. This study is a randomized descriptive study that, primarily, aimed to investigate that impacts of iron deficiency on the serum levels of gonadal hormones levels among females on reproductive age. To achieve so, a total of one hundred and twenty-three Female were included and were categorized by the menstrual phase based on obtained information form included subjects. Furthermore, based on their serum iron levels, subjects were subcategorized into iron sufficient (IS) and iron deficient (ID) females. Serum levels of iron, ferritin, Follicle-stimulating factor (FSH), Luteinizing hormone (LH), Progesterone, and estrogen were measured.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eNo remarkable change in serum hormones levels during the follicular phase. During midcycle phase, ID subjects had significantly lower LH (17.49\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14 vs. 45.22\u0026thinsp;\u0026plusmn;\u0026thinsp;6.31 IU/L, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and higher progesterone (11.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5 nmol/L vs. 1.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 nmol/L, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Though it was statistically insignificant, the average serum estrogen among ID subjects was 128.4\u0026thinsp;\u0026plusmn;\u0026thinsp;14.9 IU/L as compared to 202.4\u0026thinsp;\u0026plusmn;\u0026thinsp;52.5 IU/L among IS females, P\u0026thinsp;=\u0026thinsp;0.212. Serum LH and progesterone were significantly associated with serum iron levels (0.581, P\u0026thinsp;\u0026lt;\u0026thinsp;0.005 and \u0026minus;\u0026thinsp;0.481, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, respectively). Among ID females on luteal phase, significantly higher serum levels of estrogen (170.6\u0026thinsp;\u0026plusmn;\u0026thinsp;9.5 pg/mL vs. 121.1\u0026thinsp;\u0026plusmn;\u0026thinsp;13.1 pg/mL, P\u0026thinsp;\u0026lt;\u0026thinsp;0.005) and progesterone (43.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3 nmol/L vs. 28.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1 nmol/L, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Furthermore, they were significantly associated with serum iron (-0.421, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and \u0026minus;\u0026thinsp;0.400, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, respectively).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eour data offers initial insights into the dysregulatory impacts of iron deficiency on the dynamic balance of gonadal hormones via the disruption of the hypothalamic-pituitary-gonadal-axis. Considering the positive feedback interaction, the main findings are the reduced levels of LH and estrogen below the optimal levels, during the luteal phase, in the context of iron deficiency. The reported impaired or delayed LH surge during the midcycle may reveal clinical impacts of iron deficiency on fertility and ability to conceive among females on reproductive age. Further investigation is required to validate our findings on a larger sample size and multiple time-points measurements throughout the menstrual phases of study subjects.\u003c/p\u003e","manuscriptTitle":"Assessment of Gonadal Hormones levels in Iron Deficient Females of Reproductive Age","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-16 06:27:59","doi":"10.21203/rs.3.rs-5822617/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":"ededd956-f7a0-48a6-addc-8b1471dc80d1","owner":[],"postedDate":"January 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-07T15:53:13+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-16 06:27:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5822617","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5822617","identity":"rs-5822617","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}