Assessment of ovarian reserve in adenomyosis and ovarian endometriosis patients via 3D Transvaginal Ultrasound with AMH, FSH, and Inhibin

Assessment of ovarian reserve in adenomyosis and ovarian endometriosis patients via 3D Transvaginal Ultrasound with AMH, FSH, and Inhibin | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Assessment of ovarian reserve in adenomyosis and ovarian endometriosis patients via 3D Transvaginal Ultrasound with AMH, FSH, and Inhibin Fengrong Yang, Qin Qin, Futian Yan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7345581/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 This study aims to evaluate the effect of adenomyosis (AM) and ovarian endometriosis (OEM) on ovarian reserve in infertile patients by combining three-dimensional vaginal ultrasound and AMH, FSH and inhibition levels. From March 2023 to May 2025, 69 patients with infertility in our hospital were collected, including 21 patients as the AM group, 18 patients as the OEM group and 30 healthy patients as the healthy group. The AMH, FSH, inhibition and AFC levels were collected for each patient. AMH in the AM group and OEM group was lower than that in the healthy group (P < 0.001). The FSH and inhibin levels in the AM group were lower than those in the healthy group (P < 0.05). AFC in the healthy group was significantly higher than that in the OEM group, and higher in the OEM group than in the AM group (P < 0.05). There is a moderate positive correlation between AMH and AFC (P < 0.05). Both AM and OEM can reduce ovarian reserve. AM can reduce the ovarian reserve indicators AMH, FSH, inhibition and AFC of infertile patients, whereas OEM can reduce AMH and AFC. AFC and AMH are the best indexes to evaluate ovarian reserve. Health sciences/Diseases Health sciences/Endocrinology Health sciences/Medical research adenomyosis ovarian endometriosis Anti-Müllerian Hormone Antral Follicle Count Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Adenomyosis (AM) and ovarian endometriosis (OEM) are common gynecological diseases, which are characterized by the presence of endometrioid glands and stroma in the myometrium as well as outside the uterus [ 1 ]. Although they are benign diseases, they seriously affect the quality of life of patients. In addition to its clinical manifestations of dysmenorrhea and menorrhagia, a number of studies have shown that AM and OEM are also closely related to infertility [ 2 – 4 ]. Decreased ovarian reserve is one of the common causes of infertility [ 5 , 6 ]. The detection indexes of ovarian reserve include follicle-stimulating hormone (FSH), anti-Müllerian hormone (AMH), and antral follicle count (AFC). The serum level of AMH is correlated with AFC [ 7 , 8 ]. Transvaginal ultrasound imaging is capable of identifying follicles with a diameter of at least 2 mm, making it a simple and highly reproducible technique. A number of studies have demonstrated that ultrasound-measured AFC can be used to evaluate ovarian reserve function [ 9 – 11 ]. There are few studies on the evaluation of ovarian reserve function in AM and OEM via ultrasound combined with AMH and FSH levels. Therefore, this study aimed to evaluate the diagnostic value of transvaginal ultrasound imaging in ovarian reserve function of AM and OEM patients. Methods Study Population A total of 69 female patients who visited our hospital for infertility from March 2023 to May 2025 and received ultrasound examination were included in this study. All patients underwent transvaginal two-dimensional and three-dimensional ultrasound examination. Inclusion criteria were: (1) married women, aged 20-42 years; (2) no history of anti-estrogen medications; (3) no history of pelvic surgery. Exclusion criteria were: (1) history of pelvic surgery; (2) patients with recent use of hormonal medicine; (3) patients with bilateral fallopian tube obstruction; (4) Pregnancy; (5) Lack of clinical data. According to the results of ultrasound examination, the patients were divided into three groups: healthy group (30 cases), AM group (21 cases) and OEM group (18 cases). This study complied with the ethical principles of the Declaration of Helsinki and was granted a waiver for informed consent and ethics approval by the Ethics Committee of Guangyuan Central Hospital. Diagnostic Criteria The ultrasound diagnosis of AM was based on the diagnostic consensus on AM published by the International Morphological Uterus Sonographic Assessment (MUSA) group [12]. The ultrasonic diagnosis of AM should meet the following two or more (Fig 1): (1) uniform enlargement of the uterus; (2) fan-shaped shadows; (3) asymmetric thickening of the anterior and posterior walls of the myometrium; (4) myometrial cyst; (5) hyperechoic island; (6) subendometrial echoic linear and bud-like objects; (7) cross-lesion blood vessels; (8) irregular JZ bands. The inclusion criteria of OEM patients should meet the following conditions [13]: (1) Infertility; (2) Chronic pelvic pain; (3) OEM diagnosed by ultrasound or magnetic resonance imaging (MRI) (Fig 2); (4) Tumor marker CA125 < 200 IU /mL; (5) Exclude other malignant signs. Equipment and Procedures In this study, two ultrasound systems were used: the Resona R9S (Mindray Medical Company, Shenzhen, China) equipped with a three-dimensional probe (DE10-3wu) and the Voluson E8 (GE Healthcare, Austria) equipped with a two-dimensional probe (IC5-9-D) and a three-dimensional probe (RIC5-9-D). Two-dimensional and three-dimensional vaginal ultrasonography were performed on the 3rd to 5th day of the menstrual cycle. The early follicular phase of the menstrual cycle was selected to reduce the impact of menstrual cycle fluctuations and the possibility of identifying ovarian cysts or corpus luteum as antral follicles [14]. Patients were informed to be in the lithotomy position after emptying the bladder. The condom was placed over the ultrasound probe before inserting into the vagina. First, two-dimensional examination was performed to evaluate the size, shape and internal echo characteristics of the uterus and ovary. According to the ultrasonic manifestations, they were divided into healthy group, AM group and OEM group. Then, the maximum diameter section of the ovary was selected in the two-dimensional scanning mode, and three-dimensional ultrasound examination was performed. The number of follicles in the bilateral ovaries was detected, and the average value was calculated via built-in automatic measurement software (Fig 3). At the same time, manual adjustments were used to reduce the measurement deviation. Blood Biochemical Testing 5 ml fasting venous blood samples were collected from all participants in the early follicular phase of the menstrual cycle. The levels of AMH, FSH and inhibin in serum were determined by enzyme-linked immunosorbent assay. Statistical Analysis SPSS 23.0 software was used for data analysis. Data conforming to the normal distribution are expressed as mean ± standard deviation ( ±s). The parameters among the three groups were compared by one-way analysis of variance (ANOVA), and the differences between groups were compared by post-hoc t-tests. Pearson correlation analysis was applied to analyse the correlation between serum levels and AFC. The correlation coefficient (r) indicated a strong correlation between 0.7 and 1.0, a moderate correlation between 0.4 and 0.7, and a weak correlation between 0 and 0.4. A two-tailed P-value <0.05 was considered statistically significant. Results Tab 1 Comparison of age among the three groups Group Healthy group AM Group OEM Group P AGE 29.63±3.82 31.29±2.22 30.89±3.09 P＞0.05 * Significantly different, p < 0.05 Relationship between blood biochemical and ultrasonographic examination results Pearson correlation showed that AFC and AMH were moderately positively correlated (r=0.539, P < 0.001). There were no statistically significant correlations among other indicators. Discussion The results of our study showed that there were significant differences in AFC among the three groups. The healthy group exhibited significantly higher AFC than the OEM group, while the OEM group showed higher AFC than the AM group. Both AM and OEM groups had lower AMH levels than the healthy group. The AM group showed reduced FSH and inhibin levels compared to the healthy group. A moderate positive correlation was observed between AMH and AFC. AM and OEM are common reasons for infertility [ 15 ]. There are multiple theories to explain the pathogenesis of AM, but they all suggest that it is related to the expression of local estrogen, and the increase of local inflammatory factors and cells [ 16 – 18 ]. The pathogenesis of OEM is similar to that of AM, which is related to local estrogen expression and pro-inflammatory environment [ 19 ]. The local inflammatory environment may cause anatomical deformation, hinder the release of folliculars from the ovary, inhibit the movement of folliculars to the fallopian tube, and inhibit the migration of sperm through the fallopian tube [ 20 ]. The most important examination index of infertility is ovarian reserve function. Conventional indicators include AMH, FSH, and AFC [ 14 , 21 ]. Compared with serum indicators, measurement of AFC by transvaginal ultrasonography is simple, cost-effective, and practical. Some literature has shown that OEM can impair ovarian reserve function [ 22 , 23 ], but there is little literature on the effect of AM on ovarian reserve function indexes, including AMH, FSH and AFC. A review has shown decreased AMH levels in AM patients [ 24 ]. Our study found a low level of AMH in the AM group and OEM group relative to healthy group, though there was no significant difference between the AM group and OEM group (Fig. 4 ). The pathogenesis of AM and OEM is analogous, as both conditions are associated with hormonal secretion and inflammation expansion [ 18 , 19 ]. AMH is produced by granulosa cells around growing follicles, indicating the transition from primordial to growing follicles [ 25 ]. In the estrogen imbalance and pro-inflammatory microenvironments, the ovarian cortex is over-oxidised, causing cortical fibrosis, damaging ovarian blood vessels [ 20 ]. This eventually leads to a decrease in AMH, although this is inconsistent with other research results [ 26 ]. Emerging studies have suggested that AM and OEM may relate to the trauma mechanism of the uterus itself [ 27 ]. Uterine peristalsis itself contribute to transport sperm toward the fallopian tube, but at the same time, the mechanical contraction of the endometrium compresses the uterine cavity and both sides of the uterine horn, destroying normal anatomy, causing the displacement of the endometrial fragments in the basal layer, resulting in local estrogen production and inflammatory response. As the disease progresses, fragments of the endometrium migrate to the abdominal cavity and further afield [ 28 ]. Increased estrogen promotes abnormal follicular development and granulosa cell differentiation, affecting AMH expression [ 29 ]. Our data confirm that both AM and OEM can significantly reduce ovarian function. These findings highlight the importance of ovarian reserve assessment in these patients. In addition to AMH, follicle development is also associated with FSH and inhibin. Early follicles grow slowly under local regulatory factors and FSH [ 14 ]. FSH also promotes angiogenesis around follicles by up-regulating the expression of VEGF. The blood vessels around the follicle provide important nutrients, hormones and oxygen for follicle formation [ 30 ]. Inhibin, produced by granulosa cells, is a marker of the growth of antral follicles and inhibits FSH production [ 25 ]. Studies have shown that as AFC decreases, inhibin decreases and FSH increases [ 31 ]. In this study, it was found that the levels of FSH and inhibin in the AM group were lower than those in the healthy group (Fig. 4 ). There was a weak negative correlation between FSH and inhibin, though not statistically significant (r=-0.150, P = 0.219). This may be because the effects of granulosa cell damage on inhibin and FSH are different and complex under local estrogenic and inflammatory conditions. Notably, studies have found that both Inhibin and activin belong to the TGF-β cytokine family, but demonstrate different effects on FSH. Inhibin inhibits FSH secretion, and activin promotes it. However, the effect of the inflammatory environment on Inhibin and activin is not clear [ 32 ]. In addition to blood biochemical examination, ultrasound is a simple and cost-effective method for monitoring antral follicles. Existing studies have shown that AFC is the best indicator for predicting ovarian response in assisted reproductive technology and is often used as a preoperative consultation [ 33 – 35 ]. In this study, it was found that the AFC of the healthy group was more than that of the OEM and AM groups, and the OEM group was more than the AM group; however, the P value between the OEM and AM groups was only 0.044 (Fig. 4 ). It may be because the partial fence-like echo of AM weakens the ability of ultrasound penetration and affects the display of ovarian antral follicles, especially when the ovary is deep and disturbed by the surrounding intestinal air, resulting in an underestimation of AFC in the AM group. Compared with FSH and inhibin, several studies have proved that there is a high correlation between AMH and AFC. In this study, it was also found that AMH was only moderately positively correlated with AFC (r = 0.539, P < 0.001). FSH is produced by the pituitary gland and acts on the surface of ovarian granulosa cells. Inhibin is also affected by pituitary hormones and regulated by FSH negative feedback. Notably, there are two peaks of inhibin throughout the menstrual cycle, which not only affect the FSH level but are also disturbed by the measurement time [ 25 , 36 ]. In contrast, the AMH level is stable during the menstrual cycle and is not disturbed by the measurement time. Among these three serum indicators, AMH is the most sensitive and specific biochemical indicator for evaluating ovarian reserve function [ 37 – 39 ]. There is no apparent difference in AMH between the AM group and the OEM group. Although both of them involve the presence of endometrial glands and stroma, one located in the muscular layer and the other located outside the uterus, there was no obvious influence on infertility. Limitations There are still several limitations in this study: 1. This study is a retrospective study, and there are few cases collected due to missing clinical data. 2. It is difficult to completely rule out the impact of individual differences in ovarian aging. AM is correlated with other benign gynecological diseases, including endometriosis and uterine fibroids [ 40 ]. Previous studies have demonstrated that endometriosis is often complicated with AM, and vice versa [ 27 ]. Therefore, AM is a disease that is difficult to study alone. In real clinical studies, it remains challenging to completely exclude the influence of AM with benign gynecological diseases. 3.Future studies should expand the study period to increase the sample size and reduce the impact of poor ovarian visualization due to AM's fence-like echo, thereby improving the reliability of the study. Conclusions Both AM and OEM can reduce ovarian reserve function. AM decreases multiple ovarian reserve markers in infertile patients, including AMH, FSH, inhibin, and AFC. OEM reduces AMH and AFC in infertile patients. Among them, AFC and AMH are the most reliable indicators to evaluate ovarian reserve. The difference in the above results may be related to variations in local estrogen expression and the degree of inflammatory environment. Declarations Funding: The authors declare that no funds, grants were received during the preparation of this manuscript. Competing Interests: The authors have no relevant financial or non-financial interests to disclose. Author Contribution: All authors contributed to the Project development. Material preparation, data collection and analysis were performed by Fengrong Yang. Part of the data collection was completed by Qin qin. The first draft of the manuscript was written by Fengrong Yang. All authors read and approved the final manuscript. Ethics approval: This study was performed in line with the principles of the Declaration of Helsinki and granted ethics approval by the Ethics Committee of Guangyuan Central Hospital ( number 202520, Guangyuan, China). Consent to participate: This study was granted a waiver for informed consent. Data availability statements: All data supporting the findings of this study are available from the authors. Acknowledgement We gratefully acknowledge the technical support provided by the after-sales service teams at Mindray Medical Company and GE Healthcare. References Khan, K. N., Fujishita, A. & Mori, T. Pathogenesis of Human Adenomyosis: Current Understanding and Its Association with Infertility. J. Clin. Med. 11, 4057 (2022). Zhang, M. et al. MEKK4-mediated Phosphorylation of HOXA10 at Threonine 362 facilitates embryo adhesion to the endometrial epithelium. Cell Death Discov. 8, 415 (2022). Sun, M. et al. 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12:10:17","extension":"html","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":82103,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7345581/v1/a9e35a4b9e8841ab324500ef.html"},{"id":91987486,"identity":"3ba4b02b-65c8-4f37-9244-610bd05dec18","added_by":"auto","created_at":"2025-09-23 12:10:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":495078,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUterine longitudinal section in the AM group\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7345581/v1/473c39fec3854962495c4d5b.png"},{"id":91987505,"identity":"972589cc-a7ee-412e-aa2e-40d945fe8c19","added_by":"auto","created_at":"2025-09-23 12:10:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":660658,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEndometriosis in the left ovary of the OEM group\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7345581/v1/558003b7b48b001eac440e82.png"},{"id":91987506,"identity":"e516eac6-50ee-4198-98e9-7cd985a2cdfa","added_by":"auto","created_at":"2025-09-23 12:10:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":557403,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAFC in the right ovary of the AM group. \u003c/strong\u003eThe Mindray built-in counting software was used to analyse AFC, with identified follicles marked in different colours.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7345581/v1/f08f5725220a6667429f1ebc.png"},{"id":91987502,"identity":"8bd43585-cfdd-4344-ae59-372da3d504b9","added_by":"auto","created_at":"2025-09-23 12:10:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":164236,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of blood biochemical and ultrasonographic examination results among the three groups. \u003c/strong\u003eComparison of FSH, inhibition, AFC and AMH among the three groups. Figure a: FSH in Healthy group was higher than that in AM group; figure b: The inhibition of Healthy group was higher than AM group; figure c: AFC of Healthy group was higher than OEM group, AFC of OEM group was higher than AM group; figure d: AMH in AM group and OEM group was lower than that in Healthy group. * Significantly different, p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7345581/v1/957387b0c21b3d8f24297010.png"},{"id":93805077,"identity":"8de68973-4b62-4e41-8ade-32f7d912a4a2","added_by":"auto","created_at":"2025-10-17 17:53:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2680010,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7345581/v1/24564ef0-1049-4c3d-9b42-15979d30624a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Assessment of ovarian reserve in adenomyosis and ovarian endometriosis patients via 3D Transvaginal Ultrasound with AMH, FSH, and Inhibin","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAdenomyosis (AM) and ovarian endometriosis (OEM) are common gynecological diseases, which are characterized by the presence of endometrioid glands and stroma in the myometrium as well as outside the uterus [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Although they are benign diseases, they seriously affect the quality of life of patients. In addition to its clinical manifestations of dysmenorrhea and menorrhagia, a number of studies have shown that AM and OEM are also closely related to infertility [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDecreased ovarian reserve is one of the common causes of infertility [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The detection indexes of ovarian reserve include follicle-stimulating hormone (FSH), anti-M\u0026uuml;llerian hormone (AMH), and antral follicle count (AFC). The serum level of AMH is correlated with AFC [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTransvaginal ultrasound imaging is capable of identifying follicles with a diameter of at least 2 mm, making it a simple and highly reproducible technique. A number of studies have demonstrated that ultrasound-measured AFC can be used to evaluate ovarian reserve function [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. There are few studies on the evaluation of ovarian reserve function in AM and OEM via ultrasound combined with AMH and FSH levels. Therefore, this study aimed to evaluate the diagnostic value of transvaginal ultrasound imaging in ovarian reserve function of AM and OEM patients.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eStudy Population\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 69 female patients who visited our hospital for infertility from March 2023 to May 2025 and received ultrasound examination were included in this study. All patients underwent transvaginal two-dimensional and three-dimensional ultrasound examination.\u0026nbsp;Inclusion criteria were:\u0026nbsp;(1) married women, aged 20-42 years; (2) no history of anti-estrogen medications; (3) no history of pelvic surgery.\u0026nbsp;Exclusion criteria were: (1) history of pelvic surgery; (2) patients with recent use of hormonal medicine; (3) patients with bilateral fallopian tube obstruction; (4) Pregnancy; (5) Lack of clinical data. According to the results of ultrasound examination, the patients were divided into three groups: healthy group (30 cases), AM group (21 cases) and OEM group (18 cases). This study complied with the ethical principles of the Declaration of Helsinki and was granted a waiver for informed consent and ethics approval by the Ethics Committee of \u0026nbsp;Guangyuan Central Hospital.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiagnostic Criteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ultrasound diagnosis of AM was based on the diagnostic consensus on AM published by the International Morphological Uterus Sonographic Assessment (MUSA) group [12]. The ultrasonic diagnosis of AM should meet the following two or more (Fig 1): (1) uniform enlargement of the uterus; (2) fan-shaped shadows; (3) asymmetric thickening of the anterior and posterior walls of the myometrium; (4) myometrial cyst; (5) hyperechoic island; (6) subendometrial echoic linear and bud-like objects; (7) cross-lesion blood vessels; (8) irregular JZ bands. The inclusion criteria of OEM patients should meet the following conditions [13]: (1) Infertility; (2) Chronic pelvic pain; (3)\u0026nbsp;OEM diagnosed by ultrasound or magnetic resonance imaging (MRI) (Fig 2); (4) Tumor marker CA125 \u0026lt; 200 IU /mL; (5) Exclude other malignant signs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEquipment and Procedures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, two ultrasound systems were used: the Resona R9S (Mindray Medical Company, Shenzhen, China) equipped with a three-dimensional probe (DE10-3wu) and the Voluson E8 (GE Healthcare, Austria) equipped with a two-dimensional probe (IC5-9-D) and a three-dimensional probe (RIC5-9-D). Two-dimensional and three-dimensional vaginal ultrasonography were performed on the 3rd to 5th day of the menstrual cycle. The early follicular phase of the menstrual cycle was selected to reduce the impact of menstrual cycle fluctuations and the possibility of identifying ovarian cysts or corpus luteum as antral follicles\u0026nbsp;[14]. Patients were informed to be in the lithotomy position after emptying the bladder. The condom was placed over the ultrasound probe before inserting into the vagina.\u003c/p\u003e\n\u003cp\u003eFirst, two-dimensional examination was performed to evaluate the size, shape and internal echo characteristics of the uterus and ovary. According to the ultrasonic manifestations, they were divided into healthy group, AM group and OEM group. Then, the maximum diameter section of the ovary was selected in the two-dimensional scanning mode, and three-dimensional ultrasound examination was performed. The number of follicles in the bilateral ovaries was detected, and the average value was calculated via built-in automatic measurement software (Fig 3). At the same time, manual adjustments were used to reduce the measurement deviation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBlood Biochemical Testing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e5 ml fasting venous blood samples were collected from all participants in the early follicular phase of the menstrual cycle. The levels of AMH, FSH and inhibin in serum were determined by enzyme-linked immunosorbent assay.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSPSS 23.0 software was used for data analysis. Data conforming to the normal distribution are expressed as mean \u0026plusmn; standard deviation ( \u0026plusmn;s). The parameters among the three groups were compared by one-way analysis of variance (ANOVA), and the differences between groups were compared by post-hoc t-tests. Pearson correlation analysis was applied to analyse the correlation between serum levels and AFC. The correlation coefficient (r) indicated a strong correlation between 0.7 and 1.0, a moderate correlation between 0.4 and 0.7, and a weak correlation between 0 and 0.4. A two-tailed P-value \u0026lt;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eTab 1 Comparison of age among the three groups\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 112px;\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003eHealthy group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 112px;\"\u003e\n \u003cp\u003eAM Group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 112px;\"\u003e\n \u003cp\u003eOEM Group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003eP\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 112px;\"\u003e\n \u003cp\u003eAGE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e29.63\u0026plusmn;3.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 112px;\"\u003e\n \u003cp\u003e31.29\u0026plusmn;2.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 112px;\"\u003e\n \u003cp\u003e30.89\u0026plusmn;3.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003eP＞0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e* Significantly different, p \u0026lt; 0.05\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRelationship between blood biochemical and ultrasonographic examination results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePearson correlation showed that AFC and AMH were moderately positively correlated (r=0.539, P \u0026lt; 0.001). There were no statistically significant correlations among other indicators.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe results of our study showed that there were significant differences in AFC among the three groups. The healthy group exhibited significantly higher AFC than the OEM group, while the OEM group showed higher AFC than the AM group. Both AM and OEM groups had lower AMH levels than the healthy group. The AM group showed reduced FSH and inhibin levels compared to the healthy group. A moderate positive correlation was observed between AMH and AFC.\u003c/p\u003e\u003cp\u003eAM and OEM are common reasons for infertility [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. There are multiple theories to explain the pathogenesis of AM, but they all suggest that it is related to the expression of local estrogen, and the increase of local inflammatory factors and cells [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The pathogenesis of OEM is similar to that of AM, which is related to local estrogen expression and pro-inflammatory environment [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The local inflammatory environment may cause anatomical deformation, hinder the release of folliculars from the ovary, inhibit the movement of folliculars to the fallopian tube, and inhibit the migration of sperm through the fallopian tube [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The most important examination index of infertility is ovarian reserve function. Conventional indicators include AMH, FSH, and AFC [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Compared with serum indicators, measurement of AFC by transvaginal ultrasonography is simple, cost-effective, and practical. Some literature has shown that OEM can impair ovarian reserve function [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], but there is little literature on the effect of AM on ovarian reserve function indexes, including AMH, FSH and AFC.\u003c/p\u003e\u003cp\u003eA review has shown decreased AMH levels in AM patients [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Our study found a low level of AMH in the AM group and OEM group relative to healthy group, though there was no significant difference between the AM group and OEM group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The pathogenesis of AM and OEM is analogous, as both conditions are associated with hormonal secretion and inflammation expansion [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. AMH is produced by granulosa cells around growing follicles, indicating the transition from primordial to growing follicles [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In the estrogen imbalance and pro-inflammatory microenvironments, the ovarian cortex is over-oxidised, causing cortical fibrosis, damaging ovarian blood vessels [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This eventually leads to a decrease in AMH, although this is inconsistent with other research results [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Emerging studies have suggested that AM and OEM may relate to the trauma mechanism of the uterus itself [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eUterine peristalsis itself contribute to transport sperm toward the fallopian tube, but at the same time, the mechanical contraction of the endometrium compresses the uterine cavity and both sides of the uterine horn, destroying normal anatomy, causing the displacement of the endometrial fragments in the basal layer, resulting in local estrogen production and inflammatory response. As the disease progresses, fragments of the endometrium migrate to the abdominal cavity and further afield [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Increased estrogen promotes abnormal follicular development and granulosa cell differentiation, affecting AMH expression [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Our data confirm that both AM and OEM can significantly reduce ovarian function. These findings highlight the importance of ovarian reserve assessment in these patients.\u003c/p\u003e\u003cp\u003eIn addition to AMH, follicle development is also associated with FSH and inhibin. Early follicles grow slowly under local regulatory factors and FSH [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. FSH also promotes angiogenesis around follicles by up-regulating the expression of VEGF. The blood vessels around the follicle provide important nutrients, hormones and oxygen for follicle formation [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Inhibin, produced by granulosa cells, is a marker of the growth of antral follicles and inhibits FSH production [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Studies have shown that as AFC decreases, inhibin decreases and FSH increases [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In this study, it was found that the levels of FSH and inhibin in the AM group were lower than those in the healthy group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). There was a weak negative correlation between FSH and inhibin, though not statistically significant (r=-0.150, P\u0026thinsp;=\u0026thinsp;0.219). This may be because the effects of granulosa cell damage on inhibin and FSH are different and complex under local estrogenic and inflammatory conditions. Notably, studies have found that both Inhibin and activin belong to the TGF-β cytokine family, but demonstrate different effects on FSH. Inhibin inhibits FSH secretion, and activin promotes it. However, the effect of the inflammatory environment on Inhibin and activin is not clear [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn addition to blood biochemical examination, ultrasound is a simple and cost-effective method for monitoring antral follicles. Existing studies have shown that AFC is the best indicator for predicting ovarian response in assisted reproductive technology and is often used as a preoperative consultation [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In this study, it was found that the AFC of the healthy group was more than that of the OEM and AM groups, and the OEM group was more than the AM group; however, the P value between the OEM and AM groups was only 0.044 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). It may be because the partial fence-like echo of AM weakens the ability of ultrasound penetration and affects the display of ovarian antral follicles, especially when the ovary is deep and disturbed by the surrounding intestinal air, resulting in an underestimation of AFC in the AM group.\u003c/p\u003e\u003cp\u003eCompared with FSH and inhibin, several studies have proved that there is a high correlation between AMH and AFC. In this study, it was also found that AMH was only moderately positively correlated with AFC (r\u0026thinsp;=\u0026thinsp;0.539, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). FSH is produced by the pituitary gland and acts on the surface of ovarian granulosa cells. Inhibin is also affected by pituitary hormones and regulated by FSH negative feedback. Notably, there are two peaks of inhibin throughout the menstrual cycle, which not only affect the FSH level but are also disturbed by the measurement time [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In contrast, the AMH level is stable during the menstrual cycle and is not disturbed by the measurement time. Among these three serum indicators, AMH is the most sensitive and specific biochemical indicator for evaluating ovarian reserve function [\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. There is no apparent difference in AMH between the AM group and the OEM group. Although both of them involve the presence of endometrial glands and stroma, one located in the muscular layer and the other located outside the uterus, there was no obvious influence on infertility.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eLimitations\u003c/h2\u003e\u003cp\u003eThere are still several limitations in this study: 1. This study is a retrospective study, and there are few cases collected due to missing clinical data. 2. It is difficult to completely rule out the impact of individual differences in ovarian aging. AM is correlated with other benign gynecological diseases, including endometriosis and uterine fibroids [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Previous studies have demonstrated that endometriosis is often complicated with AM, and vice versa [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Therefore, AM is a disease that is difficult to study alone. In real clinical studies, it remains challenging to completely exclude the influence of AM with benign gynecological diseases. 3.Future studies should expand the study period to increase the sample size and reduce the impact of poor ovarian visualization due to AM's fence-like echo, thereby improving the reliability of the study.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eBoth AM and OEM can reduce ovarian reserve function. AM decreases multiple ovarian reserve markers in infertile patients, including AMH, FSH, inhibin, and AFC. OEM reduces AMH and AFC in infertile patients. Among them, AFC and AMH are the most reliable indicators to evaluate ovarian reserve. The difference in the above results may be related to variations in local estrogen expression and the degree of inflammatory environment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding:\u003c/h2\u003e\n\u003cp\u003eThe authors declare that no funds, grants were received during the preparation of this manuscript.\u003c/p\u003e\n\u003ch2\u003eCompeting Interests:\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution:\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eAll authors contributed to the Project development. Material preparation, data collection and analysis were performed by Fengrong Yang. Part of the data collection was completed by Qin qin. The first draft of the manuscript was written by Fengrong Yang. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eEthics approval:\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThis study was performed in line with the principles of the Declaration of Helsinki and granted ethics approval by the Ethics Committee of Guangyuan Central Hospital ( number 202520, Guangyuan, China).\u003c/p\u003e\n\u003ch2\u003eConsent to participate:\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThis study was granted a waiver for informed consent.\u003c/p\u003e\n\u003ch2\u003eData availability statements:\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eAll data supporting the findings of this study are available from the authors.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eWe gratefully acknowledge the technical support provided by the after-sales service teams at Mindray Medical Company and GE Healthcare.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKhan, K. 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An Online Tool Using Basal or Activated Ovarian Reserve Markers to Predict the Number of Oocytes Retrieved Following Controlled Ovarian Stimulation: A Prospective Observational Cohort Study. \u003cem\u003eFront. Endocrinol.\u003c/em\u003e 13, 881983 (2022).\u003c/li\u003e\n\u003cli\u003ePinola, P. et al. Anti-M\u0026uuml;llerian hormone: correlation with testosterone and oligo- or amenorrhoea in female adolescence in a population-based cohort study. \u003cem\u003eHum. Reprod. Oxf. Engl. \u003c/em\u003e29, 2317\u0026ndash;2325 (2014).\u003c/li\u003e\n\u003cli\u003eBrucker, S. Y. et al. Clinical characteristics indicating adenomyosis coexisting with leiomyomas: a retrospective, questionnaire-based study. \u003cem\u003eFertil. Steril. \u003c/em\u003e101, 237-241.e1 (2014).\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":"adenomyosis, ovarian endometriosis, Anti-Müllerian Hormone, Antral Follicle Count","lastPublishedDoi":"10.21203/rs.3.rs-7345581/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7345581/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study aims to evaluate the effect of adenomyosis (AM) and ovarian endometriosis (OEM) on ovarian reserve in infertile patients by combining three-dimensional vaginal ultrasound and AMH, FSH and inhibition levels. From March 2023 to May 2025, 69 patients with infertility in our hospital were collected, including 21 patients as the AM group, 18 patients as the OEM group and 30 healthy patients as the healthy group. The AMH, FSH, inhibition and AFC levels were collected for each patient. AMH in the AM group and OEM group was lower than that in the healthy group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The FSH and inhibin levels in the AM group were lower than those in the healthy group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). AFC in the healthy group was significantly higher than that in the OEM group, and higher in the OEM group than in the AM group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). There is a moderate positive correlation between AMH and AFC (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Both AM and OEM can reduce ovarian reserve. AM can reduce the ovarian reserve indicators AMH, FSH, inhibition and AFC of infertile patients, whereas OEM can reduce AMH and AFC. AFC and AMH are the best indexes to evaluate ovarian reserve.\u003c/p\u003e","manuscriptTitle":"Assessment of ovarian reserve in adenomyosis and ovarian endometriosis patients via 3D Transvaginal Ultrasound with AMH, FSH, and Inhibin","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-23 12:08:31","doi":"10.21203/rs.3.rs-7345581/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":"56818f9b-5a8c-413d-b24a-e495c6277cff","owner":[],"postedDate":"September 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54876840,"name":"Health sciences/Diseases"},{"id":54876841,"name":"Health sciences/Endocrinology"},{"id":54876842,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2025-10-17T17:53:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-23 12:08:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7345581","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7345581","identity":"rs-7345581","version":["v1"]},"buildId":"B-jG_2CBjPDmsCi4Wdhf-","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}