Prevalence of diminished ovarian reserve in Chinese women with follicular cysts and menstrual disorders

Abstract

Objectives

To assess the prevalence of diminished ovarian reserve (DOR) in Chinese women with follicular cysts and menstrual disorders and relationship to hormonal markers.

Methods

117 women with follicular cysts and menstrual disorders, aged 24 ∼ 53 (39.19 ± 6.61) years; measurements of height, weight, follicle-stimulating hormone (FSH), luteinizing hormone (LH), E2, progesterone (Po), prolactin (PRL), total testosterone, AMH, follicular cyst diameter, endometrial thickness. Three age groups were compared: 1) 21 ∼ 30 years, 2) 30 ∼ 40 years, 3) > 40 years.

Results

Total prevalence of DOR 86.3%, in the groups 50%, 81.6%, and 98.4%, in group-3 significantly higher than in group-1 and 2. 34.2% of the 117 patients complained of cessation of regular menstruations or amenorrhea, 65.8% of abnormal uterine bleeding. Follicular cysts disappeard in cycle-1 for 98 (83.8%) and in cycle-2 for 117 (100%) patients. AMH decreased with age, significantly different between the three groups. Total testosterone in group-1 and 2 was significantly higher than in group-3. In total AMH had a negative correlation with age and E2 (p < 0.01) and positive correlation with total testosterone (p < 0.05).

Conclusions

Assessing ovarian reserve with follicular cysts and menstrual disorders is important because often pointing to DOR. The overall prevalence of DOR was high; even young women (<40 years) with follicular cysts and menstrual disorders had a low level of AMH. So AMH can be used as a marker to define DOR with higher sensitivity than other markers like FSH and E2. Primarily, these results only apply to Chinese women and should be confirmed in further studies.

Keywords:

• Diminished ovarian reserve
• functional ovarian cyst
• follicular cyst
• anti-müllerian hormone
• menstrual disorders

Introduction

Ovarian reserve (OR), the functional potential of the ovary, reflects the quantity and quality of oocytes at any given time and constitutes the resting, non-growing ovarian primordial follicle population. Diminished ovarian reserve (DOR) refers to the loss of the normal reproductive capacity of the ovary due to a decrease in the numbers and quality of remaining follicles [1]. It is distinct from menopause and premature ovarian insufficiency (POI) [2] but represents an intermediate situation between normal reproductive biology and POI. DOR that occurs after age 40 is usually physiological, while women’s early experience of DOR leads to an early decline in their reproductive function. DOR’s most common clinical manifestations are menstrual disorder, an endocrine disorder, poor response to ovarian stimulation, and infertility [3].

To define DOR, the antral follicular count (AFC) and anti-müllerian hormone (AMH), and follicle-stimulating hormone (FSH) serum levels are the most estimated and frequently used criteria [4]. AMH is a glycoprotein that belongs to the transforming growth factor-beta superfamily. In women, it is secreted exclusively by ovarian granulosa cells starting in utero at about 36 weeks of gestation [5]. In recent years, serum AMH has proven effective in evaluating ovarian disorders in women from childhood to menopause. A significant advantage of serum AMH is its low intra-cycle and inter-cycle variability, as it is produced from preantral and small antral follicles, which are gonadotropin independent [6]. AMH better reflects the age-related decrease in the follicular pool than other markers, giving the most reliable reflection of reproductive aging for any particular woman [7]. In China, AMH < 1.1 ng/ml is used for diagnostic standards of DOR, which was used in our study [8].

The most common ovarian masses in premenopausal women are functional ovarian cysts (FOC) [9], a common gynecological problem among women of reproductive age worldwide. Functional cysts are not true neoplasms but should be considered a variation of a normal physiologic process. Functional cysts include follicular, corpus luteum, and theca-lutein, all benign and usually self-limiting [10]. Follicular cysts are the most common functional ovarian cysts [11], with smooth, thin walls and unilocular. Follicular cysts can form when the mature follicle involutes and may occur in women of different ages, especially in women with menstrual disorders after menarche or perimenopause [11]. However, no consensus has been reached yet on the exact mechanism of follicular cyst formation.

In clinical practice, some patients may present with menstrual disorders, and pelvic ultrasound may reveal follicular cysts. While there were some studies on the clinical characteristics and treatment of FOC developed after ovulation induction, there is limited research on spontaneous follicular cysts. Our study aimed to explore the prevalence of DOR in Chinese women with follicular cysts and presenting menstrual disorders and the value of AMH as a sensitive marker for defining DOR related to other serum markers like E2 and FSH, which may provide some advice for clinical practice.

Materials and methods

From March 2022 to February 2023, 117 women with follicular cysts and menstrual disorders, aged 24 ∼ 53 years (mean age 39.19 ± 6.61 years), were recruited by the Department of Gynecological Endocrinology, Beijing Obstetrics & Gynecology Hospital, Capital Medical University. This study was approved by the Human Ethics Committee of Beijing Obstetrics & Gynecology Hospital, Capital Medical University (2020-KY-008-01). All the study participants were provided with written informed consent prior to participation.

Inclusion criteria are the presence of follicular cysts and menstrual disorders.

Follicular cysts were defined as serum E2 levels ≥ 50 pg/ml, a mean cyst diameter ≥ 2.0 cm, and a serum level of Po < 3 ng/ml [12, 13]. Menstrual disorders include cessation of regular menstruation or amenorrhea and abnormal uterine bleeding (AUB) (i.e. light menstrual bleeding or vaginal spotting, prolonged menstruation, polymenorrhea, and irregular vaginal bleeding).

Exclusion criteria included: pregnancy; history of taking hormone therapy (HT) in the last three months; taking drugs (e.g. combined oral contraceptive, tamoxifen, or gonadotropin-releasing hormone(GnRH) agonist) that could influence ovarian follicular cysts or AMH; after ovulation induction; with levonorgestrel intrauterine devices; having diseases of the thyroid gland or adrenal gland; hyperprolactinemia; AUB caused by other etiology (except ovulatory dysfunction); or smoking and excessive alcohol use.

We used only AMH in this study as the definition for DOR, not in addition to other laboratory parameters like FSH or E2, because one aim of the study was to compare the sensitivity of AMH with those other serum parameters. As assessed elsewhere for Chinese women [8], patients presenting AMH < 1.1 ng/ml were defined as having DOR.

Body weight and height were measured, with the participants wearing light clothes and no shoes. Body mass index (BMI) was calculated. After an 8 ∼ 12h overnight fasting, blood samples were collected from each via venipuncture. Serums were separated by a centrifuge and stored at −20 °C until analysis. Reproductive hormones, including FSH, LH, E2, Po, PRL, and total testosterone, were measured with Siemens ADVIA Centaur XP. There are different kits of AMH. Serum AMH concentrations in our study were measured with an active MIS/AMH enzyme-linked immunosorbent assay (ELISA) DSL-10-14400 kit (Diagnostic System Laboratories, Webster, TX). The intra-assay and inter-assay coefficient of variation for FSH, LH, and E2 were all less than 5%. The detection range of FSH, LH, and E2 was 0.3 ∼ 200 IU/L, 0.07 ∼ 200 IU/L, and 11.8 ∼ 3000 pg/ml, respectively. The intra-assay and inter-assay coefficient of variation for AMH was less than 5% and 10%, respectively. The detection range of AMH was 0.06-18.0 ng/ml.

The ovarian cyst’s diameter and the endometrium’s thickness were measured by transvaginal or transrectal ultrasound scans with a 6.5-MHz vaginal transducer (Mylab 60, Esaote, S.P.A., Firenze, Italy). The diagnostic criteria of DOR was as follows: AMH < 1.1 ng/ml [8].

Statistical analyses

All Normal distribution data were expressed as Mean ± SD and Non-normal distribution data as Median (P25, P75). Statistical analyses were performed using Statistical Package for the Social Sciences (SPSS for Windows, version 26.0). The one-way analysis of variance (ANOVA) and Kruskal-Wallis tests were used to compare normal and non-normal data, respectively. The multiple comparisons were performed using the Bonferroni method for normal data, and Mann-Whitney U tests and Bonferroni correction were used for the pairwise comparison between groups for non-normal data. The chi-square test was applied to compare the prevalence of DOR between different age groups, and the Bonferroni method was used to correct the p-values of multiple comparisons. Spearman correlation was applied for the correlation analysis between AMH and other indices in each age group. The level of significance was set as p < 0.05.

Results

Characteristics of patients

A total of 117 women were included in the study. 40 out of 117 (34.2%) patients complained of cessation of regular menstruation or amenorrhea, and 77 out of 117 (65.8%) patients complained of AUB. Follicular cysts disappeard in 1 cycle for 98 (83.8%) patients and disappeard in 2 cycles for 117 (100%) patients. The baseline data of the participants are listed in Table 1.

Table 1. The baseline data of patients with follicular cysts and menstrual disorders (mean ± SD or Median (P25, P75)).

Variables	Mean ± SD or Median (P25, P75)	Min-Max
Age, years	39.19 ± 6.61	24-53
BMI, kg/m^2	23.28 ± 2.65	18.72-31.80
FSH, mIU/ml	5.52 (3.11, 7.57)	0.33-79.79
LH, mIU/ml	3.8 (1.95, 6.76)	0.95-56.17
FSH/LH ratio	1.29 (0.64, 2.48)	0.08-6.13
E2, pg/ml	126.31 (64.51, 240.46)	50.52-841.01
Po, ng/ml	0.5 (0.38, 0.69)	0.03-1.61
PRL, ng/ml	9.10 (6.60, 13.68)	2.33-28.70
Testosterone, nmol/l	0.73 ± 0.29	0.01-1.62
AMH, ng/ml	0.25 (0.13, 0.70)	0.03-11.09
Diameter of ovarian cyst, cm	2.70 (2.50, 3.10)	2.0-4.8
Endometrial thickness, cm	0.83 (0.65, 1.10)	0.3-2.0

Min: minimum; Max: maximum; BMI: body mass index; FSH: follicle stimulating hormone; LH: luteinizing hormone; E2: estradiol; Po: progesterone; PRL: prolactin; AMH: anti-Müllerian hormone.

Comparison of different parameters between different age groups

The participants were divided into three groups according to age, with 16 women in the age group of 21 ∼ 30 years (Group-1), 38 in the age group of 31 ∼ 40 years (Group-2), and 63 in the age group of >40 years (Group-3). There was a significant difference in the level of AMH between the three groups (p < 0.05). The level of AMH decreased with age. The testosterone level in Group-1 and Group-2 was higher than in Group-3 (p < 0.05). In contrast, there were no significant differences in BMI, FSH, LH, FSH/LH ratio, E2, PRL, the diameter of the ovarian cyst, and endometrial thickness between the three groups (p > 0.05) (Table 2).

Table 2. Comparison of different parameters between different age groups. (mean ± SD or Median (P25, P75)).

	Group-1 (21 ∼ 30 years) (n = 16)	Group-2 (31-40 years) (n = 38)	Group-3 (>40 years) (n = 63)	P
BMI, kg/m^2	23.04 ± 3.12	23.57 ± 2.41	23.16 ± 2.68	0.704
FSH, mIU/ml	5.61 (2.97, 7.32)	4.39 (1.97, 6.50)	6.00 (3.37, 9.71)	0.068
LH, mIU/ml	3.41 (1.89, 8.57)	2.95 (1.87, 6.14)	4.79 (2.28, 7.59)	0.542
FSH/LH ratio	1.28 (0.58, 2.48)	1.21 (0.51, 1.96)	1.35 (0.71, 2.58)	0.328
E2, pg/ml	73.09 (57.76, 187.21)	117.26 (68.54, 233.35)	145.13 (62.40, 257.21)	0.158
Po, ng/ml	0.56 (0.40, 0.65)	0.50 (0.35, 0.74)	0.49 (0.40, 0.63)	0.763
PRL, ng/ml	10.43 (8.67, 15.56)	10.39 (6.63, 15.66)	8.53 (6.44, 12.80)	0.155
Testosterone, nmol/l	0.83 ± 0.30	0.83 ± 0.33	0.65 ± 0.24^a^,b	0.006*
AMH (ng/ml)	1.05 (0.88, 1.70)	0.35 (0.22, 0.93)^a	0.15 (0.11, 0.26)^a^,b	<0.001*
Diameter of ovarian cyst, cm	2.97 ± 0.76	2.88 ± 0.58	2.81 ± 0.54	0.571
Endometrial thickness, cm	0.88 ± 0.39	0.83 ± 0.29	0.92 ± 0.33	0.389

BMI: body mass index; FSH: follicle stimulating hormone; LH: luteinizing hormone; E2: estradiol; Po: progesterone; PRL: prolactin; AMH: anti-Müllerian hormone.

^a p < 0.05 compared with group-1; ^bp < 0.05 compared with group-2.

Prevalence of DOR in participants

According to the definition above, 101 out of 117 women with follicular cysts and menstrual disorders could be identified as having DOR, i.e. the prevalence of DOR in women with follicular cysts and menstrual disorders was 86.3%. Besides, 8 out of 16 women in Group-1, 31 out of 38 women in Group-2, and 62 out of 63 women in Group-3 could be identified as having DOR, i.e. the prevalence of DOR in women in three age groups (21 ∼ 30 years, 30 ∼ 40 years and >40 years) was 50%, 81.6%, and 98.4%, respectively. The prevalence of DOR in Group-3 was significantly higher than in Group-1 and Group-2 (p < 0.05).

Correlation between AMH and other indices in participants

The correlation between AMH and other indices in each age group is listed in Table 3. AMH had a negative correlation with age and E2 (p < 0.01), while it had a positive correlation with testosterone (p < 0.05) in all the participants.

Table 3. Correlation between AMH and other indices in participants.

Variables	Group-1 (21 ∼ 30 years) (n = 16)	Group-2 (31-40 years) (n = 38)	Group-3 (>40 years) (n = 63)	All the participants (n = 117)
Age, years	−0.179	−0.386*	−0.459**	−0.672**
BMI, kg/m^2	−0.099	−0.189	0.283*	0.069
FSH, mIU/ml	−0.162	0.033	−0.052	−0.104
LH, mIU/ml	0.540*	0.271	−0.236	−0.040
FSH/LH ratio	−0.785**	−0.226	0.200	−0.053
E2, pg/ml	0.006	−0.327*	−0.201	−0.241**
Po, ng/ml	0.631**	0.066	−0.185	0.001
PRL, ng/ml	0.211	−0.295	−0.186	−0.049
Testosterone, nmol/l	0.957**	0.252	0.042	0.248*
Diameter of ovarian cyst, cm	0.550*	0.153	−0.059	0.078
Endometrial thickness, cm	0.230	−0.066	0.124	−0.023

* p < 0.05, **p < 0.01.

BMI: body mass index; FSH: follicle stimulating hormone; LH: luteinizing hormone; E2: estradiol; Po: progesterone; PRL: prolactin; AMH: anti-Müllerian hormone.

Discussion

Different types of menstrual disorders and persistence of cysts in Chinese women with follicular cysts and menstrual disorders

FOC are benign cystic structures that occur in reproductive-age women. A major problem in comparing different studies of FOC is the variation in the definition of FOC because of variations in size ranging from 20 ∼ 30 mm [13]. Our study defined the size of the follicular cyst as ≥2.0 cm. Our study found that 34.2% of patients with follicular cysts and menstrual disorders complaint of cessation of regular menstruation or amenorrhea, and 65.8% of patients complaint of AUB, which included light menstrual bleeding or vaginal spotting, prolonged menstruation, polymenorrhea, and irregular vaginal bleeding. Although FOC can be self-limiting and regress without treatment, it may cause substantial pelvic pain and menstrual dysfunction [14]. Our study also found that follicular cysts disappeard in 1 cycle for 83.8% of patients and disappeard in 2 cycles for 100% of patients. Although all the cysts persisted no more than two cycles in our study, these patients should be treated for their menstrual disorders instead of just watchful waiting for two or three cycles.

Prevalence of DOR in Chinese women with follicular cysts and menstrual disorders

AMH is a useful marker of OR in reproductive-aged women [15]. Its advantage over other fertility serum markers, such as FSH or E2, is its low variability throughout the menstrual cycle. Other studies found that AMH is more sensitive than FSH in diagnosing DOR and typically becomes abnormal before FSH levels do [16]. Compared to the AFC, which has excellent inter-observer variability and dependency on the equipment, AMH is a more objective marker [17]. There is a general guideline for age-specific serum AMH values, which provide approximate lower limits in 5-year age intervals for the following ages: 0.5 ng/mL for 45 years, 1 ng/mL for 40 years, 1.5 ng/mL for 35 years, 2.5 ng/mL for 30 years, and 3.0 ng/mL for 25 years [2]. In our study AMH < 1.1 ng/ml was used for the diagnostic criteria of DOR. Our results in terms of comparison with other markers like E2 and FSH demonstrated that it was correct only to use AMH as a diagnostic criterion of DOR, with the value <1.1 ng/ml as assessed in Chinese women elsewhere [8].

Our study found that the women with follicular cysts and menstrual disorders were most common in >40 years, and even the young women (<40 years) with follicular cysts and menstrual disorders also had a low level of AMH and a high prevalence of DOR. A previous study also showed that functional ovarian cyst formation after pituitary suppression in assisted reproductive technology (ART) did not happen randomly and might indicate patients with a poor ovarian response [12]. Another study found that women following the application of GnRH agonists who showed a higher basal FSH in their cycle were more likely to experience functional ovarian cysts [18]. Because of the existence of follicular cysts, which can secrete estrogen continuously, the E2 level is higher, and the FSH level is lower than its baseline level. This may result in the ignorance of DOR in these patients. With the accurate assessment of ovarian reserve and function, we can identify those women with DOR and help them make plans about conception and other therapies. So it is very important to evaluate the OR of women with follicular cysts and menstrual disorders, and AMH would be a better choice for these patients.

The endocrine characteristics and correlation between AMH and other indices in Chinese women with follicular cysts and menstrual disorders at different age groups

From birth onward, AMH levels increase with age, reflecting the increased rate of primordial follicle recruitment observed up to approximately 14 years of age. After reaching the plateau at 25 years of age, AMH levels start to decline with age until menopause, when AMH levels become undetectable [19]. AMH reflects the age-related decrease in the follicular pool more than other markers, giving any particular woman the most reliable reflection of reproductive aging [7]. Our study also found a significant difference in the level of AMH between the three age groups, and the level of AMH negatively correlated with age in all participants, especially for women >30 years.

A previous study showed a positive relationship between androgenic profiles and AMH during adolescence and reproductive years, implying a synchronism between androgens and OR [20]. Our study showed that the total testosterone level in Group-1 and Group-2 was higher than in Group-3, and AMH positively correlated with total testosterone in all participants.

Besides, this study showed no significant differences between the three groups in FSH, LH, FSH/LH ratio, E2, and PRL. AMH negatively correlated with E2 for all the participants. As FSH, LH, and E2 levels vary with the menstrual cycle, the sex hormones in our study were not tested at the baseline level, and they cannot reflect the OR accurately. However, AMH is produced by small developing, mostly antral follicles. As these follicles are not yet responsive to FSH, AMH levels remain relatively stable throughout the menstrual cycle [21].

Strengths and limitations

1. Because most follicular cysts will disappear within several months, it was argued that neither further diagnostics nor treatment is necessary. However, there is only limited research on clinical characteristics of follicular cysts. Our study showed a very high prevalence of DOR in Chinese women with follicular cysts and menstrual disorders, two thirds with abnormal uterine bleeding, so we argue that this indeed needs further medical care, especially in young women and nullipara. As we found a striking high and reproducible correlation to low AMH, we can recommend this as a first predictive marker.

2. Retrospective design and limited sample size may be limitations. However, our study aimed to assess the prevalence of DOR in Chinese women with follicular cysts and menstrual disorders, and we have not found similar published reports from other studies. We thus cannot assess the appropriate sample size based on the data from other comparable studies. As a supplement, we calculated that 93 subjects would provide 80% power of statistics analyses according to a 2-sided alpha level of 0.05, δ of 0.1, according to the overall prevalence of DOR in patients with follicular cysts and menstrual disorders was 86.3%. Moreover, because comparable studies are missing, our study is an exploratory study which does not compulsatory require a statistically sample size calculation but needs to verify whether the research direction is reasonable. Our study confirms that our research is reasonable and valuable. Based on these preliminary results we now can recommend a prospective study with a larger sample size (specially <40 years) using besides AMH to evaluate OR additional methods (e.g. AFC) to elucidate these women’s clinical characteristics further and discuss effective therapies.

Conclusions

The overall prevalence of DOR in Chinese women with follicular cysts and menstrual disorders was high, and even these young women (<40 years) also had a low level of AMH, which can be used as a marker to define DOR at least in Chinese women with higher sensitivity than other markers like FSH and E2, and a high prevalence of DOR. To what extent this also applies to non-Chinese women is unclear. Definitely, women with follicular cysts and menstrual disorders should receive special medical attention and evaluation from a healthcare professional, who may recommend the assessment of AMH to get more data on its value as a predictive marker also for other populations, including non-Chinese women.

Acknowledgements

We thank all our colleagues in the Department of Gynecological Endocrinology at Beijing Obstetrics and Gynecology Hospital, Capital Medical University, for their valuable assistance in coordinating this study and all the study participants. Additionally, we would like to acknowledge the editing and proofreading contributions of Pooja Dhungel, a native English speaker.

Disclosure statement

All authors declare that they have no conflict of interest.

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article.

Additional information

Funding

This study was supported by the National Menopause Health Care Specialist Construction Unit of China [(2020)30]; the Beijing Municipal Administration of Hospitals’ Ascent Plan (No. DFL20181401).