Menstrual cycles, lifespan, and anti-Müllerian hormone in cynomolgus monkeys.

Cynomolgus monkeys ( Macaca fascicularis ) share close similarities with humans in terms of ovarian morphology, endocrinology, and unilateral single-oocyte ovulation. These similarities make them valuable models for various fundamental research areas, including assisted reproductive technology, longevity science, and neurological disorders [ 1 , 2 , 3 , 4 , 5 ]. However, whether non-human primates such as cynomolgus monkeys experience menopause and, if so, when this event occurs within their lifespan, remains unclear. Therefore, we investigated the lifespan and age at menopause in a group of cynomolgus monkeys observed daily for a long duration. Furthermore, although cynomolgus monkeys, like humans, have menstrual cycles, ovarian reserve trends—which are important for evaluating ovarian function—throughout their lifespan are poorly understood. The transition from ovarian reserve to menopause is also unclear. Anti-Müllerian hormone (AMH) and antral follicle count (AFC) are commonly used to assess ovarian reserve in humans [ 6 ]. However, assessing ovarian reserve using AFC in cynomolgus monkeys is not feasible because of the small size of the ovaries, which cannot be detected via abdominal ultrasonography. Additionally, the anal and vaginal tracts are too narrow for transvaginal ultrasound. Conversely, AMH levels remain relatively stable throughout the menstrual cycle, similar to patterns observed in humans [ 7 ]. The lack of significant fluctuations during the menstrual cycle makes AMH a useful marker for assessing ovarian reserve [ 8 , 9 , 10 ]. AMH is involved in the development of ovarian follicles, particularly in primordial, primary, and secondary follicles. Furthermore, AMH levels are significantly correlated with the number of these follicles, suggesting that AMH measurement is an effective indicator of ovarian follicle count and overall ovarian function in monkeys [ 11 , 12 , 13 ]. To our knowledge, no studies have been published analyzing ovarian function over the lifetime of cynomolgus monkeys. The purpose of this study was to investigate the age of menarche and menopause in cynomolgus monkeys, analyze changes in AMH levels throughout their lifespan, and evaluate their usefulness as experimental animals in ovarian function research.

This study was approved by the Institutional Review Boards of the Shiga University of Medical Science (registration number: 2023-4-12 [H2]) and the National Institutes of Biomedical Innovation, Health and Nutrition (registration number: DSR05-30R1)

This work was supported by JSPS KAKENHI Grant Number 24K1255.

The results showed a mean age at menarche of 3.69 ± 2.51 years, whereas menopause occurred at 27.00 ± 2.50 years, and the mean age at death was 32.04 ± 5.33 years. These findings indicate an average 5-year survival period after menopause. To analyze the lifelong changes in AMH, the plasma of 74 monkeys aged 0–33 years was investigated. Pearson’s correlation analysis showed a negative correlation between AMH levels and age ( Fig. 1 Fig. 1. AMH levels throughout the lifespan of the cynomolgus monkeys. Pearson’s correlation analysis showed a negative correlation between AMH levels and age (n=74). Analysis of AMH levels across all age groups revealed a moderately negative correlation with age (r=−0.46811, P< 0.0001). AMH, anti-Müllerian hormone; measured as ng/ml. ). Analysis of AMH levels across all age groups revealed a moderately negative correlation with age (r=−0.46811, P <0.0001). AMH levels throughout the lifespan of the cynomolgus monkeys. Pearson’s correlation analysis showed a negative correlation between AMH levels and age (n=74). Analysis of AMH levels across all age groups revealed a moderately negative correlation with age (r=−0.46811, P< 0.0001). AMH, anti-Müllerian hormone; measured as ng/ml. The animals were categorized into three groups as previously reported: Group 1 (0–3 years), Group 2 (4–11 years), and Group 3 (≥12 years). In Group 1, a strong positive correlation was observed between AMH levels and age (r=0.870331, P =0.00493), whereas in Group 2 (r=−0.00161, P =0.993), the levels remained stable. In Group 3, a moderate negative correlation was observed (r=−0.5899, P <0.0001) ( Fig. 2 Fig. 2. AMH levels by age groups. The monkeys were categorized into three groups: (a) Group 1 (0–3 years): n=8, (b) Group 2 (4–11 years): n=29, and (c) Group 3 (≥12 years): n=37. In Group 1, a strong positive correlation was observed between AMH levels and age (r=0.870331, P =0.00493), whereas in Group 2 (r=−0.00161, P =0.993) the levels remained stable. In Group 3, a moderate negative correlation was observed (r=−0.5899, P <0.0001). ). Furthermore, the analysis was conducted by dividing the subjects into three groups: premenarchal (0–2 years), menstruating (3–31 years), and postmenopausal (≥31 years). A monkey that reached menopause at age 27 was excluded as a case of premature menopause from menstruating group. The results showed no significant correlation between age and AMH in the premenarcheal group (r=0.51561, P =0.37388). In the menstruating group, a significant negative correlation was observed with age (r=−0.46275, P =0.00011). No significant correlation was found in the postmenopausal group (r=−0.3286, P =0.589269) ( Fig. 3 Fig. 3. AMH levels by menstrual status. The monkeys were categorized into three groups: (a) premenarchal (0–2 years): n=5, (b) menstruating (3–31 years): n=63, and (c) postmenopausal (≥31 years): n=5. A monkey that reached menopause at age 27 was excluded as a case of premature menopause from Fig. 3 (b) . The results showed no significant correlation between age and AMH in the premenarcheal group (r=0.51561, P =0.37388). In the menstruating group, a significant negative correlation was observed with age (r=−0.46275, P =0.00011). No significant correlation was found in the postmenopausal group (r=−0.3286, P =0.589269). ). AMH levels by age groups. The monkeys were categorized into three groups: (a) Group 1 (0–3 years): n=8, (b) Group 2 (4–11 years): n=29, and (c) Group 3 (≥12 years): n=37. In Group 1, a strong positive correlation was observed between AMH levels and age (r=0.870331, P =0.00493), whereas in Group 2 (r=−0.00161, P =0.993) the levels remained stable. In Group 3, a moderate negative correlation was observed (r=−0.5899, P <0.0001). AMH levels by menstrual status. The monkeys were categorized into three groups: (a) premenarchal (0–2 years): n=5, (b) menstruating (3–31 years): n=63, and (c) postmenopausal (≥31 years): n=5. A monkey that reached menopause at age 27 was excluded as a case of premature menopause from Fig. 3 (b) . The results showed no significant correlation between age and AMH in the premenarcheal group (r=0.51561, P =0.37388). In the menstruating group, a significant negative correlation was observed with age (r=−0.46275, P =0.00011). No significant correlation was found in the postmenopausal group (r=−0.3286, P =0.589269). From the 74 monkeys, ten that were ≥27 years of age were analyzed. Four were premenopausal, and six were postmenopausal. The oldest monkey with a menstrual cycle was 31 years old, and all monkeys aged ≥32 years were postmenopausal. AMH was slightly secreted even in postmenopausal monkeys ( Fig. 4 Fig. 4. AMH levels at ≥27 years. Twelve monkeys aged ≥27 years were analyzed. Four were premenopausal, and six were postmenopausal. The oldest monkey with a menstrual cycle was 31 years old, and all those >32 years were postmenopausal. Filled circles represent postmenopausal monkeys. Open circles represent premenopausal individuals. ). AMH levels at ≥27 years. Twelve monkeys aged ≥27 years were analyzed. Four were premenopausal, and six were postmenopausal. The oldest monkey with a menstrual cycle was 31 years old, and all those >32 years were postmenopausal. Filled circles represent postmenopausal monkeys. Open circles represent premenopausal individuals. Analysis of 63 individuals for whom weight at blood collection was known revealed no correlation between body weight and AMH levels (r=−0.06361, P =0.620356) ( Fig. 5 Fig. 5. AMH levels and body weight. 63 individuals have no correlation between body weight and AMH levels (r=−0.06361, P =0.620356). ). AMH levels and body weight. 63 individuals have no correlation between body weight and AMH levels (r=−0.06361, P =0.620356).

In this study, the menstruation, lifetime AMH levels, and life span of cynomolgus monkeys were investigated. To our knowledge, this is the first report to document the menstruation and lifespan of cynomolgus monkeys. The results showed that menstruation begins at the age of 3.69 ± 2.51 and menopause at the age of 27.00 ± 2.50. Life expectancy was 32.04 ± 5.33 years. The mean survival after menopause was 5.0 ± 4.3 years. This corresponds to approximately 14.3% of their lifetime. The average life expectancy of a human female in Japan is 86.9 years. Menopause occurs at approximately 50 years of age, and the lifespan after menopause is extremely long, accounting for approximately 42.5% of the lifetime. Similar to humans, cynomolgus monkeys have a postmenopausal survival period. Although this survival period is shorter than that in humans, this indicates that older cynomolgus monkeys, particularly those over 27 years old, can be used as experimental animals in investigating aging and menopause. We also investigated the reproductive age and lifespan of males. The age at which males last impregnated a female was approximately 19 years (6,898 ± 1,209 days), and the lifespan calculated from 37 individuals that died at the age of 20 or older was approximately 27 years (9,974 ± 1,529 days). This indicates that males continue to survive for a long time even after losing their reproductive capacity, and it was also found that females live longer than males. The lifetime AMH levels in cynomolgus monkeys were also examined to determine changes in ovarian reserve over their lifespan. Our findings suggest that the trajectory of AMH throughout the lifetime of cynomolgus monkeys is similar to that of humans. A similar study by Long et al. , who analyzed age-related AMH levels in cynomolgus and rhesus macaques ( Macaca mulatta ) monkeys aged between 1 and 19 years, reported that the correlation between AMH levels and age was not statistically significant (r=−0.044) [ 14 ]. Their study was limited to subjects aged 1 to 19 years. In our study, the age of menopause onset was 27 years, and other papers also report menopause onset in rhesus macaques around 25 years. In other words, their study lacks data around the time of menopause [ 15 ]. This may explain why they did not find a significant correlation between age and AMH levels. In contrast to Long et al.’s study, our study included subjects aged 0–33 years. To our knowledge, this is the first study to analyze AMH levels in cynomolgus monkeys across all age groups, from infancy to post menopause. We observed a decline in AMH levels with age, which showed a weak negative correlation. Additionally, we categorized the animals into three age groups (0–3 years, 4–11 years, and ≥12 years) and investigated the correlation between age and AMH levels, as previously reported [ 14 ]. In the 0–3-year group, which corresponds to the pre-pubertal period in humans, AMH levels increased with age, showing a strong correlation. In the 4–11-year group, which corresponds to the prime fertility period, no correlation was observed. In the ≥12-year age group, which corresponds to the post-fertility period, AMH levels declined with age and showed a moderate negative correlation. These patterns of AMH changes are similar to those observed in humans when categorized by age into three groups: 0–15 years, 16–25 years, and ≥26 years [ 16 ]. A systematic review by Bhide et al. reported that AMH levels in humans tend to be elevated during childhood [ 17 ], suggesting the gonadotropin-independent replenishment of oocytes in the follicular pool [ 18 ]. A slight decrease in serum AMH levels during puberty has been attributed to the redistribution of the follicular pool [ 17 ]. Complex patterns of AMH levels during childhood and adolescence have been described, which deviate from the steady decline observed in primordial follicles during this period, from childhood to puberty. In cynomolgus monkeys, the variations in AMH levels follow a similar pattern to that observed in humans, suggesting that the follicular pool may function similarly in both species. AMH levels after puberty decrease similarly in both humans and cynomolgus monkeys. This is due to a reduction in the number of ovarian follicles. Furthermore, they were divided into three groups: premenarchal (0–2 years), menstruating (3–31 years), and postmenopausal (≥31 years). Only in the menstruating group, a significant negative correlation was observed with age (r=−0.46275, P =0.00011). However, the premenarchal and postmenopausal groups are too small to evaluate (premenarchal group: n=5, postmenopausal group: n=5). Another notable result of our study is that AMH was detected in the blood of newborn cynomolgus monkeys. This suggests that, similar to humans, AMH is produced by granulosa cells in fetal cynomolgus monkeys. The human ovary is composed of millions of germ cells or primordial follicles that form around the fifth month of gestation. These primordial follicles constitute the true ovarian reserve, which steadily decreases from birth to menopause through ovulation and follicular atresia. AMH is a glycoprotein secreted by the granulosa cells of small growing follicles and indirectly reflects the primordial follicle pool [ 19 ]. The detection of serum AMH at birth suggests that it may have been secreted by fetal granulosa cells in the uterus. The results of the present study suggest that the progression of ovarian function throughout the lifespan of cynomolgus monkeys is similar to that of humans. The similarity of ovarian structure as well as ovarian function of cynomolgus monkeys to humans may inform research in reproductive medicine, such as ovarian tissue freezing for fertility preservation in children with cancer, nephrotic syndrome [ 20 ], Turner syndrome [ 21 ], and systemic lupus erythematosus [ 22 ]. As the medulla comprises 10–30% of the ovary in younger children [ 23 ], separating the ovarian cortex from the medulla is challenging [ 24 ]. Future studies in experimental animals are needed to determine whether this method is also indicated or applicable for the freezing of ovarian tissue in human children. For this purpose, the ovaries of cynomolgus monkeys under 3 years of age may be useful as models for pediatric ovaries. We also analyzed cynomolgus monkeys aged ≥27 years in the present study, as the aforementioned data showed that menopause occurs at this age. The oldest monkey with menstruation was 31. A small amount of AMH was also found to be secreted by the postmenopausal monkeys. AMH was low in all monkeys aged 27 years and over, and nearly the same between premenopausal and postmenopausal monkeys. This indicates that while AMH levels can be used to infer a decline in the ovarian reserve, predicting menopause based solely on AMH levels may be challenging. Previous reports comparing AMH levels in cynomolgus monkeys before and after bilateral ovariectomy showed an average AMH concentration of (5.8 ± 0.42 ng/ml vs. <0.05 ng/ml, P <0.001) [ 25 ]. That is, AMH levels fell below the detection limit after bilateral ovariectomy. The detection of trace amounts of AMH even in postmenopausal individuals in this study suggests that primordial and primary follicles may still be present in small numbers within the ovaries after menopause. Nevertheless, menopause may occur because the hypothalamic–pituitary–gonadal axis ceases to function. Additionally, the correlation between body weight and AMH was analyzed, but no significant correlation was observed. Previous report also indicated no correlation between BMI and AMH in cynomolgus monkeys [ 14 ]. The present study showed that cynomolgus monkeys, like humans, have pre-menstrual and postmenopausal periods. Results also showed that AMH remained high during reproductive age, when the menstrual cycle was present; however, AMH declined with age. These findings suggest that cynomolgus monkeys can be useful as experimental animals throughout all ages. The limitations of this study include the large inter-individual variability in the AMH levels among the studied monkeys and small sample size. The sample size of 74 subjects, while relatively small compared to large-scale epidemiological studies in humans, is considered substantial for studies involving non-human primates, particularly due to the ethical, logistical, and financial constraints inherent in such research. For example, Susan et al. reported an association between AMH and ovarian reserve using a cohort of only 29 cynomolgus monkeys [ 25 ]. In this context, our study’s sample size is relatively large. Furthermore, our cohort spans a wide age range from 0 to 33 years, which enables a comprehensive evaluation of age-related changes in AMH across the lifespan. Therefore, despite the numerical limitations, the sample size is scientifically valid and meaningful within the context of non-human primate research, and the study offers unique insights that are rarely achievable in this field. In addition, the Lumipulse® G was designed for human serum samples. We also tested several samples using the AMH ELISA kit for Squirrel Monkey Serum (AMH ELISA, AL-105, Ansh Labs, Webster, TX, USA). Following the methodology of a previous study in which this kit was used on cynomolgus monkeys, the present experiment was conducted using this kit [ 14 ]. Three samples were analyzed using a monkey AMH assay kit and 2-fold, 4-fold, 8-fold, and 16-fold dilutions were made, respectively. The results showed that the values decreased in proportion to dilution. In addition, results obtained using the human AMH assay kit values were detectable at half the dilution needed as that in using the kit for monkeys. Therefore, we considered that analyzing the changes in AMH levels using the human AMH assay kit was appropriate, mechanically simple, and easy. These results are based on analyses of cynomolgus monkeys in captivity and may differ from those of wild monkeys. The cynomolgus monkeys used in this study were kept in captivity, and their lifespans were not natural. Furthermore, it cannot be confirmed that the environment—including nutrition, stress, and pathogens—was optimized to maximize the monkeys’ lifespans. In conclusion, to our knowledge this is the first report to assess ovarian reserve by analyzing changes in AMH levels and menstruation over the lifetime in cynomolgus monkeys. Their similarity to humans suggests that cynomolgus monkeys can be useful as experimental animals at all ages.

All authors report no conflicts of interest to be disclosed.

Twenty-one cynomolgus monkeys with known dates of first menstrual periods and 22 cynomolgus monkeys that were confirmed to have reached menopause and lived out their natural lifespan, were included in this study. Age at menopause and death was ascertained from retrospective records. Menopause was defined as no menstruation for more than one year. Another 74 cynomolgus monkeys, ranging in age from 0–33, were included for the analysis of lifespan changes in AMH. Serum samples were isolated from blood samples collected via the femoral or radial veins of the monkeys regardless of their menstrual cycle. The monkeys were maintained at the Research Center for Animal Life Science, Shiga University of Medical Science, and the Tsukuba Primate Research Center. The two facilities were nearly identical with regards to housing the monkeys. All monkeys were housed in stainless steel cages at 23°C to 28°C with 50% to 70% humidity, 12 air changes/h, and a 12/12-h light/dark cycle, and they were fed 70 g of commercial monkey chow (CMK-2; CLEA Japan, Inc., Tokyo, Japan) and 200 g of fruit daily. These monkeys were individual housing at this experimental period. All animal experiments were conducted in accordance with the guidelines of the Ethics Committee for Biomedical Research at Shiga University of Medical Science and National Institutes of Biomedical Innovation, Health and Nutrition. Blood samples were collected from the femoral vein between April 2023 and February 2024, regardless of the menstrual cycle. This study excluded individuals with endometriosis, as well as those who were pregnant or lactating. No experiments affecting reproductive function have been conducted in the past. The blood samples were transferred to tubes for centrifugation, and serum AMH concentrations were measured using a chemiluminescent enzyme immunoassay with Lumipulse ® G (Fujirebio Diagnostics Japan, Inc., Tokyo, Japan). The intra-assay coefficient of variation (CV) was 3.42%, and the inter-assay coefficient of variation (CV) was 6.83%. Linear regression models and Spearman’s rank correlation coefficients were used to examine the association between serum AMH levels and age. All analyses were performed using SPSS software (version 29; IBM Corp., Armonk, NY, USA).