Menstrual cycle characteristics of young females with occult primary ovarian insufficiency at initial diagnosis and one-year follow-up with serum amh level and antral follicle count.

Primary ovarian insufficiency (POI) is a condition in which women under the age of 40 experience oligomenorrhea or amenorrhea for 4 months or more, in association with serum follicle stimulating hormone (FSH) levels in the menopausal range on two occasions obtained at least one month apart according to the most recent ESHRE guidelines [ 1 , 2 ]. POI affects 1 in 10,000 women by age 20, 1 in 1000 by age 30, and 1 in 100 by age 40 [ 3 ]. The term primary ovarian insufficiency appears to be a more appropriate to describe the condition that was previously referred to as premature menopause or premature ovarian failure because it differs from menopause in that there is a varying degree of residual ovarian function in nearly half of the cases allowing 5–10% of women to conceive and deliver a child [ 2 ]. In fact, the term POI describes a continuum of impaired ovarian function rather than a specific end point of permanent loss of ovarian function. It is also less stigmatizing than the other terms premature ovarian failure or premature menopause [ 4 , 5 ]. Occult form of POI was initially described as the triad of infertility, regular menses and elevated plasma FSH concentrations [ 6 ]. There is not an established serum AMH level to identify women with occult POI. But, AMH level (<1.1ng/mL) constitutes one of the Bologna criteria developed to define poor responding IVF patients as an hormone marker of diminished ovarian reserve (DOR) [ 7 ]. AMH level<1.1ng/ml in women ≤ age 30 was used as an indicator of DOR in infertile populations in other studies as well [ 8 , 9 ]. The diagnosis of occult POI is problematic as it may develop insidiously without development of oligo/amenorrhea and there is no a proper diagnostic method in its early recognition as mentioned above. This is particularly true for young females who are otherwise healthy, not interested in childbearing and have regular menses. Thus, any subfertility problem in these women might be hidden and occult POI as an underlying cause of it may escape from professional attention and remain undiagnosed until it progresses into more advanced stages of low ovarian function and finally becomes symptomatic as menstrual abnormality. Limited data is available in the literature regarding the prevalence of occult POI, the preceding menstrual characteristics and its natural course in otherwise healthy young females. We therefore aimed in this prospective observational study to determine the prevalence of occult POI in young females (< age 30) screened with serum AMH measurement; and analyze the patterns of change in their menstruation at initial assessment and at one-year follow-up in relation to quantitative assessment of ovarian reserve with AMH and AFC.

Demographic characteristics of the 963 participants are summarized in the Table 1 . In brief, the mean±SD of age and AMH of the participants were 20.1±2.4 and 3.7±1.8ng/mL respectively. 43 of them (4.5%) had serum AMH levels ≤ 1.1ng/mL. These cases therefore were diagnosed as having occult primary ovarian insufficiency (POI) according to the previous classification [ 8 ]. The remaining 920 participants had AMH levels>1.1ng/mL. The mean age, BMI, day of menstrual cycle at the time of blood sampling, and age at menarche were comparable between the participants with and without occult POI ( Table 1 ). The mean age, BMI, day of menstrual cycle at the time of blood sampling, and age at menarche were comparable between the participants with and without occult primary ovarian insufficiency (POI). The mean cycle length reported by the participants with occult POI were significantly shorter than those without it. Significantly higher number of participants with occult POI reported a positive family history of premature menopause in their mothers and/or sisters compared to those without occult POI. AMH: Anti-mullerian hormone BMI: Body mass index There was an inverse relation between age and serum AMH level on linear and non-linear regression models when all participants were included. Based on the adjusted R 2 statistics, non-linear quadratic regression model (R 2 = 0.26, p<0.0001) explained this association more optimally than does a linear type regression model (R 2 = 0.12, p<0.001) ( Fig 1 ). This is in line with the results of the previous studies showing that quadratic regression model fits better the decline in AMH with age than linear type [ 8 , 11 , 12 ]. Quadratic regression model explains this relation better than linear type. At initial assessment, thirty-eight (88.4%) of the females diagnosed with occult POI denied any menstrual irregularity in the last 12 months including those with more drastically diminished ovarian reserve (AMH1.1ng/mL who reported at least one abnormal non-specific menstrual cycle in the last year (p = 0.36). However, when the cycle length was questioned, it appeared that significantly higher proportion of females with occult POI were complaining of shortening of their cycles in the last twelve months compared to those without occult POI (60.4% vs. 4.4% respectively, p<0.0001). The cycle length was found to be significantly shorter in the participants with occult POI compared to those without it (25.1±3.2 vs. 31.2±2.8 days respectively, p<0.01)( Table 1 ). Overall, there was a positive association with AMH level and cycle length on the correlation (r = 0.32, p<0.01) and linear regression analyses (R 2 = 0.11, p<0.001). This association was more significant for the participants with occult POI (R 2 = 0.52, p<0.0001) compared to those without it (R 2 = 0.046, p<0.01) ( Fig 2 ). 26 of 43 participants (60.4%) with occult POI had a cycle length ≤25 days compared to only 41 of 920 participants without it (4.4%, p<0.0001). 6 out of 43 with occult POI (13.9%) were menstruating regularly at every 21 days and did not report any menstrual abnormality in the last year other than shorter cycles despite their critically decreased AMH level (the mean±SD: 0.59±0.2 range: 0.3–0.8 ng/mL). By contrast, none of the participants with AMH>1.1ng/mL had a cycle length of ≤ 21 days (p1.1ng/mL (20.9% vs. 7.2% respectively, p<0.01) ( Table 1 ). The association was more significant for the participants with occult POI compared to those without it. The participants who had been diagnosed with occult POI in the initial assessment were re-evaluated in a second visit 1.3±0.7 months later. Repeat AMH measurements at early follicular phase (0.78±0.2ng/mL range: 0.3–1.2) were comparable to the first readings (0.83±0.2ng/mL range: 0.33–1.1, p = 0.33) ( Table 2 , Fig 3 ). There was only one participant, whose AMH level increased from 1.01 at initial assessment to 1.2 in the second visit, exceeding the threshold level of 1.1 ng/mL. The mean antral follicle count (2.4±0.9) was significantly associated with AMH level (R 2 = 0.19, p = 0.003). Only two participants had elevated FSH (>25 mIU/mL) at early follicular phase with the remaining having normal FSH, LH and E 2 levels ( Table 2 ). Anti-thyroid and anti-adrenal antibodies were within their normal ranges (data not shown). Karyotype analysis was 46, XX and FMR-1 mutation screening returned normal in all of the 43 participants diagnosed with occult POI. Serum AMH level and AFC were significantly decreased and FSH level was significantly increased at one-year follow-up of the participants with occult POI compared to their corresponding values at initial assessment. The mean cycle length was significantly shortened and the number of the participants menstruating every 21 days were significantly increased compared to the initial assessment. While none of the participants diagnosed with occult POI had reported any skipped menses in the first assessment, now six of them reported one missed period at one-year follow-up. FSH: Follicle stimulating hormone LH: Luteinizing hormone AMH: Anti-mullerian hormone AFC: Antral follicle count BMI: Body mass index NS: Not significant The third visit took place 12.2±0.3 months later. All participants returned for this visit. Their repeat hormone tests revealed that AMH (0.37±0.1 vs. 0.83±0.2ng/mL respectively, p<0.001) and AFC (1.6±0.6 vs. 2.4±0.9 respectively, p<0.01) were significantly declined and FSH was significantly elevated (12.9±4.7 vs. 10.9±3.4 mIU/mL respectively, p<0.001) compared to their corresponding values at initial assessment ( Table 2 , Fig 4 ). There were no differences in the number of the participants who had undergone TA vs. TV USG between the second and third visits as the only four participants who had had undergone TA USG examination in the second and third visits. Review of the one-year menstrual record charts revealed that the mean cycle length (22.9±2.4 vs. 25.1±3.2 respectively, p<0.01) was significantly shortened and the number of the participants menstruating every 21 days were significantly increased (39.5% vs. 13.9% respectively, p = 0.013) compared to the initial assessment. While none of the participants diagnosed with occult POI had reported any skipped menses in the first assessment, now six of them (13.9%, p = 0.026) reported one missed period at one-year follow-up. Twenty-five participants underwent oocyte freezing. The others did not opt for any fertility preservation method for the following reasons: not interested in childbearing (n = 2), social (n = 8), financial (n = 6), other (n = 2).

This study underscores the importance of screening young females for possible occult POI as it may develop insidiously without any heralding signs of menstrual abnormality other than shorter cycles. It also appears that occult POI may deteriorate and menstrual abnormalities may begin as early as one year after diagnosis even though it may not fully evolve into clinically evident POI. Declining AMH levels in individuals with occult POI in a relatively short period of time as early as one year as we showed in this study contradicts with the classical description of this clinical entity as a continuum or persistence of impaired ovarian reserve or function. The option of oocyte cryopreservation increased public awareness of ovarian reserve testing in young females who are not currently interested in family building. It may help identify those women with occult POI. However, given that oocyte cryopreservation for social reasons might not be widely available or its regulations by law may change according to the countries, screening of young females with AMH for occult POI might be a valuable tool.

The study was approved by the Institutional Review Board of Istanbul Aydin University (IRB#: B.30.2.AYD.0.00.00–480.2/018). Since this study aims to identify young females with occult POI (defined as serum AMH level ≤ 1.1 ng/mL in females under age 30) all students under age 30 were invited to participate in the study using flyers in the main campus of Istanbul Aydin University. There were no pre-selection criteria for inclusion in the study. All participants who were within the required age range were evaluated regardless of their menstrual pattern. There were some exclusion criteria were as follows: the presence of metabolic-endocrine diseases, polycystic ovarian syndrome (PCOS), endometriosis and/or endometriomas, current or past use of oral contraceptive pills (OCP) or other drugs that may affect ovulatory function and hypothalamic-pituitary-ovarian axis (HPO), history of exposure to gonadotoxic chemotherapy regimens and/or radiation for malignant or non-malignant conditions, history of ovarian diseases or surgery, unilateral or partial oophorectomy. A total of 963 young female college students under age 30 voluntarily participated in this study after excluding 18 for OCP use (n = 4), PCOS (n = 12), history of surgical removal of unilateral endometrioma (n = 1), Hodgkin lymphoma survivor previously exposed to cyclophosphamide (n = 1). All participants gave informed consent. They were asked to fill a questionnaire regarding the status of their menstrual cycles, menstrual symptoms, life style, exercise, contraceptive use, personal and family history of endocrine and metabolic diseases, and premature ovarian failure (POF) ( S1 and S2 Tables (English and Turkish versions, respectively). The participants were first given information about normal patterns and length of menstrual cycle and its physiological variations that can erroneously be considered as abnormal pattern by the participants. Then, type of menstrual irregularity if any, was further detailed in the questionnaire and interviewed by the authors Y.G. and/or Y.A.A at the completion of the questionnaire. These authors have experience in the definitions and diagnoses of normal and abnormal menstrual patterns. Serum samples for hormone measurements were obtained by venipuncture and stored at -80°C until assayed. Those with AMH level ≤ 1.1 ng/mL were called for further workup and scheduled for a second visit at early follicular phase of the next cycle. In the second visit AMH test was repeated along with measurement of FSH, LH, E 2 , anti-thyroid (anti-peroxidase and anti-thyroglobulin) and anti-adrenal (21-hydroxylase, CYP-21) antibodies in the blood samples at early follicular phase. Antral follicle counts (AFC) (2-10mm) were determined, and ovarian pathology (such as endometrioma and dermoid cyst) were ruled out using ultrasound exam with Voluson E8 (General Electric Health Care, Istanbul, Turkiye) transvaginal or transabdominal probes. Transvaginal ultrasound was not performed in 4 out of 43 (9.3%) participants with occult POI due to their virginity. In these cases, transabdominal approach was performed. The ultrasonographers were blind to the AMH results. Genetic work up was also done for karyotype analysis and pre-mutation (61–199 CGG repeats) and intermediate (41–60 repeats) alleles in the FMR-1 gene. The participants were given a menstrual record chart to keep the records of menstrual cycles until the next visit one year later (no. of days from start of period to beginning of next, total duration of bleeding for each period, no. of pads used for each period, type of flow (exceptionally light, normal, exceptionally heavy, spotting), any abnormal pattern of bleeding (start day of cycle, duration, type of flow, associated symptoms, recurrence)( S3 and S4 Tables, English and Turkish versions respectively). In the third visit, one-year medical and surgical history and medications were questioned. AMH, FSH, LH, E2 and AFC were determined at early follicular phase. The cycle characteristics were obtained and mean cycle length was calculated after reviewing the records of the participant. There were no cases lost to follow-up. Serum samples were collected via venipuncture in the antecubital veins and stored in -80°C until assayed. Electrochemiluminescence immunoassay (ECLIA, Cobas) kits specific for estradiol (Elecsys ® Estradiol II), FSH (Elecsys ® FSH) and LH (Elecsys ® LH) were used according to manufacturers’s instructions. The lower detection limits for E 2, FSH and LH were 5.0 pg/mL, 0.100 mIU/mL, and 0.100 mIU/mL, respectively. All analyses were performed on Cobas ® 6000 analyzer series (Roche Diagnostics). AMH levels were determined using Active Mullerian Inhibiting Substance/ Anti-Mullerian Hormone (MIS/AMH) (Diagnostic Systems Laboratories, Inc., USA) ELISA kit. The analytical sensitivity of the kit was 0.006 ng/mL. Intra-assay repeatability and the coefficient of variations were given as 4.6% (0.144 ng/mL) and 2.4% (0.843 ng/mL), respectively. DNA isolated from peripheral blood lymphocytes by conventional techniques was used to determine the FMR1 CGG repeat length size in the participants diagnosed with occult POI. The repeat region was PCR amplified with fluorescently labeled primers and sized by capillary electrophoresis with confirmation of positives with Southern blot analysis. The following criteria suggested by ACOG was used to define the alleles: %40 repeats as normal, of 41–60 repeats as intermediate, of 61–199 repeats as pre-mutation, and of >200 repeats as full mutation alleles [ 10 ]. The data base of the all participants and those with occult POI were provided as SPSS data ( S1 and S2 Informations, respectively). Continuous data were expressed as the mean±SD and compared between the participants with and without occult POI using independent student t-test or Mann-Whitney-U test according to the distribution of the data. Categorical variables were compared using Fisher’s exact test. Two-tailed Pearson correlation test and linear and non-linear regression models were applied to explain the association of AMH with age. Paired t test was utilized to compare the continuous parameters of the participants with occult POI between the initial and one-year follow-up evaluations. The significance level was set at 5% (p< 0.05). SPSS statistical programs (version 23) were used to analyse the data and create the figures.

It includes the questions to gather information about baseline demographic and menstrual characteristics of the participants along with their personal and family history of diseases and surgeries that may affect ovarian reserve and timing of menopause. (DOCX) Click here for additional data file. It includes the questions to gather information about baseline demographic and menstrual characteristics of the participants along with their personal and family history of diseases and surgeries that may affect ovarian reserve and timing of menopause. (DOCX) Click here for additional data file. Every participant diagnosed with occult primary ovarian insufficiency (POI) was requested to keep the records of their one-year menstrual function using this record chart. (DOCX) Click here for additional data file. Every participant diagnosed with occult primary ovarian insufficiency (POI) was requested to keep the records of their one-year menstrual function using this record chart. (DOCX) Click here for additional data file. (SAV) Click here for additional data file. (SAV) Click here for additional data file.