{"paper_id":"2e5038a9-6b1c-4da6-8946-0cc8c41a5916","body_text":"Infertility is of great medical importance and produces relevant socioeconomic impact, as it affects more than 186 million people worldwide. Factors directly related to this condition range from female age and previous illnesses to the unwanted period of inability to conceive ( Jacobson  et al ., 2018 ).\nOne of the most used methods in high complexity assisted reproduction is intracytoplasmic sperm injection (ICSI), which has become a viable option with high chances of success for patients seeking conception. The level of anti-Müllerian hormone (AMH) has become an important element in the initial investigation of female patients and is of great importance throughout the process ( Jacobson  et al ., 2018 ). AMH is a glycoprotein produced by granulosa cells in primary, preantral and small antral follicles, and its clinical applicability has been demonstrated in several studies ( Ebner  et al ., 2006 ;  La Marca & Volpe, 2006 ;  Gomez  et al ., 2016 ). In the literature, there is strong evidence that AMH is a suitable indicator to assess ovarian reserve and predict the probability of achieving pregnancy ( Nelson  et al ., 2009 ;  Peluso  et al. , 2014 ;  da Silva  et al ., 2014 ;  Gomez  et al ., 2016 ). Ovarian reserve is defined as the number of ovarian follicles that might be available for use in fertilization, representing a woman's fertile ovarian potential ( Nelson  et al ., 2009 ;  Romão & Navarro, 2013 ;  Peluso  et al ., 2014 ;  da Silva  et al ., 2014 ;  Gomez  et al ., 2016 ). Thus, the assessment of ovarian reserve assumes an important role in the attempt to estimate reproductive capacity, which needs to be calculated and informed to patients before treatment initiation.\nAn association has been established between poor ovarian response and decreasing levels of AMH. AMH levels decrease substantially with age, which consequently leads to considerably lower probabilities of achieving of pregnancy ( La Marca & Volpe, 2006 ;  Visser  et al ., 2006 ;  Tal & Seifer, 2017 ). According to a study published by  Khan  et al . (2019) , AMH levels decrease by about 6% a year in older women, showing the direct relationship existing between increased female age and declines in follicular supply. However, female age is not an absolute predictor of reproductive capacity ( Khan  et al ., 2019 ). In this context, AMH appears as an excellent predictor of fertility.\nA study developed by the POSEIDON (Patient Oriented Strategies Encompassing Individualize Oocyte Number) group proposed a new stratification in assisted reproduction. In the study, the ideal AMH level used for comparison was 1.2 ng/mL, based on the idea that women with at least this level of AMH might have better outcomes in terms of number of oocytes generated. This assumption was confirmed in the study, which showed an association between AMH and female age ( Humaidan  et al ., 2016 ).\nAlthough AMH is primarily related to the quantity rather than the quality of oocytes, the higher the AMH levels, the greater are the expectations around the availability of oocytes and ultimately embryos for transfer ( Anderson  et al ., 2012 ).\nPatients must be informed at the beginning of treatment about their ovarian reserve and the potential associations with age, so that they develop more realistic expectations about their response to ovulation induction treatment and become aware of the strong relationship between AMH levels and the probability of achieving pregnancy ( Nelson  et al ., 2009 ;  Peluso  et al ., 2014 ;  da Silva  et al ., 2014 ;  Gomez  et al ., 2016 ). Research in this area is of great importance and consequence, since it may lead to better approaches based on each patient's profile. This study aimed to find whether there is an association between AMH levels, age, and number of retrieved and mature oocytes of Brazilian patients undergoing assisted reproductive technology (ART) treatments.\n\nThis descriptive retrospective observational study included about 1,500 electronic medical records of patients treated at an ART clinic in Curitiba, Paraná, Brazil, between July 2012 and April 2019. Patients not submitted to IVF and/or without AMH level records were excluded. The study included women aged 20-50 years of age submitted to IVF diagnosed with fertility issues.\nThe patients included in the study were divided into groups based on age and AMH level. The AMH cutoff level was 1.2 ng/mL, as described in the POSEIDON study ( Humaidan  et al ., 2016 ).\nThe collected data included patient age, start and duration of treatment, diagnosis and reason for infertility, AMH level, number of metaphase II (MII) oocytes retrieved, and IVF outcome. Data points were statistically analyzed based on quantitative, qualitative, partial, absolute, and comparative parameters.\n\nDuring the study period, 1,440 patients underwent ART treatment at the clinic. A total of 733 patients aged 20-48 years were enrolled ( Table 1 ) after inclusion and exclusion criteria were considered. However, ART outcomes were available for only 681 of the 733 patients. Live birth rates after 21 weeks of pregnancy were significantly higher among younger patients (aged 35 years and younger). Older patients were more likely to having no oocytes retrieved ( p =0.0224) ( Table 2 ). All other ART outcomes were similar between the groups.\nDiagnosis of patients seen at assisted human reproduction clinic Conceber during the years 2012 and 2019.\nOutcomes per age group of assisted reproductive technology treatments of patients seen at Conceber Clinic in 2012-2019.\nDifferent capital letters between lines in the same column represent significant differences between groups ( p <0.05).\nA total of 717 patients were divided into three age groups (age ≤ 35 years; age between 36 and 39 years; age ≥ 40 years). Mean AMH levels were significantly different between the groups and ranged from 2.65 to 1.35 ng/mL ( Table 3 ).\nAMH levels and number of retrieved and mature oocytes (mean ± standard error) per study group of patients seen at assisted human reproduction clinic Conceber in 2012-2019.\nDifferent capital letters between lines in the same column represent significant differences between groups ( p <0.05).\nThe mean number of retrieved oocytes was significantly different between the groups and ranged from 9.5 to 5.42 ( Table 3 ). The mean number of mature oocytes in the patient groups ranged from 7.14 to 4.58 ( Table 3 ). Patients aged ≤ 35 years had a greater number of mature oocytes when compared to individuals in the other groups. There was no significant difference in the number of mature oocytes in the groups of patients aged 36-39 years and of patients aged ≥ 40 years.\nThe correlations between AMH level, age, and number of retrieved and mature oocytes are shown in  Table 4 . A negative correlation was observed between age and number of retrieved (r= -0.3354,  p <0.0001) and mature oocytes (r= -0.2839,  p <0.0001). AMH level was negatively correlated with age (r= -0.3257,  p <0.0001). Interestingly, AMH level was positively correlated with number of retrieved (r=0.6702,  p <0.0001) and mature oocytes (r=0.5770,  p <0.0001).\nCorrelations (r) and significance level (p) between measured response variables.\n\nAMH is a glycoprotein secreted by granulosa cells of developing ovarian follicles ( Siddiqui  et al ., 2019 ). It is involved in folliculogenesis and reflects the number of primordial follicles ( Seifer & Maclaughlin, 2007 ;  Pellatt  et al ., 2010 ;  Tobler  et al ., 2015 ). AMH is believed to regulate the number of growing follicles and those that will be selected for ovulation. ( Peluso  et al ., 2014 ). AMH is considered an inhibitor of the early stages of follicular development ( La Marca & Volpe, 2006 ). This means that AMH has an inhibitory effect on initial follicular recruitment, thus preventing the premature depletion of primordial follicles ( Durlinger  et al ., 1999 ;  2001 ;  Kedem  et al ., 2013 ;  2014 ;  Pankhurst, 2017 ;  Granger & Tal, 2019 ). AMH has shown to have a strong influence on ovarian function, especially on follicle growth ( Rey  et al ., 2000 ;  Visser  et al ., 2006 ), and has a good correlation with female age, antral follicle count, and ART outcomes ( Fanchin  et al ., 2003 ;  Nikolaou & Gilling-Smith, 2004 ;  Nikolaou, 2008 ;  de Vet  et al ., 2019 ). These findings have allowed the widespread use of AMH in the field of gynecology, from IVF to the diagnosis of different ovarian diseases ( Dewailly & Laven, 2019 ;  Bedenk  et al ., 2020 ).\nOvarian stimulation is an essential step in ART treatments. The administration of exogenous hormones aims at yielding an adequate ovarian response ( Broekmans  et al ., 2014 ;  Balachandren  et al ., 2020 ). Ovarian response measured by the number of retrieved oocytes decreases with age due to progressive ovarian reserve reduction ( Hansen  et al ., 2008 ;  Wallace & Kelsey, 2010 ). Decreases in ovarian reserve are an important cause of female infertility. Low ovarian response may occur in ART in 10-20% of cases and increases with age ( Grisendi  et al ., 2019 ). Therefore, the assessment of ovarian reserve is an extremely important step in IVF cycles with ovarian stimulation, since it allows the identification of patients at risk of low or excessive ovarian response ( Alebic  et al ., 2018 ;  Bedenk  et al ., 2020 ). Once it is not possible to directly determine the number of  in vivo  follicles, indirect measurements of the ovarian reserve can be made through biochemical and ultrasound markers ( Broekmans  et al ., 2006 ). AMH is considered the most sensitive marker for ovarian reserve. AMH offers an accurate, direct measurement of the ovarian follicle pool, and can predict the ovarian response and the number of retrieved oocytes from follicular aspiration ( Grisendi  et al ., 2019 ). AMH concentration assessment offers advantages over other markers such as baseline FSH and antral follicle count, since it is not limited to one phase of the menstrual cycle, does not introduce interobserver variation, and is quite reliable, although there is no internationally standardized trial ( Fleming  et al ., 2015 ;  Grossman  et al ., 2017 ;  Tal & Seifer, 2017 ;  Granger & Tal, 2019 ;  Maged  et al ., 2020 ).\nIn 2012, the American Society of Reproductive Medicine (ASRM) concluded that AMH was a useful tool for predicting ovarian reserve in patients undergoing IVF treatments, as well as for women at risk for decreased ovarian reserve ( Gianaroli  et al ., 2012 ). The present study showed that AMH levels decrease significantly with age ( p <0.001). The negative correlation observed in the present study between AMH and age had been reported in the literature. ( Nardo  et al ., 2007 ;  Bentzen  et al ., 2013 ;  Keane  et al ., 2017 ;  Loy  et al ., 2017 ;  Massarotti  et al ., 2020 ). This result was expected, since AMH represents the pool of oocytes existing in the ovary ( Nikolaou, 2008 ;  Iliodromiti  et al ., 2014 ).  van Rooij  et al . (2004)  followed 81 patients aged 26 to 45 years for four years. All patients had regular menstrual cycles. Patients who subsequently developed irregular cycles had lower AMH levels than women with regular cycles (0.3 and 1.7 ng/mL, respectively,  p <0.001).  Hansen  et al . (2008)  observed that AMH levels were significantly higher in women of reproductive age when compared to perimenopausal women.  Fatima  et al . (2020)  described similar results in a study that looked into the differences in AMH levels in different age groups. The authors reported a significant difference in AMH levels between the studied groups. According to  Gomez  et al . (2016) , serum AMH levels decrease annually by approximately 0.384µg/L. In addition, the levels of this hormone are related to the number of oocytes available in the patient. A study by  Steiner  et al . (2011)  evaluated several candidate molecules for markers to predict fertility, including FSH, estradiol, AMH, and inhibin B (plasma levels) and FSH and estrone-3-glucuronide (E3G) in the urine of patients. This study included women aged 30-44 years and showed that only AMH was significantly associated with natural fertility. Some authors believe that the decreases seen in AMH levels with aging may be accelerated due to premature ovarian failure and exposure to gonadotoxic chemotherapy ( Méduri  et al ., 2007 ;  Dólleman  et al ., 2014 ;  Dunlop & Anderson, 2015 ).\nAccording to  Fatima  et al . (2020) , AMH reflects the ovarian reserve and is a predictor of success for several ART treatments. In patients undergoing ART therapies, serum AMH is the best endocrine indicator of follicular response to ovarian stimulation compared to other commonly used markers, such as FSH, estradiol, inhibin B, and patient age alone ( Arce  et al ., 2014 ;  Hawkins Bressler & Steiner, 2018 ). Compared to FSH, AMH seems to be more strongly associated with age ( van Rooij  et al ., 2005 ;  de Vet  et al ., 2019 ); in addition, AMH levels decrease before increases in FSH can be detected ( de Vet  et al ., 2019 ). Therefore, serum AMH has been considered a more sensitive marker of ovarian reserve than FSH. Although serum AMH levels are controversial for the clinical pregnancy rate in ART, this assessment is still useful to verify the ovarian response to stimulation and to better adjust treatments for patients ( Dewailly & Laven, 2019 ). Age and serum AMH seem to be independent predictors of ovarian reserve and ovarian stimulation outcome in infertile women ( Scheffer  et al ., 2018 ).\nAccording to the ASRM and the European Society for Human Reproduction and Embryology (ESHRE), AMH has the best sensitivity and specificity for measuring ovarian response to controlled ovarian hyperstimulation ( Gianaroli  et al ., 2012 ). A study conducted by  Daney de Marcillac  et al . (2017)  found that patients with normal serum AMH levels had better ovarian stimulation, lower cancellation rates, greater numbers of recovered oocytes after follicular puncture, and higher pregnancy rates. The authors also found that patients with low AMH levels had fewer oocytes retrieved. A study by  Melado Vidales  et al . (2017)  showed that AMH levels measured during IVF predicted patient ovarian response during the days with follicular growth. The results of the present study support the literature ( Fleming  et al ., 2006 ;  Nelson  et al ., 2007 ;  Granger & Tal, 2019 ;  Zhang  et al ., 2019 ), since patients with higher serum AMH levels showed a significant positive correlation with the total number of retrieved oocytes after follicular puncture. Our data demonstrated that AMH might help predict the number of oocytes produced.\nThe present study also demonstrated a significant positive correlation between serum AMH and number of mature oocytes. Other authors have suggested that higher serum AMH levels might indicate better oocyte quality, including maturation capacity ( Ebner  et al ., 2006 ;  Gomez  et al ., 2016 ;  Borges  et al ., 2017 ). Although a correlation between embryo quality and chance of blastocyst formation had not been observed, AMH levels were correlated with oocyte quality. In the present study, a significant negative correlation was observed between patient age and number of mature oocytes. AMH levels ranged from 1.35±0.2 to 2.65±0.16 ng/mL. Although AMH levels and number of retrieved oocytes were higher in patients aged 36-39 years than in individuals aged > 40 years, the number of mature oocytes did not differ between these individuals. However, both groups had fewer mature oocytes than patients aged ≤ 35 years. This finding may be related with the median AMH levels seen in the individuals included in this study and explained by the fact that AMH is secreted only by the granulosa cells of preantral follicles and small antral follicles ( Maged  et al ., 2020 ). The mean serum AMH level was approximately 4 ng/mL in young women with good ovarian reserve. However, recently,  La Marca  et al . (2016)  described cases with levels below 1 ng/mL and low ovarian response, and levels above 3 ng/mL and high response. According to  Ebner  et al . (2006) , oocytes obtained from patients with an intermediate AMH level (between 1.66 and 4.52 ng/mL) have better morphology. For  Tal & Seifer (2017) , the AMH reference values are age-appropriate and do not refer to a general population of women, regardless of age. These authors considered lower limits of serum AMH of 0.5 ng/mL for women aged 45 years, 1ng/mL for women aged 40 years, 1.5 ng/mL for women aged 35 years, 2.5 ng/mL for women aged 30 years, and 3 ng/mL for women aged 25 years. However, it is important to consider that the ESHRE consensus criteria considered AMH levels < 0.5-1.1 ng/mL as indication of low ovarian reserve ( Ferraretti  et al ., 2011 ). To date, several limits have been suggested for AMH in predicting ovarian response to stimulation. However, there is no uniform agreement due to differences in protocols and populations and AMH test kits ( Shahrokhi  et al ., 2018 ). This means that AMH level results should be used and interpreted with caution.\nPatient age is an important factor linked to oocyte quality in IVF cycles. The lower chance of older patients becoming pregnant after IVF is due to poorer response to ovarian stimulation, worse oocyte quality, and higher aneuploidy rates ( Franasiak  et al ., 2014 ;  Scheffer  et al ., 2017 ). Our study showed that the number of retrieved oocytes decreased with patient age (<0.001). Age was negatively correlated with the number of retrieved and mature oocytes. As age advances, an inhibitory effect of AMH on the recruitment of follicles becomes possible ( Nelson  et al ., 2009 ;  Pacheco  et al ., 2017 ).\nRegarding the quality of oocytes, some authors believe that a low ovarian reserve might increase the chances of aneuploidy. Several studies suggest that low ovarian reserve and the consequent absence of adequate follicles for selection increase the likelihood of aneuploid oocytes being harvested ( Warburton, 1989 ;  Haadsma  et al ., 2010 ;  Rosen  et al ., 2011 ;  Grande  et al ., 2015 ;  Pankhurst, 2017 ). Total chromosomal non-disjunction and early separation of sister chromatids are correlated with maternal aging. Changes in sister chromatids cohesion may be a causal mechanism that predisposes premature separation and consequently non-disjunction during meiosis. In addition, the asymmetry of the female meiotic division might favor the non-random segregation of chromosomes and chromatids ( Johnson  et al ., 2007 ;  Scheffer  et al ., 2018 ). Oocyte aging causes increased damage to mitochondrial DNA and decreased oxidative phosphorylation and ATP production. Mitochondrial mutations in follicular cells around the oocyte have been correlated with maternal age, suggesting that oxidative phosphorylation in the follicle is compromised ( Smeenk  et al ., 2007 ). Aneuploid oocyte rates increase with age. Women under 35 years of age have an embryo aneuploidy rate of 53% in IVF, while older women present significantly higher aneuploidy rates (74% in patients aged 41-42 years and 93% in women aged 42+ years) ( Harton  et al ., 2013 ).\nAMH can be considered an indirect way of measuring the number of preantral and primordial follicles. It is a useful predictor for the pool of follicles that will be ready for final development in the next 3-5 months. Since the number of \"selectable\" follicles remains approximately constant over time, it can be speculated that AMH levels might predict response to controlled ovarian stimulation. On the other hand, since the number of follicles ready to mature depends on the primordial follicle pool, AMH can be used safely to estimate the ovarian reserve. To date, there is no direct method (except for histological evaluation) for measuring the size of the primordial follicle pool ( Gasca  et al ., 2007 ). However, little is known about the relationship between variations in AMH levels and the qualitative results of ART treatments (implantation, pregnancy, and birth). A recent Brazilian study showed that age negatively affected embryo or blastocyst quality at day 3 (women aged 35+ years) in subjects with low serum AMH levels (<1ng/mL) and antral follicle counts of less than seven ( Scheffer  et al ., 2021 ).\nOur data showed that a Brazilian cohort with advanced age had significant decreases in AMH levels and low numbers of retrieved and mature oocytes. However, we found no significant differences in the number of retrieved and mature oocytes between patients aged 36 years or older. We also found a positive correlation between serum AMH levels and the total number of retrieved and mature oocytes. Although this study did not report new data compared to the literature, to our knowledge this is the largest study enrolling Brazilian IVF patients in which correlations between AMH levels, age, and total number of oocytes and mature oocytes from stimulated cycles were described.","source_license":"CC-BY-4.0","license_restricted":false}