The wave-like developmental pattern of follicles ≥1 mm in temporal relationship with follicle stimulating hormone (FSH) and the existence of 2- and 3-waves of follicular development during an interovulatory interval (IOI) have been clearly defined in cattle. However, information about the developmental pattern of antral follicles <1 mm and the repeatability of the wave pattern (2- or 3-wave IOI) is lacking. Using approaches such as immunization against GnRH (to suppress circulating concentrations of FSH) and histomorphometric study of ovarian tissues collected from cyclic heifers on different days after ovulation, the developmental pattern of antral follicles <1 mm and the role of FSH in their development were studied in heifers. Ultrasonographically acquired follicular data were used to determine the repeatability of 2- and 3-wave patterns and the effect of season on the wave patterns. The ovulatory follicle in 3-wave IOI is exposed to a shorter term high-progesterone environment than that of 2-wave IOI, and it has been argued that the less-aged ovulatory follicle of 3-wave IOI yields a more fertile oocyte than the 2-wave IOI. The developmental competence of oocytes in preovulatory follicles of 2- versus 3-wave IOI was compared using in vivo environments created to mimic short-term low- and high-progesterone environments similar to 2- and 3-wave IOI, respectively. The developmental competence of oocytes in persistent dominant-type follicles was also determined.<p>The vaccination against GnRH attenuated FSH surges but did not suppress the basal circulating concentrations of FSH. The attenuation of FSH surges suppressed the wave-like emergence of follicles ≥4 mm but not of the antral follicles <4 mm. The study revealed an inverse relationship between the mean and peak circulating concentrations of FSH and the number of follicles recruited into ≥1 mm size category. Histomorphometric study revealed that antral follicles <1 mm developed in a wave-like fashion in response to a rise in the circulating concentrations of FSH. After treatment with exogenous FSH, the growth rate of follicles in GnRH-immunized heifers was similar to controls. <p>The duration of IOI was predictive of the wave pattern (i.e., 2- or 3-wave IOI), and the pattern was repeatable within individuals throughout the year. The dominant follicle of Wave 1 in 2-wave IOI had a longer duration of dominance than in 3-wave IOI. Hence, the dominant follicle of Wave 1 may have a primary role in the regulation of 2- and 3-wave patterns. Greater attrition of follicles in 3-wave IOI, due to the emergence of an extra wave compared to 2-wave IOI, may contribute to earlier follicular depletion and onset of reproductive senescence in heifers with primarily a 3-wave pattern. The fertilization capacity of oocytes that were exposed to the short-term low-progesterone environment (i.e., similar to the early growing phase of the ovulatory follicle of 3-wave IOI) was increased, but the developmental competence post-fertilization was not different from oocytes that were exposed to a short-term high-progesterone environment (i.e., similar to the early growing phase of preovulatory follicle of 2-wave IOI). Multiple follicles developed under the prolonged-low progesterone environment, but failed to ovulate.

The wave-like developmental pattern of follicles ≥1 mm in temporal relationship with follicle stimulating hormone (FSH) and the existence of 2- and 3-waves of follicular development during an interovulatory interval (IOI) have been clearly defined in cattle. However, information about the developmental pattern of antral follicles <1 mm and the repeatability of the wave pattern (2- or 3-wave IOI) is lacking. Using approaches such as immunization against GnRH (to suppress circulating concentrations of FSH) and histomorphometric study of ovarian tissues collected from cyclic heifers on different days after ovulation, the developmental pattern of antral follicles <1 mm and the role of FSH in their development were studied in heifers. Ultrasonographically acquired follicular data were used to determine the repeatability of 2- and 3-wave patterns and the effect of season on the wave patterns. The ovulatory follicle in 3-wave IOI is exposed to a shorter term high-progesterone environment than that of 2-wave IOI, and it has been argued that the less-aged ovulatory follicle of 3-wave IOI yields a more fertile oocyte than the 2-wave IOI. The developmental competence of oocytes in preovulatory follicles of 2- versus 3-wave IOI was compared using in vivo environments created to mimic short-term low- and high-progesterone environments similar to 2- and 3-wave IOI, respectively. The developmental competence of oocytes in persistent dominant-type follicles was also determined.<p>The vaccination against GnRH attenuated FSH surges but did not suppress the basal circulating concentrations of FSH. The attenuation of FSH surges suppressed the wave-like emergence of follicles ≥4 mm but not of the antral follicles <4 mm. The study revealed an inverse relationship between the mean and peak circulating concentrations of FSH and the number of follicles recruited into ≥1 mm size category. Histomorphometric study revealed that antral follicles <1 mm developed in a wave-like fashion in response to a rise in the circulating concentrations of FSH. After treatment with exogenous FSH, the growth rate of follicles in GnRH-immunized heifers was similar to controls. <p>The duration of IOI was predictive of the wave pattern (i.e., 2- or 3-wave IOI), and the pattern was repeatable within individuals throughout the year. The dominant follicle of Wave 1 in 2-wave IOI had a longer duration of dominance than in 3-wave IOI. Hence, the dominant follicle of Wave 1 may have a primary role in the regulation of 2- and 3-wave patterns. Greater attrition of follicles in 3-wave IOI, due to the emergence of an extra wave compared to 2-wave IOI, may contribute to earlier follicular depletion and onset of reproductive senescence in heifers with primarily a 3-wave pattern. The fertilization capacity of oocytes that were exposed to the short-term low-progesterone environment (i.e., similar to the early growing phase of the ovulatory follicle of 3-wave IOI) was increased, but the developmental competence post-fertilization was not different from oocytes that were exposed to a short-term high-progesterone environment (i.e., similar to the early growing phase of preovulatory follicle of 2-wave IOI). Multiple follicles developed under the prolonged-low progesterone environment, but failed to ovulate.

Introdução: A reserva ovariana (RO) refere-se à quantidade e, para alguns autores, à qualidade de folículos presentes nos ovários em um dado momento. É a medida pela qual se avalia a produção de oócitos e consequente potencial reprodutivo. Ela pode ser inferida mediante dosagem dos níveis séricos do hormônio folículo estimulante (FSH), estradiol, inibina B e hormônio antimülleriano (HAM), e ainda, ultrassonograficamente, através da contagem de folículos antrais (CFA). Na década de 50 observou-se que mulheres submetidas à quimioterapia (QT) apresentavam falência ovariana mais precocemente, efeito atribuído à gonadotoxicidade quimioterápica. Objetivos: Estudar o HAM como marcador da RO em mulheres com câncer de mama expostas à QT gonadotóxica comparando-o com outros marcadores da RO e determinar preditores de risco da ocorrência de anovulação (amenorreia ou ciclos irregulares) nessas mulheres. Métodos: Foi realizado estudo de coorte com 52 mulheres com diagnóstico de câncer de mama e necessidade de QT com ciclofosfamida, com idade até 40 anos, ciclos menstruais regulares e sem histórico de tratamento quimioterápico prévio. As pacientes realizaram coleta de sangue e ultrassonografia pélvica transvaginal (USTV) antes da QT (T1) e 2 (T2) e 6 (T3) meses após seu término. Resultados: A idade média das pacientes estudadas foi 35,3 ± 3,8 anos e o tempo médio de seguimento foi de 14 ± 3 meses. A prevalência de anovulação foi de 40% durante a QT, 85% 2 meses após o término da QT (4 a 6 ciclos de ciclofosfamida) e de 60% 6 meses após a QT. A média de idade das pacientes que se tornaram anovulatórias foi de 36,5 ± 3,8 anos, enquanto que nas que permaneceram ovulatórias foi de 32,9 ± 3,5 anos com p = 0,02. O FSH acompanhou o status menstrual, apresentando aumento e queda significativos em T2 e T3. O HAM diminuiu significativamente de T1 (2,53 (1 - 5,31) ng/mL) para T2 (valores abaixo do detectável) com p < 0,0001 e não se modificou de T2 para T3, mesmo com uma parcela de pacientes retomando a ciclicidade menstrual. CFA em T1 foi 11 (8 - 13,5) folículos, sendo estatisticamente maior que nos tempos T2 e T3 (p < 0,0001). Entre T2 e T3 não houve diferença. As pacientes que mantiveram ciclos ovulatórios após o término da QT apresentaram no final do estudo níveis significativamente mais baixos de HAM do que previamente à QT (1,46 (< 0,08 - 4,31) ng/ml versus 6,17 (3,19 - 10,07) ng/mL) e CFA (7 (5,5 - 10) folículos versus 13 (11 - 15,5) folículos). HAM e CFA apresentaram correlação negativa e significativa com a idade. Trinta e dois anos foi a idade que apresentou sensibilidade de 96% e especificidade de 39% para predição de anovulação, mesmo que sem amenorreia, com área sob a curva (ASC) ROC de 0,77. Os marcadores de RO e os respectivos pontos de corte com poder de predizer ocorrência de anovulação em pacientes expostas à QT foram HAM < 3,32 ng/ml (sensibilidade de 85%, especificidade de 75% e ASC de 0,86) e CFA < 13 folículos antrais (sensibilidade de 81%, especificidade de 62% e ASC de 0,81). Para a predição de amenorreia exclusivamente, o HAM teve como ponto de corte o valor de 1,87 ng/ml (sensibilidade de 82%, especificidade de 83% e ASC de 0,84) e a CFA valor de 9 folículos (sensibilidade de 71%, especificidade de 78% e ASC de 0,73 ). As avaliações dos marcadores de RO não foram influenciadas pelo número de ciclos de QT (4 ou 6 ciclos), nem pela dose de quimioterápico utilizado por área corporal. Conclusão: O HAM e a CFA são igualmente capazes de determinar a queda da RO em pacientes submetidas à QT gonadotóxica. Pacientes com diagnóstico de câncer de mama que necessitam de QT com ciclofosfamida devem ser alertadas para o risco de amenorreia especialmente quando a idade for de 32 anos ou mais, dosagens séricas de HAM abaixo de 3,32 ng/ml, CFA < 13, devendo receber informações a respeito da preservação da fertilidade. Dentre esses marcadores, o HAM foi o de maior poder em predizer a ocorrência de amenorreia. / Introduction: Ovarian reserve (OR) refers to quantity and, to some authors, quality of follicles present in ovaries at a given time. It is the measure used to assess the capacity of the ovary to produce oocytes. Its evaluation is trough serum analysis of FSH, estradiol, inhibin and anti-Müllerian hormone (AMH) and trough ultrassonography to count de antrals follicles (AFC). In the 50s, it was observed that women exposed to chemotherapy experienced premature ovarian failure, effect attributed to chemotherapy. Objectives: To ascertain OR by means of AMH in young women with breast cancer exposed to chemotherapy comparing them with another ovarian reserve tests. To define risk predictors of anovulation (oligomenorrhea or amenorrhea) in those women. Methods: A cohort study with 52 eumenorrheic patients (age < 40years) with breast cancer who received chemotherapy with cyclophosphamide. Assessment was carried out with serum samples and pelvic ultrasonography before chemotherapy (T1), and 2 (T2) and 6 (T3) months after chemotherapy. Results: Mean age was 35.3 ± 3.8 years. Mean duration of follow-up was 14 ± 3 months. Anovulation was present in 40% of women during the chemotherapy, 85% 2 months after and 60% 6 months after chemotherapy. Mean age of anovulatory women in T3 was 36.5 ± 3.8 years. Women with regular cycles was 32.9 ± 3.5 years (p = 0.02). FSH levels rises and decreased significantly in T2 and T3. AMH levels declined significantly, down to undetectable levels at T2 from a median of 2.53 (1 –5.31 ng/mL) at T1 (p < 0.0001) and remained unchanged from T@ and T3, even though some patients resumed normal menses. Median AFC was 11 ( 8.0 – 13.5) follicles at T1 and significantly lower at T2 and T3 (p < 0.0001). No difference was found between T2 and T3 in patients who resumed ovulation cycles after completion of chemotherapy, AMH and AFC levels were significantly lower as compared with baseline: 1.46 (< 0.08 – 4.31) ng/mL vs. 6.17 (3.19 – 10.07) ng/mL and 7 (5.5 - 10) follicles vs. 13 (11 – 15.5) follicles. In patients who remained ovulatory during chemotherapy or resumed normal menses, FSH and estradiol levels remained unchanged relative to baseline. AMH and AFC presented significantly negative correlation with age. The age of thirty-two years presented 96% of sensitivity and 39% of specificity to predict anovulation with ROC area under the curve (AUC) of 0.77. The ovarian reserve (OVR) tests with power to predict anovulation in women exposed to CT were AMH < 3.32 ng/mL (sensitivity of 85%, specificity of 75% and AUC of 0.87) and AFC < 13 follicles (sensitivity of 81%, specificity of 62% and AUC of 0.81). The AMH cut off to predict amenorrhea was 1,87 ng/mL (sensitivity of 82%, specificity of 83% and AUC of 0,84) and the AFC cut off was 9 follicles (sensitivity of 71%, specificity of 78% and AUC of 0.73 ). The analysis was not influenced by the number of cycles or dose of CT. Conclusions: AMH and AFC are equally able to determine the OVR decline in chemotherapy exposed women. FSH is not adequate for this purpose, except in women who become amenorrheic. Thirty-two year old or older women, AMH levels < 3.32 ng/mL and AFC < 13 follicles determined significantly higher risk of anovulation after CT with cyclophosphamide. These women should be encouraged to preserve their fertility. Among the OVR tests, AMH was the powerful to predict the anovulation.

Made available in DSpace on 2014-06-11T19:35:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2005Bitstream added on 2014-06-13T18:46:34Z : No. of bitstreams: 1 nogueira_mfg_dr_botfmvz.pdf: 892396 bytes, checksum: 471e1cec0bdbf86073d92bc94c87f460 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Em células da teca e da granulosa, de folículos bovinos, foram detectados quatro transcritos alternativos do receptor do hormônio luteinizante (LHR). Apenas dois deles são traduzidos em proteínas funcionais com afinidades distintas em relação aos ligantes. Em humanos e símios, a isoforma completa (full-length) tem afinidade pelo LH e hCG, enquanto que a isoforma que apresenta deleção do exon 10 tem afinidade somente pelo hCG. Além disso, isoformas com deleção do exon 3 foram observadas em ratos, embora nenhum outro estudo tenha investigado essa região do gene bovino. Objetivouse com este trabalho caracterizar o padrão da expressão do gene do LHR nas células da teca e da granulosa de folículos antrais bovinos. Ovários foram coletados em matadouro, os folículos (5-14 mm) foram dissecados e as células da teca e da granulosa separadas para extração de RNA total com Trizol. As concentrações de esteróides no fluido folicular foram determinadas por radioimunoensaio (RIE). A expressão gênica do LHR foi mensurada por RTPCR semiquantitativo com oligonucleotídeos iniciadores (primers) específicos para amplificar o fragmento entre o final da região extracelular e o final da intracelular (LHRBC; primers posicionados nos exons 9 e 11). A ocorrência de transcritos alternativos oriundos do início da região extracelular (LHRA; primers posicionados nos exons 2 e 9) foi investigada mediante amplificação por RT-PCR. Como controle interno no PCR, utilizou-se a expressão da GAPDH. Paralelamente, células da granulosa cultivadas in vitro foram tratadas com 1 ou 10 ng de FSH no meio de cultura. Mediante RT-PCR, foi investigada a expressão das isoformas do LHRBC nas células da granulosa cultivadas e tratadas com FSH... / Growth of dominant follicle in the absence of circulating FSH and the events following the LH surge that culminate in ovulation, are dependent on the interaction between LH and its receptor (LHR). Four LHR alternative transcripts were described in theca and granulosa cells from bovine follicles. Only two of them can be translated to functional proteins (receptors coupled with G protein) with different affinities to their ligands. In humans and marmosets, the full-length isoform has affinity to both LH and hCG molecules, whereas the isoform with deletion of only exon 10 has affinity to hCG exclusively. Additionally, isoforms with deletion of exon 3 were observed in rats, although no previous report have investigated this region of the bovine gene. The objective of this study was to characterize the pattern of gene expression of the LHR in theca and granulosa cells from bovine antral follicles. Additionally, LHR expression was determined in cultured granulosa cells under FSH treatment. From ovaries collected in abattoir, antral follicles were dissected (5 to 14mm of diameter), and samples of theca and granulosa cells were obtained to total RNA extraction (Trizol protocol). Steroids concentrations in the follicular fluid were determined by RIA. Gene expression of LHR was measured by semiquantitative RT-PCR with specific primers to amplify part of extracellular region (LHRA; primers annealing on exons 2 and 9) and the fragment from the end of extracellular region, including the transmembrane domain and finishing near the end of intracellular region (LHRBC; primers annealing on exons 9 and 11). As internal control of the PCR, it was used GAPDH expression. Cultured granulosa cells were treated with 0, 1 or 10 ng of FSH (3 replicates each dose). As in vivo and positive control, theca cell sample was utilized to comparison... (Complete abstract, access undermentioned electronic address)

Orientador: Ciro Moraes Barros / Resumo: Em células da teca e da granulosa, de folículos bovinos, foram detectados quatro transcritos alternativos do receptor do hormônio luteinizante (LHR). Apenas dois deles são traduzidos em proteínas funcionais com afinidades distintas em relação aos ligantes. Em humanos e símios, a isoforma completa ("full-length") tem afinidade pelo LH e hCG, enquanto que a isoforma que apresenta deleção do exon 10 tem afinidade somente pelo hCG. Além disso, isoformas com deleção do exon 3 foram observadas em ratos, embora nenhum outro estudo tenha investigado essa região do gene bovino. Objetivouse com este trabalho caracterizar o padrão da expressão do gene do LHR nas células da teca e da granulosa de folículos antrais bovinos. Ovários foram coletados em matadouro, os folículos (5-14 mm) foram dissecados e as células da teca e da granulosa separadas para extração de RNA total com Trizol. As concentrações de esteróides no fluido folicular foram determinadas por radioimunoensaio (RIE). A expressão gênica do LHR foi mensurada por RTPCR semiquantitativo com oligonucleotídeos iniciadores ("primers") específicos para amplificar o fragmento entre o final da região extracelular e o final da intracelular (LHRBC; "primers" posicionados nos exons 9 e 11). A ocorrência de transcritos alternativos oriundos do início da região extracelular (LHRA; "primers" posicionados nos exons 2 e 9) foi investigada mediante amplificação por RT-PCR. Como controle interno no PCR, utilizou-se a expressão da GAPDH. Paralelamente, células da granulosa cultivadas in vitro foram tratadas com 1 ou 10 ng de FSH no meio de cultura. Mediante RT-PCR, foi investigada a expressão das isoformas do LHRBC nas células da granulosa cultivadas e tratadas com FSH... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Growth of dominant follicle in the absence of circulating FSH and the events following the LH surge that culminate in ovulation, are dependent on the interaction between LH and its receptor (LHR). Four LHR alternative transcripts were described in theca and granulosa cells from bovine follicles. Only two of them can be translated to functional proteins (receptors coupled with G protein) with different affinities to their ligands. In humans and marmosets, the full-length isoform has affinity to both LH and hCG molecules, whereas the isoform with deletion of only exon 10 has affinity to hCG exclusively. Additionally, isoforms with deletion of exon 3 were observed in rats, although no previous report have investigated this region of the bovine gene. The objective of this study was to characterize the pattern of gene expression of the LHR in theca and granulosa cells from bovine antral follicles. Additionally, LHR expression was determined in cultured granulosa cells under FSH treatment. From ovaries collected in abattoir, antral follicles were dissected (5 to 14mm of diameter), and samples of theca and granulosa cells were obtained to total RNA extraction (Trizol protocol). Steroids concentrations in the follicular fluid were determined by RIA. Gene expression of LHR was measured by semiquantitative RT-PCR with specific primers to amplify part of extracellular region (LHRA; primers annealing on exons 2 and 9) and the fragment from the end of extracellular region, including the transmembrane domain and finishing near the end of intracellular region (LHRBC; primers annealing on exons 9 and 11). As internal control of the PCR, it was used GAPDH expression. Cultured granulosa cells were treated with 0, 1 or 10 ng of FSH (3 replicates each dose). As in vivo and positive control, theca cell sample was utilized to comparison... (Complete abstract, access undermentioned electronic address) / Doutor

Introdução: A reserva ovariana (RO) refere-se à quantidade e, para alguns autores, à qualidade de folículos presentes nos ovários em um dado momento. É a medida pela qual se avalia a produção de oócitos e consequente potencial reprodutivo. Ela pode ser inferida mediante dosagem dos níveis séricos do hormônio folículo estimulante (FSH), estradiol, inibina B e hormônio antimülleriano (HAM), e ainda, ultrassonograficamente, através da contagem de folículos antrais (CFA). Na década de 50 observou-se que mulheres submetidas à quimioterapia (QT) apresentavam falência ovariana mais precocemente, efeito atribuído à gonadotoxicidade quimioterápica. Objetivos: Estudar o HAM como marcador da RO em mulheres com câncer de mama expostas à QT gonadotóxica comparando-o com outros marcadores da RO e determinar preditores de risco da ocorrência de anovulação (amenorreia ou ciclos irregulares) nessas mulheres. Métodos: Foi realizado estudo de coorte com 52 mulheres com diagnóstico de câncer de mama e necessidade de QT com ciclofosfamida, com idade até 40 anos, ciclos menstruais regulares e sem histórico de tratamento quimioterápico prévio. As pacientes realizaram coleta de sangue e ultrassonografia pélvica transvaginal (USTV) antes da QT (T1) e 2 (T2) e 6 (T3) meses após seu término. Resultados: A idade média das pacientes estudadas foi 35,3 ± 3,8 anos e o tempo médio de seguimento foi de 14 ± 3 meses. A prevalência de anovulação foi de 40% durante a QT, 85% 2 meses após o término da QT (4 a 6 ciclos de ciclofosfamida) e de 60% 6 meses após a QT. A média de idade das pacientes que se tornaram anovulatórias foi de 36,5 ± 3,8 anos, enquanto que nas que permaneceram ovulatórias foi de 32,9 ± 3,5 anos com p = 0,02. O FSH acompanhou o status menstrual, apresentando aumento e queda significativos em T2 e T3. O HAM diminuiu significativamente de T1 (2,53 (1 - 5,31) ng/mL) para T2 (valores abaixo do detectável) com p < 0,0001 e não se modificou de T2 para T3, mesmo com uma parcela de pacientes retomando a ciclicidade menstrual. CFA em T1 foi 11 (8 - 13,5) folículos, sendo estatisticamente maior que nos tempos T2 e T3 (p < 0,0001). Entre T2 e T3 não houve diferença. As pacientes que mantiveram ciclos ovulatórios após o término da QT apresentaram no final do estudo níveis significativamente mais baixos de HAM do que previamente à QT (1,46 (< 0,08 - 4,31) ng/ml versus 6,17 (3,19 - 10,07) ng/mL) e CFA (7 (5,5 - 10) folículos versus 13 (11 - 15,5) folículos). HAM e CFA apresentaram correlação negativa e significativa com a idade. Trinta e dois anos foi a idade que apresentou sensibilidade de 96% e especificidade de 39% para predição de anovulação, mesmo que sem amenorreia, com área sob a curva (ASC) ROC de 0,77. Os marcadores de RO e os respectivos pontos de corte com poder de predizer ocorrência de anovulação em pacientes expostas à QT foram HAM < 3,32 ng/ml (sensibilidade de 85%, especificidade de 75% e ASC de 0,86) e CFA < 13 folículos antrais (sensibilidade de 81%, especificidade de 62% e ASC de 0,81). Para a predição de amenorreia exclusivamente, o HAM teve como ponto de corte o valor de 1,87 ng/ml (sensibilidade de 82%, especificidade de 83% e ASC de 0,84) e a CFA valor de 9 folículos (sensibilidade de 71%, especificidade de 78% e ASC de 0,73 ). As avaliações dos marcadores de RO não foram influenciadas pelo número de ciclos de QT (4 ou 6 ciclos), nem pela dose de quimioterápico utilizado por área corporal. Conclusão: O HAM e a CFA são igualmente capazes de determinar a queda da RO em pacientes submetidas à QT gonadotóxica. Pacientes com diagnóstico de câncer de mama que necessitam de QT com ciclofosfamida devem ser alertadas para o risco de amenorreia especialmente quando a idade for de 32 anos ou mais, dosagens séricas de HAM abaixo de 3,32 ng/ml, CFA < 13, devendo receber informações a respeito da preservação da fertilidade. Dentre esses marcadores, o HAM foi o de maior poder em predizer a ocorrência de amenorreia. / Introduction: Ovarian reserve (OR) refers to quantity and, to some authors, quality of follicles present in ovaries at a given time. It is the measure used to assess the capacity of the ovary to produce oocytes. Its evaluation is trough serum analysis of FSH, estradiol, inhibin and anti-Müllerian hormone (AMH) and trough ultrassonography to count de antrals follicles (AFC). In the 50s, it was observed that women exposed to chemotherapy experienced premature ovarian failure, effect attributed to chemotherapy. Objectives: To ascertain OR by means of AMH in young women with breast cancer exposed to chemotherapy comparing them with another ovarian reserve tests. To define risk predictors of anovulation (oligomenorrhea or amenorrhea) in those women. Methods: A cohort study with 52 eumenorrheic patients (age < 40years) with breast cancer who received chemotherapy with cyclophosphamide. Assessment was carried out with serum samples and pelvic ultrasonography before chemotherapy (T1), and 2 (T2) and 6 (T3) months after chemotherapy. Results: Mean age was 35.3 ± 3.8 years. Mean duration of follow-up was 14 ± 3 months. Anovulation was present in 40% of women during the chemotherapy, 85% 2 months after and 60% 6 months after chemotherapy. Mean age of anovulatory women in T3 was 36.5 ± 3.8 years. Women with regular cycles was 32.9 ± 3.5 years (p = 0.02). FSH levels rises and decreased significantly in T2 and T3. AMH levels declined significantly, down to undetectable levels at T2 from a median of 2.53 (1 –5.31 ng/mL) at T1 (p < 0.0001) and remained unchanged from T@ and T3, even though some patients resumed normal menses. Median AFC was 11 ( 8.0 – 13.5) follicles at T1 and significantly lower at T2 and T3 (p < 0.0001). No difference was found between T2 and T3 in patients who resumed ovulation cycles after completion of chemotherapy, AMH and AFC levels were significantly lower as compared with baseline: 1.46 (< 0.08 – 4.31) ng/mL vs. 6.17 (3.19 – 10.07) ng/mL and 7 (5.5 - 10) follicles vs. 13 (11 – 15.5) follicles. In patients who remained ovulatory during chemotherapy or resumed normal menses, FSH and estradiol levels remained unchanged relative to baseline. AMH and AFC presented significantly negative correlation with age. The age of thirty-two years presented 96% of sensitivity and 39% of specificity to predict anovulation with ROC area under the curve (AUC) of 0.77. The ovarian reserve (OVR) tests with power to predict anovulation in women exposed to CT were AMH < 3.32 ng/mL (sensitivity of 85%, specificity of 75% and AUC of 0.87) and AFC < 13 follicles (sensitivity of 81%, specificity of 62% and AUC of 0.81). The AMH cut off to predict amenorrhea was 1,87 ng/mL (sensitivity of 82%, specificity of 83% and AUC of 0,84) and the AFC cut off was 9 follicles (sensitivity of 71%, specificity of 78% and AUC of 0.73 ). The analysis was not influenced by the number of cycles or dose of CT. Conclusions: AMH and AFC are equally able to determine the OVR decline in chemotherapy exposed women. FSH is not adequate for this purpose, except in women who become amenorrheic. Thirty-two year old or older women, AMH levels < 3.32 ng/mL and AFC < 13 follicles determined significantly higher risk of anovulation after CT with cyclophosphamide. These women should be encouraged to preserve their fertility. Among the OVR tests, AMH was the powerful to predict the anovulation.

Introdução: A reserva ovariana (RO) refere-se à quantidade e, para alguns autores, à qualidade de folículos presentes nos ovários em um dado momento. É a medida pela qual se avalia a produção de oócitos e consequente potencial reprodutivo. Ela pode ser inferida mediante dosagem dos níveis séricos do hormônio folículo estimulante (FSH), estradiol, inibina B e hormônio antimülleriano (HAM), e ainda, ultrassonograficamente, através da contagem de folículos antrais (CFA). Na década de 50 observou-se que mulheres submetidas à quimioterapia (QT) apresentavam falência ovariana mais precocemente, efeito atribuído à gonadotoxicidade quimioterápica. Objetivos: Estudar o HAM como marcador da RO em mulheres com câncer de mama expostas à QT gonadotóxica comparando-o com outros marcadores da RO e determinar preditores de risco da ocorrência de anovulação (amenorreia ou ciclos irregulares) nessas mulheres. Métodos: Foi realizado estudo de coorte com 52 mulheres com diagnóstico de câncer de mama e necessidade de QT com ciclofosfamida, com idade até 40 anos, ciclos menstruais regulares e sem histórico de tratamento quimioterápico prévio. As pacientes realizaram coleta de sangue e ultrassonografia pélvica transvaginal (USTV) antes da QT (T1) e 2 (T2) e 6 (T3) meses após seu término. Resultados: A idade média das pacientes estudadas foi 35,3 ± 3,8 anos e o tempo médio de seguimento foi de 14 ± 3 meses. A prevalência de anovulação foi de 40% durante a QT, 85% 2 meses após o término da QT (4 a 6 ciclos de ciclofosfamida) e de 60% 6 meses após a QT. A média de idade das pacientes que se tornaram anovulatórias foi de 36,5 ± 3,8 anos, enquanto que nas que permaneceram ovulatórias foi de 32,9 ± 3,5 anos com p = 0,02. O FSH acompanhou o status menstrual, apresentando aumento e queda significativos em T2 e T3. O HAM diminuiu significativamente de T1 (2,53 (1 - 5,31) ng/mL) para T2 (valores abaixo do detectável) com p < 0,0001 e não se modificou de T2 para T3, mesmo com uma parcela de pacientes retomando a ciclicidade menstrual. CFA em T1 foi 11 (8 - 13,5) folículos, sendo estatisticamente maior que nos tempos T2 e T3 (p < 0,0001). Entre T2 e T3 não houve diferença. As pacientes que mantiveram ciclos ovulatórios após o término da QT apresentaram no final do estudo níveis significativamente mais baixos de HAM do que previamente à QT (1,46 (< 0,08 - 4,31) ng/ml versus 6,17 (3,19 - 10,07) ng/mL) e CFA (7 (5,5 - 10) folículos versus 13 (11 - 15,5) folículos). HAM e CFA apresentaram correlação negativa e significativa com a idade. Trinta e dois anos foi a idade que apresentou sensibilidade de 96% e especificidade de 39% para predição de anovulação, mesmo que sem amenorreia, com área sob a curva (ASC) ROC de 0,77. Os marcadores de RO e os respectivos pontos de corte com poder de predizer ocorrência de anovulação em pacientes expostas à QT foram HAM < 3,32 ng/ml (sensibilidade de 85%, especificidade de 75% e ASC de 0,86) e CFA < 13 folículos antrais (sensibilidade de 81%, especificidade de 62% e ASC de 0,81). Para a predição de amenorreia exclusivamente, o HAM teve como ponto de corte o valor de 1,87 ng/ml (sensibilidade de 82%, especificidade de 83% e ASC de 0,84) e a CFA valor de 9 folículos (sensibilidade de 71%, especificidade de 78% e ASC de 0,73 ). As avaliações dos marcadores de RO não foram influenciadas pelo número de ciclos de QT (4 ou 6 ciclos), nem pela dose de quimioterápico utilizado por área corporal. Conclusão: O HAM e a CFA são igualmente capazes de determinar a queda da RO em pacientes submetidas à QT gonadotóxica. Pacientes com diagnóstico de câncer de mama que necessitam de QT com ciclofosfamida devem ser alertadas para o risco de amenorreia especialmente quando a idade for de 32 anos ou mais, dosagens séricas de HAM abaixo de 3,32 ng/ml, CFA < 13, devendo receber informações a respeito da preservação da fertilidade. Dentre esses marcadores, o HAM foi o de maior poder em predizer a ocorrência de amenorreia. / Introduction: Ovarian reserve (OR) refers to quantity and, to some authors, quality of follicles present in ovaries at a given time. It is the measure used to assess the capacity of the ovary to produce oocytes. Its evaluation is trough serum analysis of FSH, estradiol, inhibin and anti-Müllerian hormone (AMH) and trough ultrassonography to count de antrals follicles (AFC). In the 50s, it was observed that women exposed to chemotherapy experienced premature ovarian failure, effect attributed to chemotherapy. Objectives: To ascertain OR by means of AMH in young women with breast cancer exposed to chemotherapy comparing them with another ovarian reserve tests. To define risk predictors of anovulation (oligomenorrhea or amenorrhea) in those women. Methods: A cohort study with 52 eumenorrheic patients (age < 40years) with breast cancer who received chemotherapy with cyclophosphamide. Assessment was carried out with serum samples and pelvic ultrasonography before chemotherapy (T1), and 2 (T2) and 6 (T3) months after chemotherapy. Results: Mean age was 35.3 ± 3.8 years. Mean duration of follow-up was 14 ± 3 months. Anovulation was present in 40% of women during the chemotherapy, 85% 2 months after and 60% 6 months after chemotherapy. Mean age of anovulatory women in T3 was 36.5 ± 3.8 years. Women with regular cycles was 32.9 ± 3.5 years (p = 0.02). FSH levels rises and decreased significantly in T2 and T3. AMH levels declined significantly, down to undetectable levels at T2 from a median of 2.53 (1 –5.31 ng/mL) at T1 (p < 0.0001) and remained unchanged from T@ and T3, even though some patients resumed normal menses. Median AFC was 11 ( 8.0 – 13.5) follicles at T1 and significantly lower at T2 and T3 (p < 0.0001). No difference was found between T2 and T3 in patients who resumed ovulation cycles after completion of chemotherapy, AMH and AFC levels were significantly lower as compared with baseline: 1.46 (< 0.08 – 4.31) ng/mL vs. 6.17 (3.19 – 10.07) ng/mL and 7 (5.5 - 10) follicles vs. 13 (11 – 15.5) follicles. In patients who remained ovulatory during chemotherapy or resumed normal menses, FSH and estradiol levels remained unchanged relative to baseline. AMH and AFC presented significantly negative correlation with age. The age of thirty-two years presented 96% of sensitivity and 39% of specificity to predict anovulation with ROC area under the curve (AUC) of 0.77. The ovarian reserve (OVR) tests with power to predict anovulation in women exposed to CT were AMH < 3.32 ng/mL (sensitivity of 85%, specificity of 75% and AUC of 0.87) and AFC < 13 follicles (sensitivity of 81%, specificity of 62% and AUC of 0.81). The AMH cut off to predict amenorrhea was 1,87 ng/mL (sensitivity of 82%, specificity of 83% and AUC of 0,84) and the AFC cut off was 9 follicles (sensitivity of 71%, specificity of 78% and AUC of 0.73 ). The analysis was not influenced by the number of cycles or dose of CT. Conclusions: AMH and AFC are equally able to determine the OVR decline in chemotherapy exposed women. FSH is not adequate for this purpose, except in women who become amenorrheic. Thirty-two year old or older women, AMH levels < 3.32 ng/mL and AFC < 13 follicles determined significantly higher risk of anovulation after CT with cyclophosphamide. These women should be encouraged to preserve their fertility. Among the OVR tests, AMH was the powerful to predict the anovulation.