the use of combined chemo- and radiotherapy for childhood cancer treatment has led to an increased survival rate and posed new challenges concerning health problems , organ damage , and quality of life after anticancer therapy .
gonads are particularly exposed to the deteriorating effects of certain chemotherapeutics and radiotherapy ; on the other hand , when survivors reach adulthood , they wish to have their own biological children [ 13 ] .
fertility after anticancer therapy is a very important problem known by oncologists and endocrinologists as well as by cancer survivors themselves . in women ,
gonadotoxic therapy damages the primordial follicles in the ovaries , which can lead to premature menopause .
very aggressive therapy such as myeloablative therapy prior to bone marrow transplantation ( bmt ) or surgery / ovariectomy can lead to total sterility , whereas indirect irradiation of the ovaries and chemotherapy can result in a lowered ovarian reserve [ 46 ] . in the last years
, the measurement of anti - mllerian hormone ( amh ) has been used as an informative marker of the ovarian reserve .
amh is a product of granulosa cells of preantral and early antral follicles , capable of growing . in healthy women ,
amh measurement is useful for the determination of the reproductive life span and the time of future menopause [ 7 , 8 ] .
the aim of our study was to determine the ovarian reserve in young women after anticancer treatment during childhood and adolescence , using the protocols with different degrees of gonadotoxicity .
we recruited 83 young women , cancer survivors , from the department of pediatric oncology and hematology ( outpatient clinic ) , medical university of bialystok . at diagnosis , they were from 0.9 to 17.8 years old ( x = 10.5  5.21 ) , and , at examination , they were 18.78  4.98 years old .
they had been treated for hodgkin lymphoma , hl ( n = 22 ) , wilms tumor ( n = 11 ) , soft tissue sarcoma , sts ( n = 7 ) , neuroblastoma ( n = 2 ) germinal tumors ( n = 7 ) , acute myeloblastic leukemia ( n = 4 ) , acute lymphoblastic leukemia ( n = 22 ) chronic myeloblastic leukemia ( n = 2 ) , and non - hodgkin lymphoma ( n = 6 ) .
they were all treated according to international protocols ; 20 received irradiation for the infradiaphragmatic area ( 10 with hl ) , irradiation for the central nervous system ( cns)9 patients with leukemia , 6 received bone marrow transplantation ( 1total body irradiation , tbi12  gy ) .
all patients were classified according to a possible degree of gonadotoxicity proposed by wallace et al . ; the risk of disturbed fertility or infertility depends on diagnosis , stage , and type of treatment ( alkylating agents , radiotherapy to the pelvis / ovaries ) . the probability of infertility in the low risk group is less than 20% and in the medium risk group between 20 and 80% , whereas , in the high risk group , is greater than 80% . at diagnosis ,
forty - five patients were in tanner stage t1 - 2 , n = 5 in t3 , and n = 33 in t4 - 5 . on examination , one female presented with primary amenorrhea , 65 had normal , regular menses , and 17 had irregular menses or oligomenorrhea ( 11 were classified as high risk group , 2 as low risk , and 4 as middle risk group ) .
six patients have their biological children ( five treated for hl , one treated for non - hodgkin lymphoma , nhl ) .
details concerning diagnosis , age at the time of therapy , type of therapy , the interval between the end of therapy , and measurements of hormone levels are presented in table 1 , taking into consideration the risk groups proposed by brougham and wallace .
serum concentrations of fsh , lh , and e2 were measured in the same laboratory using the commercially available immunoenzymatic kits ; serum amh levels were determined with the eia amh / mis kit ( immunotech , beckman coulter company / marseille , france ) .
all hormonal measurements were performed in the early follicular phase ( 24 days of menstrual cycle ) and stored at 80c .
we found higher fsh and lower amh levels in the entire group as compared to the control group ( p = 0.001 ; p = 0.001 , resp . ) , whereas the mean levels of estradiol and lh were normal .
when the study group was subdivided according to the risk of gonadotoxicity , the levels of fsh were elevated only in the high risk group ( 18.11  28.7  miu / ml versus 5.36  1.89  miu / ml , p = 0.005 ) , whereas , in the middle and low risk groups , they were comparable with the control group .
amh values were lower than those in the control group in all the three risk groups ( hr group 14.14  13.26  pmol / l ( p = 0.001 ) ; mr group 14.82  16.2  pmol / l ( p = 0.019 ) ; lr group 19.44  13.96  pmol / l ( p = 0.053 ) ) .
mean serum lh and estradiol values did not differ between the risk groups and control ( see table 2 ) .
the hr group was analyzed separately : patients diagnosed with hl irradiated and nonirradiated for the infradiaphragmatic region , patients treated for solid tumors with radiation to the infradiaphragmatic area , and patients after bone marrow transplantation . in these subgroups , amh values were lower than those in the control group , being the lowest in patients after bone marrow transplantation ( 3.37  2.32  pmol / ml ) .
fsh levels were the highest in females after bmt ( 42.55    26.55  miu / ml ) and elevated in females treated for hl with inverted y irradiation .
the values of lh and estradiol did not differ between the hr group and control ( except the patients after bmt ) ( see table 3 ) .
there were 19 females ( 22.9% ) in the study group with elevated fsh levels ( > 10  miu / ml ) , amh lower than 12.5  pmol / l , and normal lh levels ; 12/19 derived from the hr group .
lowered amh levels ( yet with normal fsh and lh ) were observed in 43 patients ( 51.8% ) .
we found no influence of age at the time of treatment ( before puberty , during or after puberty ) , although amh was lower in patients treated after puberty ( 13.04  12.06  pmol / l ) than during puberty ( 15.43  13.65  pmol / l ) and before puberty ( 18.52  14.93  pmol / l ) .
combined anticancer treatment has improved the prognosis for young patients and at the same time has enabled us to recognize different late effects of the treatment .
diminished fertility or infertility and early menopause are the major side effects lowering life quality among cancer survivors . according to childhood cancer survivor study ( ccss ) , premature menopause occurs in 8% survivors and depends on age , dose of irradiation to the ovaries , and cumulative dose of alkylating agents .
brougham and wallace classified the most common cancers treated during childhood and adolescence according to the risk of subfertility resulting from gonadotoxicity .
the high risk group includes patients after tbi , megachemotherapy , tumors located in pelvis and irradiated , metastatic soft tissue sarcomas , and hodgkin lymphomas treated with alkylating agents .
the risk of impaired fertility in this group is more than 80% , as compared to less than 20% in the low risk group .
our knowledge concerning the toxicity of anticancer treatment enlarges , and treatment protocols change , not only for better outcome and improved survival but also for a reduction in side effects .
we categorized our patients according to the type of malignancy and treatment , mainly the area of radiotherapy and total dose of alkylating agents . to evaluate ovarian function
in males , it is easier to evaluate gonadal function by analyzing spermiograms and hormone measurements ; in females , assessment of oocyte depletion and premature ovarian failure is more difficult .
amh seems to be very useful to determine the ovarian reserve , better than the evaluation of the antral follicle count by vaginal ultrasonography or the measurement of fsh and inhibin b. the amh level is relatively constant from mid - childhood to early adulthood , without fluctuations between pubertal stages [ 7 , 1113 ] .
elevated fsh levels were observed only in the hr group , whereas lower amh ( compared to the control group ) was found in the total cohort independently of risk group .
more than half ( 51.8% ) of the patients had low amh levels , whereas 22.9% presented with elevated fsh values . abnormalities were most pronounced in patients after bmt , who had very low amh values and elevated fsh .
total body irradiation and/or high doses of alkylating agents , such as cyclophosphamide or busulphane , led to ovary dysfunction .
similar results have been reported by miyoshi et al . , who found low amh levels in 53% and high fsh in 30% of childhood cancer survivors .
the treatment for hl also leads to high risk of premature menopause [ 16 , 17 ] .
de bruin et al . found a 12-fold higher risk of early ( before the age of 40 ) menopause in hl survivors treated with procarbazine as an element of chemotherapy as compared to those irradiated for the supradiaphragmatic areas or paraaortic nodes . like in our study , van beek et al .
found that hormone levels were not influenced by age at treatment ( before versusduring puberty ) .
different results have been presented for women treated at an age older than 30 years since amh levels fall gradually due to a reduced oocyte pool [ 20 , 21 ] . in the patients treated for hl , lower amh values
were noted for the irradiated and nonirradiated infradiaphragmatic areas , whereas elevated fsh levels were observed only in irradiated females .
those who are irradiated for the infradiaphragmatic region received 3 or 4 cycles with procarbazine ( mopp ) , and those who are nonirradiated received only two cycles . in the former
patients treated for solid tumors , irradiated for the abdomen , also presented with lower amh levels ; in that group , only one female was irradiated ( 44  gy ) for the pelvic area 
irradiation directly for the ovaries , especially in the total dose > 15  gy , seems to be the most important factor deteriorating gonadal function , although according to wallace et al .
the ld50 for human oocyte is < 2  gy . we observed lowered amh levels not only in the hr group but also in the mr or lr group , which indicates that all types of anticancer treatment affected gonadal function , even when low doses of chemotherapeutics were used .
patients treated for acute lymphoblastic leukemia , classified to the low risk group , show subtle ovarian disorders ( lower estradiol levels )   and some of them are infertile after anticancer treatment . in a prospective study performed during and after cytotoxic treatment , brougham et al
. observed progressive lowering of amh during treatment and recovery in the low and middle risk groups between 2 and 12 months following therapy completion , thus indicating possible restoration of the pool of growing oocytes .
this recovery was not observed in the high risk group , suggesting a profound loss of the primordial follicle pool .
sixty - five out of 83 survivors had normal regular menstrual cycle , one had primary amenorrhea , and 17 had irregular menses or oligomenorrhea ; the latter group included patients after bmt after hl treatment ( hr group ) .
the group with normal menstrual cycles contained patients with lower amh levels and with a diminished ovarian reserve .
we found elevated fsh in the early follicular phase with normal lh and estradiol levels ; this situation is characteristic of premature ovarian failure and can appear even 20 years prior to menopause [ 19 , 27 , 28 ] . taken together ,
lowered mean amh values in the entire cohort and a monotropic rise in fsh indicate the possibility of premature menopause .
six patients had their biological children ; five were treated for hl and were classified to the hr group .
the peak incidence for hl is observed in older adolescents , most often over 15 years of age .
our results show the utility of amh measurement as an early , sensitive marker of a reduced ovarian reserve in young cancer survivors .
patients after conditioning therapy prior to bmt as well as patients treated for hl , independently of age at treatment ( prepuberty or puberty ) , are at the highest risk of gonadal damage and early menopause .
 we evaluated ovarian function by measuring the levels of anti - mllerian hormone ( amh ) , estradiol , and gonadotropins in 83 young women treated for cancer during childhood and adolescence , and classified according to post - treatment gonadal toxicity versus 38 healthy females . 
 results . 
 the mean amh values were lower in the entire cohort independently of the risk group as compared to the control , whereas fsh was elevated only in the high risk group . 
 the lowest amh values were noted in patients after bone marrow transplantation ( bmt ) and those treated for hodgkin lymphoma ( hl ) . 
 nineteen patients ( 22.9% ) had elevated fsh . 
 they all had low amh values . 
 lowered amh values ( but with normal fsh and lh ) were observed in 43 patients ( 51.8% ) . 
 there was no effect of age at the time of treatment ( before puberty , during or after puberty ) on amh levels . 
 conclusion . 
 our results show the utility of amh measurement as a sensitive marker of a reduced ovarian reserve in young cancer survivors . 
 patients after bmt and patients treated for hl , independently of age at treatment ( prepuberty or puberty ) , are at the highest risk of gonadal damage and early menopause . 