Non-growing follicle density is increased following adriamycin, bleomycin, vinblastine and dacarbazine (ABVD) chemotherapy in the adult human ovary

Funded by UK Medical Research Council Grants G0901839 and MR/L00299X/1.Study question: Do the chemotherapeutic regimens of ABVD (adriamycin, bleomycin, vinblastine and dacarbazine) or OEPA-COPDAC (combined vincristine, etoposide, prednisone, doxorubicin (OEPA) and cyclophosphamide, vincristine, prednisone, dacarbazine (COPDAC)) used to treat Hodgkin lymphoma (HL), affect the density, morphology and in vitro developmental potential of human ovarian follicles? Summary answer: Ovarian tissue from women treated with ABVD contained a higher density of non-growing follicles (NGFs) per cubic millimetre and increased numbers of multiovular follicles but showed reduced in vitro growth compared with patients with lymphoma who had not received chemotherapy, patients treated with OEPA-COPDAC, age-matched healthy women and age-related model-predicted values. What is known already: Chemotherapy regimens can cause a loss of follicles within the ovary that depends on the drugs given. Early stage HL is commonly treated by ABVD, a non-alkylating regimen which apparently has ovarian sparing qualities, thus it is important to investigate the histological appearance and distribution of follicles within ABVD-treated ovarian tissue. Study design, size, duration: Thirteen ovarian biopsies were obtained from HL patients (6 adolescents and 7 adults) and one biopsy from a non-HL patient. Two HL patients and the non-HL patient had received no treatment prior to biopsy collection. The remaining 11 HL patients received one of two regimens; ABVD or OEPA-COPDAC. Tissue was analysed histologically and compared to biopsies from healthy women, and in a sub-group of patients, tissue was cultured for 6 days in vitro. Participants/materials, setting, methods: Ovarian biopsies were obtained from patients undergoing ovarian cryopreservation for fertility preservation, and from healthy women at the time of Caesarian section (‘obstetric tissue’). Follicle number and maturity were evaluated in sections of ovarian cortical tissue, and compared to an age-related model of mean follicle density and to age-matched contemporaneous biopsies. The developmental potential of follicles was investigated after 6 days tissue culture. Main results and the role of chance: A total of 6877 follicles was analysed. ABVD-treated tissue contained a higher density of non-growing follicles/mm3 (230±17) (mean±SEM) than untreated (110±54), OEPA-COPDAC-treated (50±27 and obstetric tissue (20±4)(P< 0.01),with follicle density 9-21 standard deviations higher than predicted by an age-related model. Bi-ovular and binucleated non-growing follicles occurred frequently in ABVD-treated and in adolescent untreated tissue but were not observed in OEPA-COPDAC-treated or obstetric tissue, although OEPA-COPDAC-treated tissue contained a high proportion of morphologically abnormal oocytes (52% versus 23% in untreated, 22% in ABVD-treated and 25% in obstetric tissue; P< 0.001). Activation of follicle growth in vitro occurred in all groups, but in ABVD-treated samples there was very limited development to the secondary stage, whilst in untreated samples from lymphoma patients growth was similar to that observed in obstetric tissue (untreated; P< 0.01 versus ABVD-treated, ns versus obstetric). Large scale data: N/A Limitations, reasons for caution: Although a large number of follicles were analysed, these data were derived from a small number of biopsies. The mechanisms underpinning these observations have yet to be determined and it is unclear how they relate to future fertility. Wider implications of the findings: This study confirms that the number of NGFs is not depleted following ABVD treatment, consistent with clinical data that female fertility is preserved. Our findings demonstrate that immature follicle density can increase as well as decrease following at least one chemotherapy treatment. This is the first report of morphological and follicle developmental similarities between ABVD-treated tissue and the immature human ovary. Further experiments will investigate the basis for the marked increase in follicle density in ABVD-treated tissue.PostprintPeer reviewe

Measuring anti-Müllerian hormone for the assessment of ovarian reserve: when and for whom is it indicated?

Our understanding of female reproductive function has been hampered by our inability to directly assess the number of non-growing primordial follicles present in the ovary, the ovarian reserve. Female reproductive hormones (FSH and LH, the inhibins and steroids) reflect the activity of the larger growing follicles and thus are largely informative of peri-ovulatory ovarian activity. In contrast anti-Müllerian hormone (AMH) is a product of the granulosa cells of small growing follicles, whose number (and therefore circulating AMH concentrations) is reflective of the ovarian reserve. AMH declines with age in adult women, and emerging data suggest a relationship with remaining reproductive lifespan and age at the menopause. Early studies demonstrated that AMH concentrations are stable across the menstrual cycle, adding to its clinical utility. The most established role for AMH measurement is in women about to start IVF treatment, where it is predictive of the ovarian response and is of clear value in identifying women at risk of ovarian hyperstimulation syndrome or whose response will be poor and thus their expectations can be tailored. AMH is detectable in childhood, and although relationships to puberty are not yet available, it appears that AMH rises to a peak in the early 20s. Developing indications include in assessment and individualisation of the risk to fertility from chemotherapy, in the diagnosis of PCOS and as a tumour marker in granulosa cell tumours. The increasingly routine use of AMH by IVF clinics heralds much wider adoption in a range of clinical situations across the reproductive lifespan

Knemometry in childhood: a study to compare the precision of two different techniques

Knemometry is an accurate and non-invasive method of quantifying lower leg length changes. It reveals multiple fluctuations in leg length velocity over a short period of measurement, and on the basis of this it has been proposed that short-term growth is saltatory rather than a continuous phenomenon. The technical error (TE) of the technique which is generally employed, and which is subject to observer bias, ranges from 0·09 to 0·16 mm. This study was undertaken to compare the original method (OM) to a modified technique which involved measuring from a baseline value of which the operator was not aware; this technique is referred to as the random zero method (RM). Over a period of 10 months, 58 subjects were measured on 413 occasions. Overall, median TE in the RM group at 0·15 mm (P5-0, P95-0·65) was higher than the median TE in the OM group at 0·11 mm (P5-0, P95-0·37). However, the median TE over the last 3 months of 0·15 mm (P5-0·05, P95-0·87) was lower than the TE in the preceding 4 months of 0·20 mm (P5-0, P95-0·55) (WSR, p = 0·04) pointing towards the presence of an operator learning curve. The random zero method is a simple modification of the original method. It reduces observer bias but leads to a higher TE, which could explain some of the fluctuations seen between frequent knemometric measurements. Some knowledge of the length of the training period is important in the design of new studies involving knemometry; our data suggest that there should be a learning period of about 4 months if knemometry is performed as often as quoted above

The relationship between short-term changes in weight and lower leg length in children and young adults

As the knemometer is increasingly being used to study changes in lower leg length in conditions associated with weight changes it is important to clearly delineate the relationship between these two variables. Lower leg length and weight were measured in 26 children and nine adults including one pregnant woman. There was a weak but positive relationship between lower leg length and weight fluctuation in children. Daily fluctuations in weight as well as lower leg length were higher in women than men; median lower leg length fluctuation: women, 0.16 mm (P5-0, P95-0.7); men, 0.1 mm (P5-0, P95-0.48) p approximately 0.02, Wilcoxon signed-rank test. Median weight fluctuation: women 0.15 kg (P5-0, P95-0.54); men, 0.1 kg (P5-0, P95-0.5) p = 0.94 (Wilcoxon signed-rank test). Sustained weight gain in pregnancy led to a reduction in lower leg length followed by an increase which was coincident with the appearance of dependent oedema. Lower leg length changes are likely to be positively related to changes in weight when the latter are only modest in magnitude. However, greater sustained increases in weight are likely to have an opposite effect on lower leg length due to direct compression of the lower leg. Due consideration of weight is essential in longitudinal studies of lower leg length changes, especially in conditions which are associated with significant changes in weight

Effects of intensive chemotherapy on bone and collagen turnover and the growth hormone axis in children with acute lymphoblastic leukemia

To investigate the effects of disease and intensive chemotherapy on bone turnover and growth in children with acute lymphoblastic leukemia (ALL), a longitudinal prospective study was carried out in 22 children, aged 1.2–13.5 yr, enrolled in the Medical Research Council-funded randomized trial of childhood ALL treatment in the UK. We measured lower leg length and markers of bone formation [bone alkaline phosphatase (ALP) and procollagen type I C-terminal propeptide (PICP)], bone resorption [pyridinoline, deoxypyridinoline, and carboxyl-terminal telopeptide of type I collagen (ICTP)], soft tissue turnover [procollagen type III N-terminal propeptide (P3NP)], and the GH axis [IGF-I, IGF-binding protein-3 (IGFBP-3), IGFBP-2, and urinary GH] at 1- to 4-week intervals from diagnosis to week 27 of treatment. In addition, GH-binding protein was measured at diagnosis. &lt;br&gt;&lt;/br&gt; At diagnosis, mean SD scores were: bone ALP, -1.84; PICP -1.77; pyridinoline, -1.42; deoxypyridinoline, -1.66; ICTP, -0.42; P3NP, +1.45; GH, +24.4; IGF-I, -1.70; IGFBP-3, -0.88; IGFBP-2, +2.42; and GH-binding protein, -0.69. Bone ALP, PICP, and IGFBP-3 were all correlated (P 0.03). During induction and intensification, there was shrinkage of the lower leg, with decreases in PICP, pyridinoline, ICTP, and P3NP (P &#60; 0.05), whereas IGF-I and IGFBP-3 increased (P &#60; 0.05). After prednisolone was discontinued, bone ALP and collagen markers increased markedly (P &#60; 0.01), but there was no significant change in IGF-I and IGFBP-3. In 12 children who received high dose iv methotrexate, postglucocorticoid increases in bone ALP and PICP were less, whereas those in ICTP and P3NP were greater, compared to levels in children who did not receive methotrexate (P &#60; 0.05). &lt;br&gt;&lt;/br&gt; We conclude that ALL itself caused GH resistance and low bone turnover. During early intensive chemotherapy, further suppression of osteoblast proliferation and osteoclast activity occurred, not mediated through the systemic GH axis, probably by the direct action of prednisolone on bone. The postglucocorticoid increase in bone turnover was also independent of the GH axis and was modulated by high dose iv methotrexate, which depressed osteoblast recovery and enhanced osteoclast activity

Effects of intensive chemotherapy on bone and collagen turnover and the growth hormone axis in children with acute lymphoblastic leukemia

To investigate the effects of disease and intensive chemotherapy on bone turnover and growth in children with acute lymphoblastic leukemia (ALL), a longitudinal prospective study was carried out in 22 children, aged 1.2–13.5 yr, enrolled in the Medical Research Council-funded randomized trial of childhood ALL treatment in the UK. We measured lower leg length and markers of bone formation [bone alkaline phosphatase (ALP) and procollagen type I C-terminal propeptide (PICP)], bone resorption [pyridinoline, deoxypyridinoline, and carboxyl-terminal telopeptide of type I collagen (ICTP)], soft tissue turnover [procollagen type III N-terminal propeptide (P3NP)], and the GH axis [IGF-I, IGF-binding protein-3 (IGFBP-3), IGFBP-2, and urinary GH] at 1- to 4-week intervals from diagnosis to week 27 of treatment. In addition, GH-binding protein was measured at diagnosis. &lt;br&gt;&lt;/br&gt; At diagnosis, mean SD scores were: bone ALP, -1.84; PICP -1.77; pyridinoline, -1.42; deoxypyridinoline, -1.66; ICTP, -0.42; P3NP, +1.45; GH, +24.4; IGF-I, -1.70; IGFBP-3, -0.88; IGFBP-2, +2.42; and GH-binding protein, -0.69. Bone ALP, PICP, and IGFBP-3 were all correlated (P 0.03). During induction and intensification, there was shrinkage of the lower leg, with decreases in PICP, pyridinoline, ICTP, and P3NP (P &#60; 0.05), whereas IGF-I and IGFBP-3 increased (P &#60; 0.05). After prednisolone was discontinued, bone ALP and collagen markers increased markedly (P &#60; 0.01), but there was no significant change in IGF-I and IGFBP-3. In 12 children who received high dose iv methotrexate, postglucocorticoid increases in bone ALP and PICP were less, whereas those in ICTP and P3NP were greater, compared to levels in children who did not receive methotrexate (P &#60; 0.05). &lt;br&gt;&lt;/br&gt; We conclude that ALL itself caused GH resistance and low bone turnover. During early intensive chemotherapy, further suppression of osteoblast proliferation and osteoclast activity occurred, not mediated through the systemic GH axis, probably by the direct action of prednisolone on bone. The postglucocorticoid increase in bone turnover was also independent of the GH axis and was modulated by high dose iv methotrexate, which depressed osteoblast recovery and enhanced osteoclast activity