Mikkel Rosendahl

Two successful pregnancies following autotransplantation of frozen/thawed ovarian tissue

BACKGROUND Cryopreservation of the ovarian cortex with subsequent autotransplantation has, on an experimental basis, been performed to preserve fertility in women being treated for a malignant disease. The present study reports ovarian activity and pregnancies following autotransplantation of frozen/thawed ovarian tissue. METHODS One complete ovary was cryopreserved from each of six patients who were 26-35 years old prior to treatment. Tissue from three of the patients was transported 4-5 h on ice prior to cryopreservation. After a period of 17-32 months, orthotopic autotransplantation was performed replacing 20-60% of the tissue. Two patients received additional heterotopic transplants. RESULTS In all cases, the tissue restored menstrual cyclicity 14-20 weeks following transplantation. Four of the six women conceived following assisted reproduction: two women (who had the tissue transported 4-5 h prior to cryopreservation) each, based on the orthotopic transplanted tissue, delivered one healthy child (February 2007 and January 2008); one woman miscarriaged in gestational Week 7; and the other had a positive hCG test but no clinical pregnancy. The remaining two women did not become pregnant. CONCLUSIONS Two additional healthy children have been born as a result of the ovarian cryopreservation procedure. In both cases, the ovarian tissue was transported 4-5 h prior to freezing demonstrating that hospitals may offer cryopreservation without having the necessary expertise locally.

Evidence of residual disease in cryopreserved ovarian cortex from female patients with leukemia.

OBJECTIVE To systematically search for leukemic cells in cryopreserved ovarian cortex from Danish female patients with leukemia, who had ovarian cortex cryopreserved for fertility preservation before potentially sterilizing treatment. DESIGN Retrospective analysis of data in a clinical project. SETTING University hospital laboratories. PATIENT(S) In total, 26 patients diagnosed with leukemia, who had ovarian tissue cryopreserved before potentially sterilizing chemotherapy and conditioning. INTERVENTION(S) Ovarian cortex from each patient was examined with histology and immunohistochemistry. In addition, in eight cases a specific chromosomal abnormality could be used as a genetic marker for detection of malignant cells by polymerase chain reaction (PCR). MAIN OUTCOME MEASURE(S) Evidence of malignant cells by immunohistochemistry or PCR. RESULT(S) Histology and immunohistochemistry did not reveal malignant cell infiltration in the ovarian cortex of any of the patients. In six of the eight patients (75%) with chromosomal abnormalities in the malignant cells, PCR showed evidence of leukemic cells in the ovarian tissue. CONCLUSION(S) Immunohistochemistry was unable to locate leukemic cells in the ovarian cortex; however, PCR detected potentially malignant cells in the majority of cases. The viability and malignancy of these cells remains to be determined. At present, reimplantation of ovarian cortex to leukemia patients cannot be recommended.

Concentrations of AMH and inhibin-B in relation to follicular diameter in normal human small antral follicles

BACKGROUND The aim of the present study was to determine the intrafollicular concentrations of anti-Müllerian hormone (AMH), inhibin-B and steroids in normal human small antral follicles and to relate them to follicular size. METHODS A group of 103 women having one ovary removed for fertility preservation by cryopreservation prior to gonadotoxic treatment served as a source of a total of 272 human small antral follicles. Prior to cryopreservation of the ovarian cortex, fluid from small antral follicles were collected. On the basis of the follicular volume, the diameter was calculated and follicles with diameters from 3 to 12 mm were included. RESULTS Concentrations of AMH decreased significantly (P < 0.0005) from 1124 +/- 158 ng/ml (mean +/- SEM) in follicles with a diameter of 3 mm to a concentration of 392 +/- 98 ng/ml in 9 mm follicles, followed by a reduction to below 100 ng/ml in 12 mm follicles. The concentrations of inhibin-B rose from 57 +/- 10 ng/ml (mean +/- SEM) in 3 mm follicles to 142 +/- 10 ng/ml in 12 mm follicles (P < 0.0005) with a peak concentration of almost 200 ng/ml in 9-10 mm follicles. Relating hormone concentrations with age showed that even follicles from girls younger than 10 years showed the same range of AMH concentrations as those from older girls or women. CONCLUSIONS The intrafollicular concentrations of AMH become progressively lower with increasing follicle diameters. In contrast, concentrations of inhibin-B increased with increasing follicle diameter with peak values at around 9 mm in diameter. This suggests that AMH and inhibin-B undertake important intrafollicular functions around the time of normal follicular selection in the mid-follicular phase of the menstrual cycle.

Cryopreservation of ovarian tissue for a decade in Denmark: a view of the technique.

This paper presents the Danish 10-year experience (1999-2009) with cryopreservation (n=386) and autotransplantation of ovarian tissue (n=18). Before applying the technique to humans, the method was thoroughly tested and validated. The cryoprotectant solution was chosen after histological evaluation of mouse and human ovarian tissue after freezing with four different combinations of cryoprotectants. Viability was confirmed by transplantation of frozen-thawed human ovarian tissue (n=49) to oophorectomized Nude mice. Viability after transport of fresh tissue 4-5h prior to freezing had previously been validated. Overnight transport of fresh ovarian tissue prior to cryopreservation was evaluated when human ovarian tissue was kept on ice for 20h and then cryopreserved. The thawed ovarian tissue was transplanted to an oophorectomized Nude mouse and histology confirmed viability. In Denmark 12 women have received a total of 18 autotransplantations of ovarian tissue. All women resumed ovarian function and three healthy babies were born to two women. In both women, the tissue was transported on ice for 4-5h prior to cryopreservation. Ovarian tissue cryopreservation is an important method for fertility preservation; however, before applying the method clinically, each laboratory should perform thorough validation of their technique.

Dynamics and mechanisms of chemotherapy-induced ovarian follicular depletion in women of fertile age

OBJECTIVE To study ovarian follicular dynamics during chemotherapy to understand the mechanisms behind chemotherapy-induced ovarian follicular depletion and to evaluate whether pretreatment levels of ovarian reserve markers were predictive of the posttreatment levels. DESIGN Prospective clinical study. SETTING University hospital fertility center. PATIENT(S) Seventeen women (median age 30 years; range 19-35 years) undergoing chemotherapy. INTERVENTION(S) Patients were seen before, frequently during, and after chemotherapy, until 1 year after the end of treatment. Antral follicle count and levels of FSH, LH, E(2), anti-Müllerian hormone (AMH), and inhibin A and B were monitored at each visit. MAIN OUTCOME MEASURE(S) The dynamics of the ovarian reserve markers during chemotherapy and factors predictive of posttreatment ovarian function. RESULT(S) Anti-Müllerian hormone level (mean +/- 2 SEM) dropped from 2.7 +/- 1.0 to 1.1 +/- 0.6 and to 0.4 +/- 0.4 ng/mL immediately after one and two series of chemotherapy, respectively. Inhibin B and antral follicle count decreased after three series whereas FSH reached menopausal levels after four series. High pretreatment AMH levels predicted higher posttreatment AMH levels. CONCLUSION(S) Anti-Müllerian hormone and inhibin B levels immediately declined in response to chemotherapy, and the follicular target of chemotherapy appeared to be growing follicles. High pretreatment AMH levels were predictive of a higher posttreatment AMH level.

Cryopreservation of ovarian tissue for fertility preservation: no evidence of malignant cell contamination in ovarian tissue from patients with breast cancer

Cryopreserved ovarian cortical biopsies from 51 patients with breast cancer were examined by histologic and immunohistochemical analysis and showed no sign of metastases. Autotransplantation of ovarian cortex to patients with low-stage breast cancer disease appears safe, but confirmatory studies are required, including xenotransplantation studies.

Cryopreserved ovarian cortex from patients with leukemia in complete remission contains no apparent viable malignant cells

Some women suffering from leukemia require bone marrow transplantation to be cured. Bone marrow transplantation is associated with a high risk of sterility, and some patients are offered fertility preservation by cryopreservation of the ovarian cortex. Transplantation of the ovarian cortex to women cured of leukemia who became menopausal is currently not performed because of the risk of introducing the disease. In this study, individual pieces of ovarian cortex intended for reimplantation from 25 patients with leukemia were transplanted to each of 25 nude mice for 20 weeks. The ovarian cortex was examined before and after transplantation by histology and immunohistochemistry, and RT-quantitative PCR (in the 7 patients with a known marker). Seventeen patients had the ovarian cortex retrieved when they were in complete remission. Before transplantation, 4 of 7 pieces (2 from patients in complete remission) of ovarian cortex had a positive RT-quantitative PCR. After transplantation, none of the mice revealed any sign of disease, neither in the pieces of ovarian cortex transplanted nor in any of the murine organs evaluated. Thus, the ovaries from patients in complete remission do not appear to contain viable malignant cells contrasting ovarian tissue retrieved before treatment.

Autotransplantation of cryopreserved ovarian tissue in 12 women with chemotherapy-induced premature ovarian failure: the Danish experience

OBJECTIVE To describe a cohort of 12 Danish women who received autotransplantation of frozen-thawed cryopreserved ovarian tissue because of premature ovarian failure after cancer treatment. DESIGN Retrospective study. SETTING University hospitals. PATIENT(S) Twelve women with autotransplanted frozen-thawed ovarian tissue. INTERVENTION(S) Monitoring of hormonal parameters and results of 56 IVF cycles in 10 women. MAIN OUTCOME MEASURE(S) Levels of gonadotropins and sex steroids, functional life span of the grafts, and results of IVF. RESULT(S) All 12 women regained ovarian function between 8 and 26 weeks (mean 19 weeks) after transplantation. Ten women underwent a total of 56 IVF cycles, 76 follicles developed, 49 oocytes were aspirated, 18 were fertilized, and 16 embryos were transferred resulting in six pregnancies: two biochemical, one clinical that miscarried in week 7, and two ongoing resulting in the delivery of two healthy infants born at term to two women. One of these women subsequently conceived spontaneously and delivered another healthy infant. The life span of the transplanted tissue has been between 6 months and still functioning after 54 months. CONCLUSION(S) Autotransplantation consistently leads to recovery of ovarian function after treatment-induced ovarian failure. Four women became pregnant, after IVF or spontaneously, resulting in the delivery of three healthy infants.

Pretreatment anti-Müllerian hormone predicts for loss of ovarian function after chemotherapy for early breast cancer

Aim Improving survival for women with early breast cancer (eBC) requires greater attention to the consequences of treatment, including risk to ovarian function. We have assessed whether biochemical markers of the ovarian reserve might improve prediction of chemotherapy related amenorrhoea. Methods Women (n = 59, mean age 42.6 years [(range 23.3–52.5]) with eBC were recruited before any treatment. Pretreatment ovarian reserve markers (anti-Müllerian hormone [AMH], follicle-stimulating hormone [FSH], inhibin B) were analysed in relation to ovarian status at 2 years. Results Pretreatment AMH was significantly lower in women with amenorrhoea at 2 years (4.0 ± 0.9 pmol/L versus 17.2 ± 2.5, P < 0.0001), but FSH and inhibin B did not differ between groups. By logistic regression, pretreatment AMH, but not age, FSH or inhibin B, was an independent predictor of ovarian status at 2 years (P = 0.005; odds ratio 0.013). We combined these data with a similar cohort (combined n = 75); receiver–operator characteristic analysis for AMH gave area under curve (AUC) of 0.90 (95% confidence interval (CI) 0.82–0.97)). A cross-validated classification tree analysis resulted in a binary classification schema with sensitivity 98.2% and specificity 80.0% for correct classification of amenorrhoea. Conclusion Pretreatment AMH is a useful predictor of long term post chemotherapy loss of ovarian function in women with eBC, adding significantly to the only previously established individualising predictor, i.e. age. AMH measurement may assist decision-making regarding treatment options and fertility preservation procedures.

Concentration of Anti-Müllerian Hormone and Inhibin-B in Relation to Steroids and Age in Follicular Fluid from Small Antral Human Follicles

CONTEXT Ovaries surgically removed for fertility preservation served as a source of follicle fluid from human small antral follicles. OBJECTIVE The objective of the study was to measure intrafollicular concentrations of anti-Müllerian Hormone (AMH), inhibin-B, progesterone, androstenedione, testosterone, estradiol, and IGF binding protein-4. SETTING The study was conducted at a university hospital. PATIENTS Patients included 43 women having one ovary removed prior to receiving gonadotoxic treatment due to malignant disease. INTERVENTIONS Fluid from 100 follicles (diameter of 3-9 mm) were included. MAIN OUTCOME MEASURES Intrafollicular concentrations of the measured hormones, their possible intercorrelation, and correlation with age were measured. RESULTS Concentrations of AMH were unrelated to follicular fluid concentrations of androstenedione and testosterone. There was a significant negative correlation between estradiol, inhibin-B, progesterone, and AMH. In four age groups spanning 11-37 yr, levels of AMH, estradiol, androstenedione, testosterone and inhibin-B remained constant, whereas progesterone showed significant variations. IGF binding protein-4 was unrelated to any other measured hormone. CONCLUSIONS This study was unable to confirm a stimulatory effect of androgens on AMH secretion but did enforce a close intimate correlation between AMH and estradiol expressions in the developing human follicle. The insignificant variation of the AMH concentration with age, even in prepubertal girls, suggests that AMH expression is unrelated to menstrual cycle FSH cyclicity.