Kirsten Tryde Schmidt

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.

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.

Orthotopic autotransplantation of cryopreserved ovarian tissue to a woman cured of cancer–follicular growth, steroid production and oocyte retrieval

Cryopreservation of human ovarian tissue is now an option for cancer patients facing treatment with gonadotoxic regimes, as a means of preserving their fertility. So far, there have been only a few reports on autotransplantation of frozen-thawed tissue with regard to restoration of ovarian function. The present report describes a 32-year-old woman diagnosed with Hodgkins lymphoma, who had cryopreserved ovarian tissue transplanted orthotopically after secondary ovarian failure due to chemotherapy. Only 8 weeks after transplantation, ultrasonography of the remaining ovary revealed two follicles with diameters of 10 and 15 mm. Concomitantly, circulating concentrations of oestradiol increased, while concentrations of gonadotrophins decreased. In the following months, the patient menstruated three times. Subsequent pituitary down-regulation with a gonadotrophin-releasing hormone (GnRH) agonist and ovarian stimulation resulted in development of one pre-ovulatory follicle from which a metaphase II oocyte was retrieved; however, this oocyte was unable to sustain further development after intracytoplasmic sperm injection (ICSI). Intrafollicular concentrations of oestradiol and progesterone suggested a normal luteinizing response of the follicle to human chorionic gonadotrophin stimulation. A 7-month follow-up revealed continued vivid follicular activity and normal oestradiol concentrations. In conclusion, cryopreserved human ovarian tissue restored ovarian function for several cycles and sustained development of mature oocytes in a woman cured of cancer.

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.

LH-Receptor Gene Expression in Human Granulosa and Cumulus Cells from Antral and Preovulatory Follicles

CONTEXT Human granulosa cells (GC) acquire LH receptor (LHR) expression during the follicular phase of the menstrual cycle. Currently, the precise follicular stage is unknown, and specific roles of LH in the follicular development are not fully understood. OBJECTIVE Our objective was to measure LHR gene expression on GC and cumulus cells (CC) from normal human follicles with diameters form 3-20 mm. DESIGN, SETTING, AND PATIENTS At a university hospital, GC, CC, and the corresponding follicular fluid (FF) were collected from patients undergoing fertility preservation by having one ovary frozen and patients undergoing infertility treatment. INTERVENTIONS Cells and fluids were isolated from surgically excised ovaries or from aspirated preovulatory follicles. MAIN OUTCOME MEASURES We evaluated gene expression of LHR, FSHR, androgen receptor (AR), aromatase (CYP19a1), and AMHR2 normalized to the GAPDH expression and associated with FF levels of anti-Mullerian hormone, inhibin-B, and steroids. RESULTS LHR expression was maximal in GC from preovulatory follicles before ovulation induction. A majority of 150 antral follicles (3-10 mm in diameter) showed LHR expression at approximately 10% of the maximum, and LHR expression showed significant associations with FSHR, AR, CYP19a1, and AMHR2 and with FF estradiol and progesterone. Levels of FSHR continued to decline in GC as the follicular diameter increased. CONCLUSIONS The LHR gene is expressed in GC of human antral follicles throughout the follicular phase and is significantly associated with expression of the CYP19a1 gene and with the corresponding FF concentrations of estradiol and progesterone. LH appears to affect human follicular development during most the follicular phase in normal women.

Anti-Müllerian hormone initiates growth of human primordial follicles in vitro.

Survival and growth of follicles in human ovarian tissue is presently only performed with limited success. We evaluated the effect of anti-Müllerian hormone (AMH) and/or testosterone on follicular growth during a 4-week culture period using ovarian cortical tissue from six women in their reproductive years. The cortex of each biopsy was isolated and immediately cryopreserved upon collection and stored in liquid nitrogen. After thawing the tissue was placed in culture. After the culture period all follicles were counted on histological sections and classified for viability and stage of development. Based on evaluation of 6603 follicles it was found that the number of growing follicles significantly increased during the culture period as compared to the uncultured control, irrespective of the composition of the culture medium. Furthermore, significantly more follicles advanced to the primary and secondary stage (p<0.05) in tissue cultured with AMH (54%) as compared to tissue cultured in control medium (41%). The mean diameter of follicles classified as primary follicles was significantly enhanced in tissue cultured in the presence of AMH (p=0.002) and AMH plus testosterone (p<0.001) as compared to that observed in tissue cultured with control medium and medium containing testosterone alone. In contrast the mean diameter of the oocyte and its nucleus remained similar irrespective of culture medium. In conclusion, AMH seems to affect early stages of human follicular development by enhancing recruitment, survival and/or growth during a 4-week culture period.

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.

Cryopreservation of ovarian tissue for fertility preservation in young female oncological patients

Girls and women suffering from a cancer that requires treatment with gonadotoxic drugs may experience cessation of reproductive function as a side effect due to obliteration of the ovarian pool of follicles. Techniques are now available for fertility preservation, such as cryopreservation of mature oocytes, embryos or ovarian cortical tissue. Whereas collection of mature oocytes and embryos requires at least a 2-week period, ovarian tissue may on short notice be frozen prior to treatment and can be transplanted back into women with ovarian failure. Transplanted frozen/thawed tissue supports survival and growth of follicles, giving rise to menstrual cycles and hormone production for several years. Worldwide, the procedure has resulted in the birth of 15 healthy children. Many cancer patients including girls and young women want fertility preservation, and the techniques are now being further developed and implemented in several centers.

Evaluation of the ovarian reserve in women transplanted with frozen and thawed ovarian cortical tissue

OBJECTIVE To investigate ovarian reserve and ovarian function in women transplanted with frozen/thawed ovarian tissue. DESIGN Retrospective cohort study. SETTING University hospital. PATIENT(S) 18 women transplanted with their own frozen/thawed ovarian tissue. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Levels of antimüllerian hormone (AMH), duration of function of the transplanted ovarian tissue, outcome of assisted reproduction. RESULT(S) Of the 18 women who received transplanted ovarian tissue, levels of AMH were measured in 12 women; AMH never exceed a concentration of 1 ng/mL, and in several cases they were below the detection limit of the assay in combination with regular menstrual cycles. Two women with AMH below the detection limit conceived spontaneously. The duration of function of the transplants was between 9 months and 7 years and still functioning. Twelve women received assisted reproduction therapy; in 72 cycles, 65 oocytes were retrieved. The pregnancy rate and live-birth rate per cycle were 6.9% (5 of 72) and 2.8% (2 of 72), respectively. CONCLUSION(S) The relatively poor outcome of assisted reproduction in women transplanted with frozen/thawed ovarian tissue may reflect reduced follicular selection rather than defective or aged oocytes. In normal women, reduced follicular selection with age may be part of explaining the decline in female fecundity with increasing age.