The Howard and Georgeanna Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, VA 23507, USA
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Abstract |
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Key words: autologous transplantation/chemotherapy/cynomolgus monkey/ovarian physiology/ovarian preservation
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Introduction |
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At the gonadal level, chemotherapy and radiation therapy typically result in a high incidence of primary ovarian failure. Women treated with chemotherapy for Hodgkin's disease have a 69% incidence of ovarian failure if their age is <29 years, while 96% of patients developed ovarian failure if they were 30 years (Chapman et al., 1979
). Radiation therapy also has a profound effect on ovarian function, with a >60% incidence of permanent ovarian failure if the ovaries are exposed to radiation doses >300 cGy, which is far below typical therapeutic doses (Chambers et al., 1991
).
For the long term survivors, primary ovarian failure can have devastating sequelae that can include significant, progressive osteoporosis, accelerated coronary artery disease, hot flashes, mood changes, vaginal dryness, depression, decreased libido, infertility and an overall decrease in the quality of life based upon both subjective and objective quality assessments (Barrett et al., 1992; Zichella, 1993
). Both oral and transdermal hormonal replacement therapies are available; however, both are typically fraught with compliance rates of <50% at 1 year and do not address the issue of gamete preservation (Hammond, 1994
).
With minimal success, various prophylactic methods have been studied in an effort to preserve ovarian function after radiation and chemotherapy. Administration of oral contraceptives, antecedent to chemotherapy does not appear helpful in preserving ovarian function after the administration of chemotherapy (Whitehead et al., 1983). Likewise, in cases of pelvic irradiation, ovarian transposition has proved unsuccessful, with a >83% incidence of ovarian failure within 5 years (Anderson et al., 1993
; Anderson, 1995
).
Ovarian preservation through the transplantation of fresh or cryopreserved ovarian tissue has several theoretical advantages. For example, fresh autologous ovarian transplantation into a heterotopic site may allow for removal of the ovarian tissue from the field of radiation therapy, thus preserving the ovarian tissue and providing long-term estrogen therapy and preservation of gametes. In situations of chemotherapy, ovary removal, cryopreservation and autologous transplantation after the completion of chemotherapy may minimize ovarian tissue damage, thus reducing the incidence of ovarian failure.
Dissen et al. evaluated autologous ovarian transplantation in 23 day old juvenile rats and demonstrated that immature rat ovaries become revascularized and regained the ability to control gonadotrophin secretion via steroid negative feedback within 1 week without vascular anastomosis (Dissen et al., 1994). Vascular corrosion casting and scanning electron microscopy revealed that the transplanted ovarian tissue became revascularized within 48 h after transplantation and RNA blot hybridization demonstrated a 4060 fold increase in the expression of vascular endothelial growth factor (VEGF) and transforming growth factor ß1 (TGF-ß). Several authors have studied the effects of exogenous VEGF administration in ischaemic gracilis and skin flaps with resultant increased flap survival (Taub et al., 1998
; Kryger et al., 1999
).
Ovarian transplantation of cryopreserved ovarian tissue demonstrates that ovarian tissue is tolerant to freezing and thawing. Gosden et al. cryopreserved sliced cortical ovarian tissue (Gosden et al., 1994). Three weeks later, the cryopreserved ovarian tissue was thawed, the contralateral ovary removed and the thawed ovarian tissue was autologously transplanted into the pelvis. All of the sheep had peripheral plasma concentrations of FSH in the normal basal range 4 months after transplantation. The animals were allowed to mate and one of the animals with transplanted cryopreserved ovarian tissue became pregnant, yielding viable offspring.
There is a paucity of data regarding transplantation of primate ovarian tissue. In 1995, Candy et al. reported on the follicular development of fresh and cryopreserved marmoset ovarian tissue transplanted underneath the kidney capsule of ovariectomized immunodeficient mice (Candy et al., 1995). The ovarian grafts recovered, as demonstrated by estradiol production in the recipient mice in the third week after transplantation of fresh and frozen grafts. The total number of follicles and the proportion of normal follicles were similar in both the fresh and frozen grafts.
Several human cases of ovarian transplantation have reported varying degrees of short-term success (Johnson, 1998; Hartum, 1999
). Oktay in 1999 reported on a 28 year old woman who underwent autologous transplantation of cryopreserved ovarian tissue to the pelvis followed by ovarian stimulation resulting in the production of a dominant follicle and a peak estradiol concentration of 93 pg/ml (Oktay, 1999
). The following month, she was placed back on hormone replacement therapy due to symptoms of hypo-estrogenism.
Oktay et al. also reported on a case of autologous ovarian tissue transplantation to the right forearm of a 33 year old female with stage IIIB squamous cell cervical carcinoma prior to pelvic radiotherapy (Oktay and Karlikaya, 2000). Venous blood sampling of the right hand versus the right cubital fossa demonstrated an estradiol gradient consistent with estradiol production from the graft. FSH concentrations returned to normal 120 days after transplantation; however, ovulation did not occur.
More recently Radford et al. reported on a 37 year old patient with recurrent lymphoma who had ovarian tissue strips cryopreserved prior to chemotherapy and transplanted onto the left ovary after chemotherapy (Radford et al., 2001). Seven months after transplantation, the patient was found to have estradiol production that lasted for 9 months, at which time she was found to have castrate levels of estradiol.
Evidence from mouse, rat and sheep models demonstrates the potential viability of subcutaneous ovarian transplantation; however, varying techniques of tissue transplantation can have a profound impact upon post transplant viability. The study by Dissen et al. demonstrated that VEGF may also play an important role in ovarian transplant survival, and exogenous VEGF administration has been demonstrated to enhance ischaemic skin flap survival. VEGF is a member of the fibroblast growth factor family, which stimulates the formation of new vasculature through the sequential steps of endothelial cell proliferation, migration, and penetration of host stroma and extracellular matrix (Dissen et al., 1994; Taub et al., 1998
; Kryger et al., 1999
).
The study reported herein describes a prospective experimental study involving 16 cycling female cynomolgus monkeys randomly divided into three groups who underwent extrapelvic ovarian transplantation with subsequent monitoring of serum estradiol, progesterone, LH and FSH concentrations and ovarian stimulation. The effects of ovarian stimulation were investigated, and post-transplanted tissue was analysed histologically.
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Materials and methods |
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Experimental design
After oophorectomy, the 16 monkeys were randomized to three groups: (i) sham transplant group (n = 5), which underwent a sham transplantation of adipose tissue in the upper outer aspect of the right arm followed by cryopreservation of the ovarian tissue, (ii) ovarian transplantation without VEGF administration (n = 6), and (iii) ovarian transplantation followed by the daily administration of 1 µg of VEGF (Biodesign International, Kennebunk, ME, USA), s.c., for 14 days (n = 5). After completion of the fresh transplantation components of the study, the sham transplant monkeys underwent thaw and transfer of their cryopreserved ovarian tissue.
Surgical methods
Ovarian transplantation was performed by making a 1 cm skin incision followed by blunt dissection of a 1 cm subcutaneous pocket, 2 cm distal to the skin incision. The end was then cut off a standard insulin syringe and both ovaries were loaded into the insulin syringe after being sliced into 1 mm sections. The syringe was then placed through the skin incision and ovarian tissue was deposited 2 cm distal to the skin incision. Particular care was used during the transplantation to avoid trauma to the subcutaneous pocket. The skin incision was then closed with nylon.
Cryopreservation
The ovarian tissue from the bilateral oophorectomy (each ~1 cm3) in the sham transplant group was sliced into 1 mm sections and cryopreserved with a protocol previously described by Gosden (Gosden et al., 1994). Briefly, within 1 h of surgery the sliced ovarian tissue was evenly transferred into 10 cryogenic vials each containing 1 ml of M2 media (Sigma-Aldrich Corporation, St Louis, MO, USA) supplemented with fetal calf serum (Gibco, Grand Island, NY, USA) and DMSO (Sigma-Aldrich) to give a final concentration of 10% fetal calf serum and 1.5 mol/l DMSO, and held on ice for 15 min. The cryogenic vials were then transferred to a Planer programmable freezer (T.S. Scientific, Perkasie, PA, USA) and cooled at a rate of 2°C/min to -7°C, then held at -7°C for 10 min before seeding. Manual seeding was then performed by touching the cryogenic vials with forceps previously frozen in liquid nitrogen. After seeding occurred, the temperature was then lowered by 0.3°C/min to -40°C and thereafter by 10°C/min to -140°C. The vials were removed and plunged into liquid nitrogen and stored for 9 months.
Thawing
The frozen ovarian slices from the sham transplant monkeys were rewarmed in air for 2 min before being transferred to a warm water bath at room temperature to complete the thawing process. After washing in three changes of fresh medium to remove the DMSO, the ovarian tissue slices were then transferred autologously into the upper outer aspects of the left arm of the respective sham transplant monkeys in a procedure identical to that described with the fresh ovarian transplantation. These monkeys then underwent daily observations for 100 consecutive days, during which we conducted daily observations for vaginal bleeding and collected every other day femoral blood sampling. Serum was assayed for estradiol, progesterone, LH, and FSH.
Study phases
The first phase of the study involved daily observations of the sham transplant and fresh ovarian transplant groups for 120 consecutive days. During this interval, daily vaginal bleeding observations were performed along with every other day femoral artery blood sampling. Serum samples were collected and assayed for estradiol, progesterone, LH, and FSH.
After 120 days of observation, seven of the 16 monkeys were randomly assigned to ovarian stimulation (two sham transplant monkeys, three monkeys transplanted without VEGF administration and two monkeys transplanted with VEGF administration). All primates were down-regulated using a luteal Lupron protocol administering Lupron Depot (TAP Pharmaceutical Products Inc., Lake Forest, IL, USA) 0.5 mg per kg i.m. Serum estradiol and progesterone analyses were performed 2 weeks later; estradiol in all monkeys was at basal concentrations; then 75 IU of HMG (Repronex; Ferring Pharmaceuticals) was administered i.m. daily for 7 days, followed by 2000 units of HCG (Profasi; Serono Laboratories Inc.) administered i.m. The ovarian tissue and uterus was harvested ~30 h later. The follicles were aspirated with a 25-gauge needle and the follicular fluid was examined with a Nikon SMZ-10 dissecting microscope for oocyte identification and grading for morphology and maturation with an inverted Nikon Diaphot-300 microscope. A similar ovarian stimulation and oocyte retrieval procedure was employed for the primates receiving the cryopreserved ovarian tissue 100 days after transplantation, with stimulation lasting 9 days.
The six remaining monkeys, who underwent ovarian transplantation and were not selected for ovarian stimulation, underwent collection of the uterus and ovarian tissue 120 days after transplantation. The transplanted ovarian tissue was easily identified and removed via a 2 cm skin incision. While under anaesthesia the primates underwent a mid-line laparotomy and hysterectomy. The specimens were then embedded in paraffin block. Five-micron sections were collected at 50 micron intervals throughout the tissue and stained with haematoxylin and eosin.
Hormone analyses
17ß-Estradiol and progesterone assays were performed using commercial radioimmunoassay kits: 17ß-estradiol (ICN Pharmaceuticals, Costa Mesa, CA, USA); and progesterone (Diagnostic Systems Laboratories, Webster, TX, USA). The average intra- and inter-assay CVs were 6.7 and 11.4% respectively for estradiol and 8.3 and 11.2% respectively for progesterone. The assay sensitivities were 10 pg/ml and 0.3 ng/ml for estradiol and progesterone respectively. Peak follicular phase cynomolgus monkey estradiol concentrations were 150 pg/ml and luteal phase progesterone concentrations average 5 ng/ml.
FSH concentrations were determined using a rabbit antibody to ovine FSH (H-31), preparations of hFSH (NIH-FSH-3) for iodination and cynomolgus FSH (NICHD-CYN-FSH-RP1) as reference preparation. (Hodgen et al., 1976). The FSH assay sensitivity was 14 ng/ml and the average intra- and inter-assay CVs were 6.3 and 8.7% respectively. Average cynomolgus monkey FSH concentrations were 30 ng/ml.
Monkey LH was quantitated with the previously described mouse Leydig cell bioassay (Gordon et al., 1991). The LH assay sensitivity was 11 ng/ml and the average intra- and inter-assay CVs were 10 and 13% respectively. Average cynomolgus monkey LH concentrations were 30 ng/ml with concentrations at LH surge typically >100 ng/ml.
Statistical analysis
Spectral analysis was used to compare the hormone data of all four treatment groups. Hormone data lends itself to spectral analysis because the data are a time series with more or less cyclical patterns. Spectral analysis describes a stationary (no trend) time series in terms of sinusoidal waves of various frequencies, i.e. acos(wt) + bsin(wt), where t is time and w is the frequency of fluctuations and is expressed in terms of Fourier frequencies equal to 2j/n for a positive integer j/n. The coefficients, a and b, are obtained by regular regression. The plot of the spectral function to frequency is termed a periodogram.
In this case, the interest was in comparing periodograms of the functioning ovarian transplants in the four treatment groups. Averaged time series (e.g. estradiol) of the various groups were computed, and a spectral function computed for the resulting series of each group. The ratio of the treatment groups being compared was then obtained and tolerance intervals computed (F distribution) to determine if the resulting ratio of spectral functions differed significantly from 1. In addition, a trend in the ratio of spectral functions indicates a difference in cyclical patterns of the time series whereas no trend indicates similar cyclical patterns. (Diggle, 1990).
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Results |
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The interval from transplantation to detection of serum estradiol was 24 ± 5.12 days in the fresh transplant groups and 14 days in the cryopreserved group. For virtually all days, the primates with functioning ovarian transplants in the VEGF group had a higher estradiol concentration (average estradiol 73 pg/ml) than those with functioning transplants in the group without VEGF (average estradiol 52 pg/ml). However, taking the ratios of VEGF to without VEGF spectral functions and computing the spectral function of the ratios indicated that the ratios, although most were >1, none was significantly so (horizontal line), thus demonstrating no significant difference between the two groups (Figure 1).
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Discussion |
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Approximately half of the primates that had non-functional ovarian transplants demonstrated early evidence of graft function with low estradiol concentrations and mild suppression of FSH and LH. The estradiol concentrations in these primates returned to castrate levels within 45 days and remained low. Conversely, all of the primates with functioning ovarian tissue 45 days after transplantation continued to demonstrate functional ovarian tissue throughout the remainder of the study.
In an attempt to enhance angiogenesis and resultant transplant viability, VEGF was administered subcutaneously at the site of ovarian transplantation in one third of the monkeys. Although not statistically significant, the VEGF administration at the doses tested was associated with a smaller percentage of monkeys with functioning transplanted ovarian tissue compared with the transplant group without VEGF administration. The cause of decreased graft viability is unclear and does not appear to be related to trauma associated with the injections as the group without VEGF administration had subcutaneous administration of saline as a control. One monkey in the group with VEGF administration was noted to have a haematoma that developed postoperatively at the transplant site. This monkey did not have a functioning transplant.
Those primates that did have functioning ovarian transplants after VEGF administration had higher progesterone concentrations in the luteal phase and a trend toward higher estradiol concentrations in the follicular phase. This could be due to and increase in the graft vascularity and/or the percentage of surviving tissue.
The histology of transplanted ovarian tissue demonstrated viable primordial and antral follicles in both the fresh and cryopreserved groups. One monkey had a large corpus luteum with adequate progesterone production as evidenced by its secretory endometrium. The viable primordial and antral follicles support the longevity of the ovarian tissue and possible source of gametes with future ovarian stimulation.
Ovarian stimulation of the fresh transplants was performed after administration of Lupron Depot and stimulation with one ampoule of HMG for 7 days. Based upon the estradiol concentrations at HCG administration and the high percentage of immature oocytes obtained at harvest of the fresh transplanted tissue, the stimulation in the cryopreserved transplants was lengthened to 9 days; one mature egg was recovered. The fresh monkeys with fresh ovarian transplants may have been under-stimulated and lengthening the duration of ovarian stimulation may have increased the number and percentage of mature oocytes recovered. Due to documented antibody formation after one cycle of HMG administration we did not stimulate these monkeys a second time (Platia et al., 1984).
In the cryopreserved group two of four (50%) of the primates had functioning ovarian transplants 9 months after oophorectomy. Since the primary endpoint of our study was the establishment of ovarian function as demonstrated by hormonal secretion and gamete production, we chose the cryopreservation and thaw protocol published by Gosden et al. that resulted in live offspring in sheep (Gosden et al., 1994). Other cryopreservation protocols using histological endpoints show promise and may provide superior results (Gook et al., 1999
; Gook et al., 2000
).
This study demonstrates that fresh and cryopreserved autologous extrapelvic ovarian transplantation results in the restoration of ovarian function with resultant ovarian and menstrual cyclicity and the production of mature gametes. The administration of VEGF at the dose tested did not appear to improve transplant outcome in the small numbers tested. The success of ovarian transplantation in these non-human primates with menstrual cycles bodes well for the development of ovarian transplantation protocols for women at risk for ovarian failure from chemotherapy, radiation therapy or other causes.
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Notes |
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* This presentation was awarded the General Program Prize Paper at the 56th Annual Meeting of the American Society of Reproductive Endocrinology, San Diego, California, October 2000.
Submitted on April 25, 2001; resubmitted on September 25, 2001
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References |
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accepted on November 8, 2001.