TECHNIQUES AND INSTRUMENTATION

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1 FERTILITY AND STERILITY VOL. 80, NO. 1, JULY 2003 Copyright 2003 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A. TECHNIQUES AND INSTRUMENTATION A technique for transplantation of ovarian cortical strips to the forearm Kutluk Oktay, M.D., a Erkan Buyuk, M.D., a Zev Rosenwaks, M.D., a and James Rucinski, M.D. b The Center for Reproductive Medicine and Infertility, Joan and Sanford I. Weill Medical College of Cornell University, New York, New York, New York, and Methodist Hospital, Brooklyn, New York Received July 5, 2002; revised and accepted December 12, No reprints available. Correspondence: Kutluk Oktay, M.D., The Center for Reproductive Medicine and Infertility, Joan and Sanford I. Weill Medical College of Cornell University, 505 East 70th Street, HT-340, New York, New York (FAX: ; kuo9001@med.cornell.edu). No reprints available. a The Center for Reproductive Medicine and Infertility, Joan and Sanford I. Weill Medical College of Cornell University. b Department of Surgery, New York Methodist Hospital /03/$30.00 doi: /s (03) Objective: To describe a forearm heterotopic ovarian transplantation technique. Design: Case study. Setting: Academic medical center. Patient(s): One patient with stage IIIB squamous cell cervical carcinoma and one patient with recurrent benign ovarian cysts. Intervention(s): Preparation of thin ovarian cortical slices and transplantation under the skin of the forearm. Main Outcome Measure(s): Follicular development and oocyte retrieval; cyclical estradiol (E 2 ) and progesterone (P 4 ) production; restoration of serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels to reproductive age range. Result(s): Both patients were menopausal immediately after oophorectomy. The first patient developed a dominant follicle 10 weeks after transplantation, and her gonadotropin levels decreased to nonmenopausal levels. Percutaneous aspiration of ovarian follicles yielded a metaphase I (M-I) oocyte that was matured to metaphase II (M-II). The first patient s graft was functional for at least 21 months. In the second patient, ovarian follicle development was detected 6 months after transplantation, and periodic menstruation occurred thereafter. Spontaneous ovulation was confirmed by a midluteal increase in her P 4 levels. Menstruation and follicle development continued for more than 2 years after the transplant. Conclusion(s): Heterotopic transplantation of ovarian tissue to the forearm is a simple and promising technique to restore ovarian function in women who become menopausal due to chemotherapy, surgery, or radiation. (Fertil Steril 2003;80: by American Society for Reproductive Medicine.) Key Words: Ovarian transplant, cancer, fertility preservation, heterotopic, forearm Recent advances in cancer treatment modalities, including aggressive chemotherapy, radiotherapy, and bone marrow transplantation, have resulted in a significant increase in cure rates (1, 2). Such aggressive treatment strategies, however, often cause infertility and premature ovarian failure (2). Moreover, many women are faced with premature menopause due to oophorectomies performed during their reproductive lives. We have previously reported a case of laparoscopic transplantation of frozen-banked ovarian tissue to the ovarian fossa in a patient whose ovaries had been removed because of benign causes (3). Others have reported similar results with orthotopic ovarian transplantations in cancer patients (4). When even a small risk of reseeding cancer exists, however, intraabdominal replacement of ovarian tissue may be less desirable than heterotopic placement. In addition, in patients who receive radiotherapy to the pelvis, the vascular supply may not be sufficient for orthotopic transplantation. We have previously reported the results of the two cases of forearm heterotopic ovarian transplantation (5). The aim of this article is to describe the surgical technique of transplantation of ovarian cortical pieces to the forearm, and provide information on the most recent follow-up. Although these procedures were done with fresh tissue, the same technique can be used with frozen-banked tissue, as was recently demonstrated in a primate study (6). For heterotopic transplantation, we chose the forearm location because of the successful transplantation of both fresh and frozen-thawed parathyroid tissues (7, 8) to this site. The fore- 193

2 arm location offers the advantage of easy accessibility. Also, in case of a local cancer recurrence in the transplanted ovarian tissue, the forearm site may be safer for two reasons: identification of a recurrent malignancy is easier, and the forearm is located at a greater distance from the vital organs. The transplantation of ovarian tissue to the forearm may also be more desirable in benign gynecologic cases, such as recurrent ovarian cysts or endometriosis, where close surveillance of ovarian tissue is necessary. Moreover, because general anesthesia is not required, the tissue may easily be removed, or more cortical slices may be implanted when the oocyte reserve becomes low. Finally, the technique of heterotopic ovarian transplantation is less complex than orthotopic ovarian transplantation, and it does not require laparoscopy or laparotomy. FIGURE 1 A surgical technique for ovarian transplantation to the forearm. (See text for the details of the technique.) MATERIALS AND METHODS Both patients were extensively counseled, and each signed a research consent form approved by the Institutional Review Board at the New York Methodist Hospital and the New York Presbyterian Hospital of Weill Medical College of Cornell University. Hormone measurements were performed by a radioimmunoassay. Statistical analyses were performed using the paired two samples t-test. A P value of.05 was considered statistically significant. Patients Patient-I was a 35-year-old woman with stage IIIB squamous cell carcinoma of the cervix, who was referred by her gynecologic oncologist for autologous ovarian transplantation prior to pelvic radiotherapy. The patient was counseled about ovarian transposition, however, because of a 17% 100% failure rate associated with scatter radiation and vascular compromise (9 13), and because of her family history of ovarian cancer, she chose to undergo the experimental ovarian transplantation. The primary gynecologic oncologist involved in the case was concerned that transposition of the ovaries would render screening of the ovaries by palpation and transvaginal ultrasound examination more difficult. Patient-II was a 37-year-old woman who, at 18 years of age, had a left oophorectomy to treat a serous cystadenoma. Subsequently, she developed recurrent ovarian cysts in her remaining ovary and had undergone two operations. At her third recurrence, the primary gynecologist advised removal of the remaining ovary. Because of concerns regarding another recurrence, the forearm site was chosen for ovarian transplantation. Procedure After oophorectomy, ovarian cortical pieces are prepared. If the tissue will be cryopreserved first, cm strips of 1 3 mm thickness are prepared. If the transplant will be performed with fresh tissue, the strips can be longer; cm strips of 1 3 mm thickness will facilitate the transplantation procedure. In either case, frozen section biopsies are Oktay et al. Ovarian transplant to the forearm. Fertil Steril performed to confirm that the ovarian tissue is free of disease. If frozen tissues are used, thawing is performed as described previously (14). Fresh or recently thawed ovarian cortical pieces are placed in phenol-red-free Minimum Essential Medium Alpha Medium (with L-glutamine, ribonucleosides, and deoxyribonucleosides, Gibco, cat. no ), supplemented with 20% patient s own serum and 10 g/ml of cefotetan and kept on ice. Then, each piece is tagged with 4-0 vicryl by passing the needle between stroma and cortex under an operating microscope (Fig. 1A). The needle is then cut, and the pieces are left in the medium until the surgical site is prepared. Then, a 1.5-cm transverse incision is made over the brachioradialis muscle, 5 10 cm below the antecubital fossa. If the patient has cosmetic concerns, the incision and the implantation may be made more medially over the 194 Oktay et al. Ovarian transplant to the forearm Vol. 80, No. 1, July 2003

3 superficial flexor muscles of antebrachial area, as is the case in our second patient. Using blunt dissection, a pocket is created between the fascia and the subcutaneous tissue (Fig. 1B). This area is relatively vascular, and attention must be given to avoid injuring the larger veins and arteries because the ovarian tissue will acquire its blood supply from the local vessels. It is not desirable to perform extensive cauterization. Once the dissection is completed, the free end of the suture is threaded onto a reusable needle. We typically use a 1 2-circle cutting needle with chord length of mm, depending on the size of the ovarian cortical strips. This needle is inserted in the subcutaneous space as far as possible. The needle is then passed through the skin, and the cortical piece is wedged into the subcutaneous pocket by pulling on the suture (Fig. 1C). The transplanted strips are always inserted with the cortical side facing up. The needle is then removed, and the free end of the suture is held with a mosquito clamp. The purpose of this suture pull-through technique is to guide the tissue placement and to avoid overlapping the strips, instead of anchoring them. Depending on the patient s size, as many as 5 to 15 cortical pieces can be fanned out beneath the forearm skin (Fig. 1D). In order to accommodate a large number of pieces, the pocket can be expanded in the opposite direction (toward the antecubital fossa). Once the tissue placement is satisfactory, the sutures are cut. After the skin is closed using an intradermal/subcuticular suture technique, a nonpressure dressing is applied. A pressure dressing must be avoided to minimize compromise of the blood flow to the area. In keeping with evidence derived from animal studies (15), 75 IU/day of follicle-stimulating hormone (FSH) is injected directly in the subcutaneous space above the grafts for 7 days. Follicle-stimulating hormone is dissolved in 1 cc of diluent and administered using a 25-gauge, 5/8-inch hypodermic needle; a small amount of FSH is released over each graft. In addition, 80 mg of aspirin is administered for seven days. The FSH and aspirin administration is limited to 7 days because of the animal data that graft revascularization occurs within less than 7 days (15). The patient s forearm is splinted for 72 hours to prevent dislodgment of the transplanted tissue by muscle movement. Within 48 hours of the transplant, the patient is started on hormone therapy (HT) in the form of 0.1 mg of transdermal estradiol (E 2 ), in keeping with evidence from animal studies suggesting that estrogen treatment may enhance angiogenesis (16). RESULTS The detailed results of the early follow-up of these two patients have already been published (5) and cannot be discussed here. Next, we give a brief summary of the early outcome and a more detailed account of the most recent follow-up. Patient-I Despite the estrogen therapy (ET), the patient s FSH and luteinizing hormone (LH) levels demonstrated menopause 6 weeks after the procedure. Ten weeks later, she presented with a painless bulge at the site of the transplant. On ultrasound examination, multiple antral follicles were present. Hormone replacement therapy was discontinued, and subsequent observations found cyclical ovarian follicle development (Fig. 2) and estrogen production, as well as normalization of FSH and LH levels (5). Continued monitoring also revealed that the right antecubital vein had assumed the role of ovarian vein; the E 2 measurements from this site were consistent with levels obtained in previous ovarian vein catheterization studies (17). Despite cyclical E 2 production, P 4 remained below the ovulatory level in this patient. A percutaneous oocyte retrieval following controlled ovarian stimulation yielded an oocyte in M-I stage (5). The patient s graft was still functional 21 months after transplantation, as indicated by continued follicle development and normal FSH levels. This occurred despite the administration of three courses of 650 mg carboplatinum over 9 weeks, following detection of a pelvic sidewall relapse at around 10 months after the transplant. After this date, the patient did not return for follow-up. Patient-II As in patient-i, an elevated FSH level confirmed the menopausal status of patient-ii immediately after the procedure (5). Five months following the transplant, a developing lump appeared at the transplant site, and a follicle was found on ultrasound examination. After the discontinuation of HT, the patient had spontaneous luteinization as confirmed by LH and midluteal P 4 measurements (5). Thereafter, the patient developed a dominant follicle every month, which could be palpated beneath the skin, and menstruated regularly every days (mean SE: ). Her cycle day 2 FSH, LH, and E 2 levels were within the normal range 11 months after the transplant. Sixteen months after the transplant, the patient s peripheral FSH, LH, and E 2 levels were 67 miu/ml, 71 miu/ml and 57 pg/ml, respectively, which indicated menopause. After this date, the patient did not come in for follow-up until 25 months after the transplant. At the 25-month follow-up, the patient indicated that her periods had become less regular since the 16th month after the transplant, and ranged from days (mean SE: ). The patient did indicate, however, that she continued to notice the development and regression of a follicle every month (Fig. 3A). On the 11th day of that cycle, ultrasound examination revealed a mm dominant follicle (Fig. 3B), and the peripheral FSH, LH, E 2, and P 4 levels were 17 IU/mL, 12 miu/ml, 100 pg/ml, and 4.7 ng/ml, respectively, which indicated that the patient had ovulated. FERTILITY & STERILITY 195

4 FIGURE 2 Ovarian follicle growth visible after the transplantation in patient-i. Oktay et al. Ovarian transplant to the forearm. Fertil Steril In an observed difference from patient-i, we did not find a gradient between the right cubital vein (central) E 2 measurements and right-hand (peripheral) values ( pg/ml vs pg/ml, respectively; P.05) during the first 11 months after the transplant. However, measurements that were performed at 16 and 25 months after the transplant began to show a gradient in E 2 ( pg/ml vs pg/ml; P.03). Twenty-eight months after the transplant (August 2002), this patient became amenorrheic, and began complaining of hot flashes. She no longer noticed ovarian follicle growth beneath her skin, and her gonadotropin levels rose to menopausal levels. DISCUSSION This article discribed a surgical technique for heterotopic ovarian tissue transplantation to the forearm. Ovarian function was demonstrated both by follicle growth on ultrasound examination and by estrogen production. Although the first patient did not spontaneously ovulate, ovulation was demonstrated in the second patient. In the first patient, recovery of a metaphase I oocyte following ovarian stimulation, and luteinization after human chorionic gonadotropin (hcg) administration (5) indicated that the follicles in the graft were competent to undergo spontaneous ovulation and luteinization. In both patients reported here, transplantation was performed with fresh tissue. Tissues were not cryopreserved because our research protocol at the time limited cryopreservation to women 35 years of age. Recent studies, however, have shown that the majority of follicle loss occurs during the initial ischemia after transplant, not due to the cryopreservation procedure (18). Confirming this point, a recent primate study comparing fresh and frozen-thawed ovarian transplants to the forearm did not find any statistically significant differences in outcome. Ovarian function was restored in a similar fraction of subjects in both groups, and mature oocytes were recovered percutaneously (6). The medial placement of ovarian grafts over the superficial flexor muscles gives better cosmetic results. The veins in that area do not drain to the cubital vein (19), however, and no gradient was seen between the proximal and distal measurements of ovarian steroids before the 16th month. This observation may be associated with ongoing progressive revascularization of the grafts such that a gradient was established by 16 months after the transplant. The fact that these grafts continue to revascularize over the course of several years was also supported by our previous finding that 196 Oktay et al. Ovarian transplant to the forearm Vol. 80, No. 1, July 2003

5 FIGURE 3 Ovarian follicle development 25 months after the transplant in patient-ii: (A), as visible by inspection; (B), as seen on ultrasound examination. Oktay et al. Ovarian transplant to the forearm. Fertil Steril in the first patient, mean FSH and LH levels continued to decrease from miu/ml and miu/ ml, during the period from 78 to 117 days after transplantation, to miu/ml and miu/ml during the period from 120 to 227 days after transplantation (P.002) (5). Transplantation of ovarian tissue to the forearm has many advantages compared with the orthotopic technique that we reported previously (3). The forearm ovarian transplantation technique (FOT procedure) does not require general anesthesia and is relatively simple. The monitoring of the ovarian tissue is easier, and the tissue may be easily removed when FERTILITY & STERILITY 197

6 needed. When the main goal of the procedure is fertility, all of the tissue need not be transplanted at once. The simplicity of the forearm transplantation technique allows for sequential procedures to insert additional cortical strips as the previous grafts exhaust their follicle reserves. This is an important advantage because the longevity of these grafts beyond 2 years has not yet been confirmed. Because numerous centers have been banking ovarian tissue around the world, our FOT procedure may represent a less invasive experimental approach to test the feasibility of ovarian tissue banking in those patients who have already stored their tissue. References 1. Ries LAG, Percy CL, Bunin GR. Cancer incidence and survival among children and adolescents: United States SEER Program In: Ries LAG, Smith MA, Gurney JG, et al., eds. National Cancer Institute, Surveillance, Epidemiology, and End Results (SEER) Program. Bethesda, MD: National Institutes for Health (NIH) Pub. No , 1999: Meirow D, Nugent D. The effects of radiotherapy and chemotherapy on female reproduction. Hum Reprod Update 2001;7(6): Oktay K, Aydin BA, Karlikaya G. A technique for laparoscopic transplantation of frozen-banked ovarian tissue. Fertil Steril 2001;75(6): Radford JA, Lieberman BA, Brison DR, Smith AR, Critchlow JD, Russell SA, et al. Orthotopic reimplantation of cryopreserved ovarian cortical strips after high-dose chemotherapy for Hodgkin s lymphoma. Lancet 2001;357(9263): Oktay K, Economos K, Kan M, Rucinski J, Veeck L, Rosenwaks Z. Endocrine function and oocyte retrieval after autologous transplantation of ovarian cortical strips to the forearm. JAMA 2001;286(12): Schnorr J, Oehninger S, Toner J, Hsiu J, Lanzendorf S, Williams R, et al. Functional studies of subcutaneous ovarian transplants in nonhuman primates: steroidogenesis, endometrial development, ovulation, menstrual patterns and gamete morphology. Hum Reprod 2002;17(3): Wells SA Jr, Ellis GJ, Gunnels JC, Schneider AB, Sherwood LM. Parathyroid autotransplantation in parathyroid hyperplasia. N Engl J Med 1976;295: Wagner PK, Seesko HG, Rothmund M. Replantation of cryopreserved human parathyroid tissue. World J Surg 1991;15: Husseinzadeh N, Nahhas WA, Velkley DE, Whitney CW, Mortel R. The preservation of ovarian function in young women undergoing pelvic radiation therapy. Gynecol Oncol 1984;18(3): Le Floch O, Donaldson SS, Kaplan HS. Pregnancy following oophoropexy and total nodal irradiation in women with Hodgkin s disease. Cancer 1976;38(6): Thomas PR, Winstanly D, Peckham MJ, Austin DE, Murray MA, Jacobs HS. Reproductive and endocrine function in patients with Hodgkin s disease: effects of oophoropexy and irradiation. Br J Cancer 1976;33(2): Hunter MC, Glees JP, Gazet JC. Oophoropexy and ovarian function in the treatment of Hodgkin s disease. Clin Radiol 1980;31(1): Guglielmi R, Calzavara F, Pizzi GB, Polico C, Maluta S, Turcato G, et al. Ovarian function after pelvic lymph node irradiation in patients with Hodgkin s disease submitted to oophoropexy during laparotomy. Eur J Gynaecol Oncol 1980;1(2): Oktay K. Ovarian tissue cryopreservation and transplantation: preliminary findings and implications for cancer patients. Hum Reprod Update 2001;7(6): Imthurn B, Cox SL, Jenkin G, Trounson AO, Shaw JM. Gonadotrophin administration can benefit ovarian tissue grafted to the body wall: implications for human ovarian grafting. Mol Cell Endocrinol 2000; 163(1-2): Morales DE, McGowan KA, Grant DS, Maheshwari S, Bhartiya D, Cid MC, et al. Estrogen promotes angiogenic activity in human umbilical vein endothelial cells in vitro and in a murine model. Circulation 1995;91(3): Ala-Fossi SL, Maenpaa J, Aine R, Punnonen R. Ovarian testosterone secretion during perimenopause. Maturitas 1998;29(3): Baird DT, Webb R, Campbell BK, Harkness LM, Gosden RG. Longterm ovarian function in sheep after ovariectomy and transplantation of autografts stored at 196 C. Endocrinology 1999;140(1): Netter FH. In: Dalley AF, ed. Atlas of human anatomy. 2nd edition. Salt Lake City, Utah: Icon Learning Systems, 1997: Plate Oktay et al. Ovarian transplant to the forearm Vol. 80, No. 1, July 2003