Hum. Reprod. Advance Access originally published online on February 3, 2006
Human Reproduction 2006 21(6):1345-1348; doi:10.1093/humrep/del007
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OPINION |
OPINION
Spontaneous conceptions and live birth after heterotopic ovarian transplantation: is there a germline stem cell connection?
Fertility Preservation Program, Center for Reproductive Medicine & Infertility, Weill Medical College of Cornell University, New York, NY, USA
To whom correspondence should be addressed at: Fertility Preservation Program, Center for Reproductive Medicine & Infertility, Weill Medical College of Cornell University, 505 E 70th Street, New York, NY 10021, USA. E-mail: kuo9001{at}med.cornell.edu
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Abstract |
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 Top Abstract IntroductionLive birth after heterotopic...A cautionary noteAn alternative hypothesisAcknowledgementsReferences |
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Ovarian cryopreservation and transplantation is an emerging technology to preserve fertility in women and children undergoing cancer treatment. Recent reports of live births after orthotopic transplantation raised hopes for the future success of this procedure. However, doubts remained whether the reported pregnancies were as a result of resumed function in the remaining ovary. We recently performed an autologous heterotopic ovarian transplantation in a 32-year-old Hodgkin lymphoma survivor who was menopausal for 2.5 years as a result of a preconditioning chemotherapy given before a hematopoietic stem cell transplant. Subsequent to the transplantation, the patient conceived twice within 3 months and delivered a healthy female child at 40 weeks of gestation. The occurrence of spontaneous pregnancies after heterotopic ovarian transplantation highlights the need for caution when interpreting the source of pregnancies in recipients with intact ovaries. On the other hand, the temporal relationship between the ovarian transplant and the spontaneous resumption of ovarian function and pregnancies in previously menopausal women is intriguing, especially in the light of recent reports of germ cell renewal and migration from the bone marrow to the ovary in rodents.
Key words: cancer/germline stem cells/ovarian function/ovarian transplantation
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Introduction |
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TopAbstractIntroductionLive birth after heterotopic...A cautionary noteAn alternative hypothesisAcknowledgementsReferences |
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Ovarian cryopreservation and transplantation is one of the promising technologies for the preservation of fertility in cancer patients undergoing gonadotoxic chemotherapy. Building on successful animal studies, researchers have been able to restore ovarian function in a number of patients by ovarian transplantation to orthotopic and heterotopic locations (Oktay and Karlikaya, 2000
; Radford et al., 2001; Donnez et al., 2004; Oktay et al., 2004; Meirow et al., 2005). The methodology and safety issues have been reviewed (Sonmezer and Oktay, 2004). Recently, a four-cell embryo was generated from frozen–thawed ovarian tissue transplanted subcutaneously in a breast cancer survivor (Oktay et al., 2004). Simultaneously, a live birth was reported in a monkey after a similar transplantation procedure with fresh autologous ovarian tissue (Lee et al., 2004). Following the first report of ovarian function after orthotopic ovarian transplantation (Oktay and Karlikaya, 2000), Donnez et al. (2004) reported a patient who gave birth after a similar transplantation procedure using ovarian tissue which was cryopreserved when the patient was at the age of 25 years. However, because this patient had received a chemotherapy regimen that carried a relatively low probability of ovarian failure (Schilsky et al., 1981), and because she occasionally ovulated from the remaining ovary, the mechanism of this spontaneous conception was questioned (Oktay and Tilly, 2004; Wallace and Pritchard, 2004). Because the patient was not closely monitored in the cycle leading to the pregnancy, it remained possible that the fertilized oocyte had been ovulated from the pre-existing ovary, rather than the one which was grafted. More recently, a live birth was reported in a non-Hodgkin lymphoma survivor who had her previously cryopreserved ovarian tissue (Meirow et al., 2005) grafted in her menopausal ovaries. Ovarian cortical biopsies had been cryopreserved subsequent to several courses of combination chemotherapy but before the preconditioning chemotherapy for a stem cell transplant. Before ovarian transplantation, the patient was in menopause for 24 months. Within 8 months of the transplant, the patient’s ovarian function returned, as shown by inhibin-B, anti-Müllerian hormone, FSH and estradiol (E2) measurements and by ultrasound examinations. An IVF cycle resulted in a pregnancy and live birth. Although this case provided stronger evidence for the restoration of fertility after ovarian transplants, because the tissue was implanted in the pre-existing ovary, a theoretical possibility of spontaneous resumption of ovarian function, albeit with <5% probability, remained.
We recently performed a heterotopic ovarian transplantation in a patient who had received preconditioning chemotherapy and autologous stem cell transplantation (SCT) following ovarian cryopreservation. The patient conceived twice, immediately after transplantation (Oktay and Oktem, 2005). This case report carries implications for interpreting the origin of spontaneous pregnancies after ovarian transplants, and brings forward intriguing hypotheses considering the recent animal studies suggesting germ cell renewal and migration in adult mice (Johnson et al., 2004, 2005a).
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Live birth after heterotopic ovarian transplant |
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TopAbstractIntroductionLive birth after heterotopic...A cautionary noteAn alternative hypothesisAcknowledgementsReferences |
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The patient was a 32-year-old female who was initially diagnosed with Hodgkin lymphoma at the age of 28 and who received six courses of Adriamycin, bleomycin, vinblastine, dacarbazine (ABVD) along with radiotherapy to chest and spleen. This treatment did not result in ovarian failure, as it did not contain an alkylating agent. Because of a relapse a year later, the patient was then offered an autologous hematopoietic stem cell transplantation (HSCT). Before receiving chemotherapy with ifosfamide, carboplatin and etoposide (ICE) and rituximab, followed by preconditioning treatment for HSCT (high-dose cyclophosphamide, etoposide and carmustine), she underwent laparoscopic removal of her left ovary and the ovary cryopreserved as described previously (Oktay and Buyuk, 2003
). The ovarian cryopreservation and transplantation study protocol were approved by the Institutional Review Board at the Weill Medical College of Cornell University. The patient became menopausal immediately after stem cell transplant and did not menstruate for 2.5 years and did not receive HRT. During that time, she had FSH and LH levels as high as 95 and 45 mIU/ml, respectively. At the end of 2.5 years, the patient requested ovarian transplantation, as she desired to have a child. After testing one cortical piece for viability, primordial follicle density and absence of malignant cells, remaining ovarian tissue was thawed and subcutaneously transplanted to the suprapubic area, as described previously (Oktay et al., 2004). From the follicle density, it was estimated that the transplanted tissue contained 22 800 primordial follicles. Two months after the transplant, the patient reported breast tenderness, change in vaginal secretions and a tingling sensation in the area of the transplant. Out-of-town E2 and progesterone measurements were 250 pg/ml and 14 ng/ml, respectively, indicating ovulation. The patient was examined 2 weeks later in our centre. Physical and ultrasound examination of the subcutaneous graft did not reveal any antral follicles. On vaginal ultrasound, a 6-week intrauterine pregnancy was seen, but there was no fetal cardiac activity. Because a simultaneous 
-HCG measurement was 77 000 IU, a diagnosis of missed abortion was made. Her right ovary was small, measured 16.9 x 16.6 x 8 mm and contained a complex cyst measuring 4.8 mm, probably representing a regressing corpus luteum. Pregnancy was evacuated by dilation and curettage (D and C) and the cytogenetic analysis of the product of conception showed trisomy 16. Three weeks after the D and C, patient felt growing follicles in her graft and had a spontaneous menstruation a week later. An LH surge was detected by urinary testing on the 14th day of the ensuing cycle at which time the patient had intercourse. The patient felt follicle growth in her graft contemporaneously. A pregnancy test was positive 17 days later, and an ultrasound showed a viable pregnancy of 7 weeks gestational age 3 weeks later (Figure 1A). The pregnancy progressed uneventfully (Figure 1B), and the patient delivered a healthy female child at 40 weeks of gestation.
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A cautionary note |
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TopAbstractIntroductionLive birth after heterotopic...A cautionary noteAn alternative hypothesisAcknowledgementsReferences |
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Occurrence of spontaneous pregnancies in a patient who received a heterotopic ovarian transplantation, after remaining in menopause for 2.5 years, illustrates the need to be cautious about the origin of pregnancies after ovarian transplantation. Even in a patient who receives a treatment such as SCT, which is known to be associated with the highest rate of ovarian failure, spontaneous pregnancies and live birth are possible.
The most conventional and more likely explanation for the mechanism of recurrent conceptions in the patient presented here is the spontaneous recovery of ovarian function through yet-an-unexplained mechanism. The incidence of permanent ovarian failure after SCT can vary depending on the patient’s age and preconditioning chemotherapy regimen used (Meirow and Nugent, 2001). Although many studies suggested a near certainty of ovarian failure in adult patients receiving bone marrow transplants (Deeg, 1990), pregnancies have occurred even in those who appeared to have remained in menopause for several years (Sanders et al., 1998; Hershlag and Schuster, 2002). For example, in a study involving 708 postpubertal women treated with high-dose chemotherapy alone or with total-body irradiation and stem cell transplant for aplastic anaemia or hematologic malignancies, 10 recovered normal ovarian function and 32 became pregnant (Sanders et al., 1996). In addition, nine formerly prepubertal girls with normal gonadal function became pregnant. In a small series of 17 patients, even though all experienced menopause initially, 29% had recovery of gonadal functions at a median of 24 months after SCT (Schimmer et al., 1998). In that study, recovery was more pronounced in younger women. Thus, ovarian function can recover at least for a period following SCT, especially in prepubertal patients or young adults, but the likelihood of infertility is approximately 95%. It, however, remains to be explained, if chemotherapy depletes the entire ovarian reserve, how is it possible that ovarian function can resume many years later and result in normal pregnancies. It is unlikely that the E2 and/or progesterone production by the transplanted ovary can initiate follicular activity in a menopausal ovary, as previous reports did not show a difference in spontaneous pregnancy rates between women with premature ovarian failure who did and did not receive HRT (Kasteren and Schoemaker, 1999). A placebo effect from the ovarian transplantation procedure also seems implausible, given the confirmed ovarian failure in the patient reported here.
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An alternative hypothesis |
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TopAbstractIntroductionLive birth after heterotopic...A cautionary noteAn alternative hypothesisAcknowledgementsReferences |
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Is it merely a coincidence that in the case reported here, pregnancies occurred shortly after ovarian transplantation and contemporaneously with the follicular activity in the graft? This patient had not conceived despite having unprotected intercourse for over 2 years before the ovarian transplantation.
Although it is more likely that the recovery of ovarian function and spontaneous pregnancies after ovarian transplantation is a coincidence in the patient reported here, alternative hypotheses can be put forward. In a recent study by Johnson and co-workers, researchers built on their observation of discrepancy between the percentage of primordial follicles dying from apoptosis and the rate of decline in ovarian follicle reserve in several strains of mice. They observed that at the rate of apoptotic death in the mouse ovary, menopause should occur within weeks of birth. Instead, these mice continue to reproduce well over a year (Johnson et al., 2004).
Recently, the same group showed the expression of germline stem cell markers in the bone marrow and peripheral blood in rodents and women, and these researchers were able to replenish ovarian reserve by injection of bone marrow and peripheral blood samples taken from controls to animals which were sterilized by chemotherapy (Johnson et al., 2005a). The expression of the germline stem cell markers varied with the estrous cycle of the mice, and oophorectomy completely abolished the expression of these markers in the bone marrow. However, the replacement of estrogen and/or progesterone did not restore the expression of germ cell markers in the bone marrow. These findings suggested that there may be an endocrine communication between the ovary and the bone marrow and that hormones responsible for this communication are not sex steroids.
This discovery challenged the more than 50-year-old dogma that primordial follicle reserve is fixed at birth in mammals. Nevertheless, other indirect evidence from human and non-human primate studies existed for more than 80 years. On the basis of histological data, as early as 1923, Allen suggested the continuation of oogenesis in adult ovaries (Allen, 1923). A study by Vermande-Van Eck (1956) put forward an even stronger argument for the production of primordial follicles in non-human primate ovaries. In that study, based on the incidence of atresia and the time it would take for atretic follicles to be cleared from the ovaries, it was estimated that the 90% of ovarian reserve would be depleted within the first 2 years of life. Considering the fact that the monkey strain used in that study do not even experience puberty until the age of 4, and typically remain fertile for 20 years, continued oogenesis becomes a real possibility in post-natal ovaries.
Could there be an association between the transplantation of ovarian tissue and resumption of ovulation in a seemingly menopausal patient’s remaining ovary? Could recovery of ovarian function and fertility after receiving sterilizing chemotherapy and radiotherapy be due to resumption of de-novo production of primordial follicles? Is it possible that chemotherapy damages not only primordial follicles but the endocrine cells that produce the signals that recruit new germ cells from the bone marrow? Is it then possible that the transplanted ovary provides the missing signal for the remaining menopausal ovary to begin producing primordial follicles or for the bone marrow to provide new germ cells to that ovary via blood stream?
If bone marrow transplants can restore ovarian function in mice why is it that only 5% of patients receiving HSCT regain their fertility? The most obvious answer to this is that the putative germline stem cells do not exist in human bone marrow. However, there are multiple logical explanations for the low rate of return in fertility after HSCT even if germline stem cells indeed exist in the bone marrow. Initially, bone marrow transplants were performed by crude bone marrow infusions from HLA-matched but unrelated donors, commonly causing an immunological rejection reaction that could theoretically harm donor germline stem cells. In many instances, transplant donors were male. Recently, autologous hematopoietic stem cells derived from the bone marrow or, more commonly, from peripheral blood have been used for bone marrow transplantation (HSCT). This approach is more likely to leave out potential germline stem cells, as it was shown by Johnson et al. that putative bone marrow germline stem cells do not exist in the fraction of cells that contain markers for hematopoietic stem cells (Johnson et al., 2005a). Moreover, many preconditioning treatments include total-body irradiation with resultant damage to ovarian stroma and uterus, which may further reduce the likelihood of spontaneous pregnancies. Moreover, if germline stem cells indeed migrate from the bone marrow, and if their production and migration is governed by the signals emanating from yet-unknown ovarian cells, then an ovary damaged by chemotherapy or radiation may not be able to recruit or harbour these germ cells.
Although alternative interpretations to Johnson et al.’s findings (Telfer et al., 2005) have been suggested, a rebuttal to those alternative explanations has also been put forward (Johnson et al., 2005b). Regardless of the controversy, the mechanism behind spontaneous recovery of ovarian function after highly gonadotoxic chemotherapy and a possible role of ovarian transplantation in facilitating this recovery deserve further exploration.
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Acknowledgements |
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TopAbstractIntroductionLive birth after heterotopic...A cautionary noteAn alternative hypothesisAcknowledgementsReferences |
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I thank Dr. Ozgur Oktem as well as Lucinda Veeck, Richard Bodine and Nikica Zaninovic for their contributions in cryopreservation and thawing of the ovarian tissue.
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References |
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Submitted on November 22, 2005; resubmitted on December 14, 2005; accepted on January 4, 2006.
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