The female-to-male transsexual patient: a source of human ovarian cortical tissue for experimental use

Rudy Van den Broecke1,4, Josiane Van der Elst2, Jun Liu2, Outi Hovatta3 and Marc Dhont2

1 Department of Gynaecologic Oncology, 2 Infertility Centre, Ghent University Hospital, Ghent, Belgium and 3 Department of Obstetrics and Gynaecology, Karolinska Institutet, Huddinge University Hospital, Sweden


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We demonstrated that ovaries removed from female-to-male transsexuals can be used as research material for ovarian cryopreservation, grafting and culture studies. A 21 year old female-to-male transsexual individual, who had been treated with androgens for 12 months, donated her ovaries removed in the sex reversal operation. She had normal numbers of, and proportions of, primordial (98.6%) and primary (1.4%) follicles in her ovarian cortex. After freezing and thawing the ovarian tissue was grafted to immuno-incompetent mice, which were stimulated by human recombinant FSH for 14 days, 10 weeks after transfer. Initiation of growth had occurred in the grafts in which only 79.4% of follicles were primordial, 17.1% primary, and in which there were also secondary and pre-antral follicles. Because long-term androgen treatment does not appear to cause depletion of the primordial follicle pool or affect the developmental capacity of the follicles, ovaries from individuals who undergo sex-reversal operations are an excellent source of tissue for research and maybe even for oocyte donation in the future.

Key words: androgens/grafts/ovarian tissue source/ovary/transsexuality


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The use of cytotoxic drugs as well as the application of new strategies such as high dose chemotherapy in medical oncology will undoubtedly lead to a higher survival rate of young female cancer patients. Many of these women will therefore reach reproductive age but will not be able to conceive due to the damage to their ovarian follicles caused by the chemotherapeutic drugs.

Cryopreservation of ovarian cortical tissue has been introduced into reproductive medicine as a novel technique for the preservation of primordial follicles with the aim to restore fertility in young female cancer patients after systemic chemotherapy (Newton, 1998Go). Although tremendous progress has been achieved in the knowledge of ovarian physiology and reproductive biology the cryopreservation and in-vitro maturation of immature human oocytes is still in its infancy (Newton, 1998Go). Grafting of cryopreserved ovarian cortical tissue has therefore attracted much interest as a means for the restoration of fertility. Ovarian auto- or allografting has been shown to be an efficient method of restoring fertility in mice (Parrott, 1960), hamsters (Parrott, 1959) and sheep (Gosden et al., 1994Go). Normal follicular development to the antral stage was observed after xenografting cryopreserved ovarian cortical tissue of primates under the kidney capsule of nude mice (Candy et al., 1995Go). The viability of frozen human ovarian cortical tissue under the kidney capsule of severe combined immunodeficient (SCID) mice was described (Newton et al., 1996Go). Development of human primordial follicles to antral stages after grafting of ovarian tissue to FSH-stimulated SCID mice has been reported (Oktay et al., 1998Go). These successful experiments led to the first laparoscopic grafting of frozen–thawed human cortical ovarian tissue to the ovarian fossa resulting in follicular growth under gonadotrophic stimulation (Oktay et al., 1999Go). Quite recently, mature oocytes have been rescued from antral follicles of cryopreserved mouse ovarian tissue (Sztein et al., 2000Go). Also, restoration of a normal reproductive lifespan after orthotopic grafting of frozen mouse ovaries has unequivocally been demonstrated (Candy et al., 2000Go). These results will not only stimulate further research in this field but will undoubtedly also urge many centres to start banking ovarian cortical tissue of young women afflicted with malignant disease. However, many questions about the best method of cryopreservation, the ideal site or the optimal way of stimulation of grafts remain unanswered. More experimental work is needed before putting the concept of grafting of banked ovarian tissue into practice.

The scarce availability of donated cortical ovarian tissue is a limiting factor in research on follicular growth after grafting. Since ovarian tissue is often obtained as biopsy specimens from women undergoing gynaecological surgery, the age of the donor and the associated decline of follicular density by age is another limitation. Taking into account the heterogeneous distribution of the follicles around the cortex and the loss of follicles by cryopreservation and grafting, it is clear that experimental data leading to clinically relevant conclusions will require more study. In this paper we present therefore the young female-to-male transsexual patient as a potential candidate for voluntary donation of ovarian cortical tissue.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Source of ovarian tissue and cryopreservation
Informed consent was obtained from a 21 year old female patient undergoing sexual reassignment surgery. She had been treated with testosterone undecanoate (Undestor®; Organon, Oss, The Netherlands) in a daily dose of 240 mg for a period of 1 year. Immediately after removal, the ovaries were placed in Leibovitz L-15 medium and transported to the laboratory. The ovaries were bisected and the medulla was removed. The cortex was scraped until all medullary fragments were removed and only a 1 mm thick cortical slice remained. A fragment of the ovarian cortex was fixed in phosphate-buffered formaldehyde and sent to the department of pathology for histological processing. The remaining tissue was cut into blocks of approximately 2 mm by 1 mm under sterile conditions. The blocks were prepared for cryopreservation with 1.5 mol/l dimethylsulphoxide as described by Newton et al. (1996). Three tissue blocks were frozen per vial.

Grafting
Non-obese-diabetic (NOD)–SCID mice were bred in specific pathogen-free breeding facilities and were treated according to the guidelines of the Laboratory Animal Ethical Commission of the Ghent University Hospital.

Seventeen female animals were anaesthetized with an intra-peritoneal injection of 100 µl of sodium pentobarbital (Nembutal®; Sanofi, Brussels, Belgium) diluted 1:4 in phosphate-buffered saline (PBS; Sigma, Bornem, Belgium). A skin incision was made in the dorsal region with scissors and the skin was dissected free from the underlying muscles. Three frozen–thawed ovarian cortical grafts were inserted deep in the subcutaneous space using fine watchmakers forceps. Haemostasis was carefully controlled. The dorsal wound was closed with resorbable sutures (Dexon 4/0 sertix; Davis and Geck, Wayne, NY, USA). The contents of one vial (three pieces of frozen–thawed tissue) was placed in phosphate-buffered formaldehyde for histological processing.

Gonadotrophic stimulation
Ten weeks after grafting, stimulation by daily intra-peritoneal injection of 5 IU recombinant FSH (Puregon®; Organon) per mouse was started. After a stimulation period of 14 days, the animals were killed by neck dislocation.

Graft recovery
The dorsal skin wound was re-opened and the dorsolateral side of the animals closely inspected. Grafts were easily relocated at the former insertion site and were dissected from the surrounding tissues and fixed immediately in phosphate-buffered formaldehyde.

Microscopy
Histological examination was performed on paraffin-embedded tissue cut into 5 µm thick sections and stained with haematoxylin–eosin. Follicles were classified as primordial, primary, secondary, pre-antral or antral following Gougeon (1986). In order to avoid double counting of oocytes, the counting was done only when the nucleus was clearly visible.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Forty-four of 51 grafts were recovered at the 17 sites (86% graft recovery rate). All 44 grafts showed a reduction in size of ~50% in comparison with the original dimensions. All grafts appeared as well vascularized pieces of tissue either on the surface of the abdominal muscles or on the inner side of the skin.

Histological examination of the freshly collected ovarian biopsy revealed a majority of primordial follicles (n = 127; 98.6%) and only two primary stage follicles (n = 2; 1.4%). Only one vial containing three tissue blocks was thawed for histological processing before grafting. This tissue contained only primordial follicles (n = 9). In the frozen–thawed grafts transferred to the recipient animals and stimulated with recombinant FSH, a shift from primordial follicles (n = 110; 79.4%) to further developed stages (n = 29; 20.6%) was seen. Among these, three secondary (2.1%) and two pre-antral follicles (1.4%) were seen in the transplants. No antral follicles were observed in the recovered grafts.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Follicular growth can be obtained in ovarian cortical grafts originating from young female-to-male transsexual donors who have been on androgen therapy for prolonged periods of time. In the present study proof is only given that primordial follicles can resume early growth after a period of androgen treatment. Definitive proof of full follicle development can only be obtained by fertilization and embryo formation.

Our hospital serves as a referral centre for patients with gender identity disorders. At the moment of entering the programme, the patient is thoroughly investigated by an experienced endocrinologist in order to exclude any form of intersexuality and to make sure the patient has no medical disorders contra-indicating hormonal therapy. During a full year, the patient is studied by a psychiatrist and a psychologist in order to exclude any other diagnosis such as transvestic fetishism, effeminate homosexuality or borderline personality disorders with gender identity problems. In the second stage, hormonal down-regulation is started. From that stage onwards, real life tests must be carried out. This is followed by a `reality-diagnosis'. In the third year of the procedure, cross-sex hormones are administered. At the end of the process, the patient is referred for sex reassignment surgery. During this procedure, the female patient undergoes radical hysterectomy with bilateral adnexectomy. Analysing the data from 1985 to 1993 from the Ghent University Hospital Gender Identity Clinic, it was found that the female applicants and the female-to-male transsexuals are significantly younger than the male counterparts (De Cuypere, 1995Go), the mean age being <30 years. The use of ovarian cortical tissue of young women certainly has advantages over that of older patients as the former contains higher numbers of primordial follicles (Faddy et al., 1992Go).

Transfer to SCID mice, whether under the kidney capsule or subcutaneous, can only be achieved by the use of small grafts. Immediately after transfer a period of ischaemia, before vascularization of the grafts, may be responsible for the marked reduction in size of the grafts. Furthermore, the cryopreservation procedure itself may result in the loss of some follicles. Therefore the higher the number of follicles in the original tissue the higher the chance of finding surviving follicles in the transplanted tissue. From the literature it is clear that the best results are achieved by using tissue from young women. In their study on grafting of cryopreserved human ovarian tissue to SCID mice, Newton et al. (1996) used tissue of patients aged 17–31 years, whereas Oktay et al. (1998) obtained consent from a 17 year old patient to use strips of healthy ovarian cortical tissue for experimental use. Our own experience with the use of ovarian cortical tissue obtained from patients >30 years of age has been rather disappointing (Van den Broecke et al., 1999Go). In the future the use of healthy ovarian tissue of female transsexual patients could lead to a major source of donor oocytes.

A major concern in the proposed model was however that the female-to-male transsexual applicants had been treated with high dose testosterone before undergoing sex reassignment surgery. In monkey ovaries, androgens promote the initiation of follicle development (Vendola et al., 1999Go). In the ovaries of the individual we studied, who had received androgens for 1 year, we could not see any increased activation or depletion of the primordial follicles. Her ovaries contained similar amounts and proportions of primordial and primary follicles as women who had not received androgens (Lass et al., 1997Go). Our results show evidence that the administration of androgens probably does not affect the capability of ovarian follicles to resume growth. There was a good number of follicles in the cortical tissue, and after 2 weeks of FSH stimulation of the mice, secondary and pre-antral follicles could be recovered. The follicles had hence maintained their normal developmental capacity. We therefore present the use of ovarian tissue of young female-to-male transsexuals as a source of follicles either for experimental use or, maybe in the future, as donor oocytes.


    Acknowledgments
 
The authors want to express their gratitude to Prof. Plum, head of the Laboratory of Microbiology and Virology of the Ghent University Hospital, for allowing us the use of sterile mouse housing and operating facilities. Mrs M.Desmet is most sincerely thanked for breeding the NOD–SCID mice. We also thank Prof. Dr S.Monstrey, head of the Department of Plastic Surgery, for his kind cooperation. Our special thanks furthermore go to Prof. Dr C.De Potter of the Goormaghtigh Institute of Pathology for help with the histological processing of the ovarian tissue.


    Notes
 
4 To whom correspondence should be addressed at: Department of Gynaecologic Oncology, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium. E-mail: rudy.vdb{at}pronet.be Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Candy, C.J., Wood, M.J. and Whittingham, D.G. (1995) Follicular development in cryopreserved marmoset ovarian tissue after transplantation. Hum. Reprod., 10, 2334–2338.[Abstract]

Candy, C.J., Wood, M.J. and Whittingham, D.G. (2000) Restoration of a normal reproductive lifespan after grafting of cryopreserved mouse ovaries. Hum. Reprod., 15, 1300–1304.[Abstract/Free Full Text]

De Cuypere, F. (1995) Transseksualiteit. Psychiatrische Aspecten in Het Kader van de Geslachtsaanpassende Behandeling. Proefschrift tot het verkrijgen van de graad van Doctor in de Biomedische Wetenschappen. Rijksuniversiteit, Ghent.

Faddy, M.J., Gosden, R.G., Gougeon, A. et al. (1992) Accelerated disappearance of ovarian follicles in mid-life: implications for forecasting menopause. Hum. Reprod., 7, 1342–1346.[Abstract]

Gosden, R.G., Baird, D.T., Wade, J.C. and Webb, R. (1994) Restoration of fertility to oophorectomized sheep by ovarian autografts stored at –196°C. Hum. Reprod., 9, 597–603.[Abstract]

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Oktay, K., Karlikaya, G. and Aydin, B.A. (1999) Ovarian Transplantation Now a Reality? Abstract Book of the International Symposium on Storing Reproduction, October 7–8, 1999, Bologna, Italy, p. 23.

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Submitted on June 12, 2000; accepted on September 29, 2000.





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