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Original Article |
Departments of Obstetrics and Gynecology (J.G.H., M.F., C.R., M.M., O.H.) and Pediatrics (B.B.), Karolinska Institutet, Huddinge University Hospital, S-141 86 Stockholm, Sweden; Department of Obstetrics and Gynecology (M.L.L., N.S.), University Hospital of Uppsala, S-751 85, Uppsala, Sweden; Program for Developmental and Reproductive Biology (M.O., L.D.), Biomedicum Helsinki, and Hospital for Children and Adolescents, University of Helsinki, FIN-00290 Helsinki, Finland; and The Family Federation of Finland (T.T.), Kalevagatan 16, FIN-00100 Helsinki, Finland
Address all correspondence and requests for reprints to: Julius G. Hreinsson, Fertility Unit K-59, Huddinge University Hospital, S-141 86 Stockholm, Sweden. E-mail: . Julius.Hreinsson{at}obgyn.hs.sll.se
Abstract
Infertility caused by ovarian failure is a characteristic feature in Turner’s syndrome. Spontaneous pregnancies are seen in 2–5% of these women, and up to 30% have at least some pubertal development, indicating the presence of follicles in their ovaries in adolescence. It has not been clear at which age the follicles disappear.
We analyzed the numbers and densities of follicles in ovarian cortical tissue from nine adolescent girls with Turner’s syndrome who came to our clinics after having been informed about the study, with an aim to preserve ovarian tissue for possible infertility treatment later in life. A quarter to one whole ovary was laparoscopically removed for the procedure.
Follicles were seen in the biopsy tissue in eight of nine subjects from whom ovarian tissue was laparoscopically obtained, the highest numbers being seen in the youngest girls and in those with mosaicism. In one 17-yr-old girl, no ovarian tissue was found. Follicle density was correlated with serum levels of FSH; individuals with the lowest FSH levels had the highest follicle density. One to 190 follicles were counted in the approximately 0.1–2.0 mm3 of tissue analyzed, giving a density of 1.5–499 follicles/mm3 of ovarian cortical tissue. Girls up to the age of 17 had primordial follicles in their ovaries. Three girls, two aged 15 yr and one aged 19, had only secondary follicles, with many being atretic.
Our finding that adolescent girls with Turner’s syndrome still have follicles in their ovarian cortical tissue raises the possibility of future fertility through cryopreservation of ovarian tissue. However, before such procedures can be recommended for clinical management, it is essential that future studies be performed to determine whether the oocytes retrieved from girls with Turner’s syndrome have a normal chromosomal complement.
INFERTILITY CAUSED BY ovarian failure is one of the features of Turner’s syndrome. Spontaneous pregnancies occur in 2–5% of these women, and up to 30% achieve at least some pubertal development (1). Pregnancies are more common in subjects with chromosomal mosaicism, but they also occur in nonmosaic Turner’s syndrome (2, 3). The number of primordial follicles in the ovaries has been estimated to be normal up to the 18th week of fetal life (4, 5). It has not been clear at which age the follicles begin to disappear.
Because cryopreservation of ovarian tissue is now feasible (6, 7), it might be possible to preserve the fertility of girls with Turner’s syndrome by cryopreservation of primordial follicles within the ovarian cortical tissue, before their disappearance. Such an approach has been previously used for preservation of fertility in patients at risk of premature menopause because of chemo- or radiotherapy in cancer treatment. Replantation of cryopreserved and thawed ovarian cortical tissue has resulted in restoration of ovarian function and follicular development (8, 9). The promising data obtained so far in these studies has resulted in the advocation of this method for selected groups of patients before cancer treatment (10).
Up to 30% of girls with Turner’s syndrome undergo some pubertal development (1), indicating the presence of follicles in the ovaries of some girls until adolescence. Because there is hardly any information regarding the numbers of follicles at this stage, we analyzed the numbers and densities of follicles in ovarian cortical tissue from 9 of 10 adolescent girls who came to our clinics with their parents after having expressed a desire to have ovarian tissue frozen for possible infertility treatment in later life.
Subjects and Methods
Subjects
Ovarian tissue from nine girls, one aged 12 yr, three aged 13 yr, three aged 15 yr, one 17 yr, and one 19 yr, was cryopreserved to store the possible ovarian follicles still left in their ovaries. One of the girls, aged 15 yr, had a marker Y-chromosome, and she underwent oophorectomy because of the risk of cancer. As part of our management program for young girls with Turner’s syndrome in counseling them and their families, the issue of infertility and hormone replacement therapy (HRT) is carefully explained. All of the participating girls and their parents expressed their desire to have ovarian tissue cryopreserved, and they gave informed consent for participation in the study, knowing the experimental nature of the procedure. A quarter to one whole ovary was laparoscopically removed for the procedure. In addition to these nine girls, one 17-yr-old girl underwent laparoscopy, but no ovarian tissue was found.
All of these girls had received GH therapy, starting at the age of 7.9 ± 2.9 yr (mean ± SD), with a range of 2.8–11.9 yr. Subjects 1–7 were receiving GH therapy at the time of biopsy. Estradiol substitution had been started when necessary, between the ages of 13 and 15, depending on individual subjects. Six of the girls had had spontaneous onset of puberty and development at least up to Tanner stage 4, and four of them were on HRT when the biopsy sample was taken. Spontaneous pubertal development before HRT, according to Tanner’s classification (11 ; mean of breast stage and pubic hair stage), is given in Table 1
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. Three of the girls, aged 13, 15, and 19 yr, already had high serum concentrations of FSH (67, 94, and 69 IU/liter, respectively); the others had slightly elevated or normal FSH levels (taken without HRT). The serum FSH levels were analyzed at the time of biopsy for subjects 1, 2, 3, 5, 8, and 9, and within 6 wk of the biopsy for subjects 6 and 7; for subject 10, the analysis was performed 5 months earlier, and for subject 4 it was performed 8 months before the biopsy was taken. Four of the girls had nonmosaic Turner’s syndrome, with all of the analyzed cells displaying karyotype 45X; the others had varying degrees of mosaicism. One 13-yr-old girl had karyotype 45X/46XXp+, in which p+ depicts uncharacterized chromosomal material, and another 13-yr-old had karyotype 45X/46XX/47XXX. This study was approved by the Ethics Committees of the Karolinska Institutet at Huddinge University Hospital and the Hospital for Children and Adolescents, University of Helsinki.
Cryopreservation
The ovarian tissue was collected into pre-equilibrated HEPES-buffered culture medium containing human serum albumin (Gamete, Vitrolife, Gothenburg, Sweden), or 0.9% NaCl, pH 4.7 (Orion Diagnostica, Espoo, Finland) and immediately transported to the laboratory. After removing a 0.5- to 3-mm3 piece of the ovarian cortex for histological analysis, we cut the cortical tissue into small pieces, equilibrated it with cryoprotectants, and cryopreserved it according to the method for cryopreservation of ovarian tissue described by Hovatta et al. (6).
Fixation and histological analysis
The samples taken for histology were fixed in Bouin’s solution for 24 h at 2–8 C, in Histochoice Tissue Fixative (Amresco, Solon, OH) or in formalin, and then dehydrated in 70% ethanol. After being embedded in paraffin (Paraplast, Sherwood Medical, St. Louis, MO), 4-µm sections were cut. Double counting of follicles was avoided by disregarding 10–15 slides between counts. Staining was performed with hematoxylin/eosin for histological examination.
To measure the areas of the analyzed tissue pieces, we used a digital image analysis system (Easy Image Mätning, Bergström Instrument, Stockholm, Sweden) connected to an inverted microscope (Nikon, Bergström Instrument). The volume was then calculated by multiplying the area of the tissue piece by the known thickness of 4 µm.
Follicles were judged to be in the (latent) primordial stage when all the granulosa cells were flat; primary follicles were defined as those containing one or more cuboidal follicular cells, and secondary follicles were defined as those in which at least part of the follicle had two or more layers of granulosa cells. Only follicles with viable cells were counted. Pyknotic granulosa cells, eosinophilia of the ooplasm, and contraction and clumping of chromatin were regarded as signs of atresia (12).
Hormone assays
The assays in Sweden were performed as follows. Unconjugated 17ß-estradiol in serum was measured by a sensitive solid-state RIA coated tube kit (ESTR-US-CT, Orion Diagnostica) for direct quantitative determination. The detection limit for the assay was 5 pmol/liter, with an intra-assay coefficient of variation (CV) of 13% at a mean value of 22.6 pmol/liter. The CV was less than 4% at higher estradiol concentrations. The interassay CV was 17.6% at a mean value of 12 pmol/liter, 5.8% at a mean value of 94 pmol/liter, and 8.1% at 260 pmol/liter. The standards used were 0, 10, 25, 100, 500, and 2000 pmol/liter in human serum.
FSH and LH in serum were measured by immunometric assays (Immulite, Diagnostic Products Corporation, Los Angeles, CA). The analytical sensitivity of the FSH assay was 0.1 IU/liter, with intra-assay CV of 5.4% at a mean of 7.8 IU/liter, 6.1% at 19.7 IU/liter, and 7.7% at 42.5 IU/liter. The interassay CV was 8.1% at 8.3 IU/liter, and it was somewhat lower at higher concentrations. For the LH assay, the analytical sensitivity was 0.1 IU/liter, with an intra-assay CV of 4.8–6.5%. The interassay CV was 26.0% at a mean concentration of 0.1 IU/liter and 12.6% at 0.27 IU/liter; it ranged from 7–11% at higher concentrations.
The assays in Finland were performed as follows. Serum 17ß-estradiol concentrations were determined by a modified RIA using coated tube technology (Spectria E2, Orion Diagnostica) after diethylether extraction. The detection limit of the assay was 6 pmol/liter.
Serum LH and FSH concentrations were measured in single samples by time-resolved immunofluorometric assays (Wallac, Inc., Turku, Finland). The sensitivity of the LH and FSH assays was 0.05 IU/liter, with interassay CV for LH of 3.2% at a concentration of 1.96 IU/liter, 4.6% at 17.51 IU/liter, and 3.7% at 53.95 IU/liter. For FSH, the interassay CV was 2.6% at 6.63 IU/liter, 2.7% at 13.17 IU/liter, and 3.3% at 35.14 IU/liter. The intra-assay CV for LH were 4.1% at 0.35 IU/liter, 2.0% at 2.98 IU/liter, and 1.6% at 8.71 IU/liter; and for FSH they were 4.4% at 0.25 IU/liter, 1.8% at 2.94 IU/liter, and 1.6% at 6.66 IU/liter (13).
Results
Follicles were seen in the biopsy specimens in eight of nine subjects from whom ovarian tissue was laparoscopically found, the highest numbers being seen in the youngest girls. One additional girl underwent laparoscopy, but no ovarian tissue was obtained. The results of follicle counting are given in Table 2. One to 190 follicles were counted in the approximately 0.1–2.0 mm3 of tissue analyzed, giving a density of 1.5–499 follicles/mm3 of ovarian cortical tissue.
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. We also observed the highest follicular counts in the individuals with the lowest degree of mosaicism (Fig. 1), but even girls with nonmosaic Turner’s syndrome still had follicles in their ovaries, although the counts were lower than in the nonmosaic subjects. Within this narrow age range, we were not able to see any clear correlation between follicle density and age, even when the presence of mosaicism was taken into account.
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). Three of the older girls had only follicles that had already begun growth; only follicles at the secondary stage of development were found in these cases. In one girl aged 19 yr, a single secondary follicle was found; the oocyte in this follicle did not appear normal (Fig. 3). Normal early antral follicles were seen in the tissue from subject 9 during histological analysis (Fig. 4, A and B).
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Discussion
We showed that 8 of 10 adolescent girls with Turner’s syndrome had follicles in their ovaries. This could be expected, because many of these girls undergo at least some degree of pubertal development, and 2–5% of Turner’s syndrome subjects are fertile (3).
Follicular density correlated relatively well with levels of FSH in serum, individuals with elevated FSH levels showing the lowest follicular density. Hence, high FSH concentrations predicted ovarian failure among these girls, although several months had passed between FSH measurement and the biopsy, in some cases. The calculated numbers of follicles in these girls match well with estimated follicular densities among premenopausal women, as calculated by Faddy and Gosden (17). The proportion of secondary follicles appeared to be high in the ovarian cortical tissue in these cases, when compared with that in non-Turner’s syndrome women (14). The initiation of growth of the follicles has been related to the numbers of follicles that are left in the ovaries (18). Our subjects had relatively low numbers of follicles left, which may explain the high proportion of follicles at more advanced stages of development.
The correlation between follicle density and karyotype is less clear; the highest numbers of follicles were indeed seen in the subjects with mosaic Turner’s syndrome and a low percentage of analyzed cells with the karyotype 45X. However, we also showed that follicles were present in the girls with nonmosaic Turner’s syndrome at a density of 50–128 follicles/mm3, at least in the younger subjects.
Four of the 10 girls had nonmosaic Turner’s syndrome. Spontaneous pregnancies have also been encountered among women with nonmosaic Turner’s syndrome (3); hence the follicular loss among these women appears to occur later than earlier suspected and is not as complete as has been thought.
The present findings are encouraging as regards the cryopreservation of ovarian tissue (6, 7) from 12- to 13-yr-old and younger girls with Turner’s syndrome. Transplantation of cryopreserved human ovarian tissue has already resulted in functioning ovarian tissue in women with other indications (8, 9). Retrieval of mature oocytes for in vitro fertilization from transplanted tissue after cryopreservation and thawing may be feasible for these girls.
The optimal time to obtain as many follicles as possible for cryopreservation is at a young age, and according to our findings at the latest when girls with Turner’s syndrome are 12–13 yr old, at least in cases of nonmosaic Turner’s syndrome. We performed this study with adolescents because they were already old enough to understand the purpose of the procedure and they could give their own informed consent. The girls and their families had expressed sadness over the prospect of future infertility and wished to participate in the study to retain some possibility of having children with the same genetic background, even after the experimental nature of the project was carefully explained. In addition, one girl had her ovaries removed because of cancer risk.
On the basis of our findings and the promising results of transplantation studies, it might be justified to start offering even younger girls the possibility of cryopreserving ovarian biopsy samples. The diagnosis of Turner’s syndrome is, for the time being, made relatively early, on the basis of growth failure (19, 20).
Laparoscopy bears some risks, and although they are not high, they have to be weighed in this context. The latest stage for biopsy is when the concentration of serum FSH starts to rise, but considering the optimal numbers of follicles, this is probably too late.
The amount of tissue to be taken is another open question. Unilateral oophorectomy is easy to carry out, and it bears a relatively low risk of bleeding. However, in the 2–5% of possibly fertile women with Turner’s syndrome, it might reduce the likelihood of pregnancy in later life. For some of our adolescent subjects, it appeared psychologically important not to remove a whole ovary.
A high frequency of miscarriage has been reported among the spontaneous pregnancies of women with Turner’s syndrome (1, 3). It has been suspected that this is due to abnormal oocytes in Turner’s syndrome. In a Swedish survey, 77 pregnancies occurred among 25 women with Turner’s syndrome, two of these women with karyotype 45X. The miscarriage rate was 29 of 66 (21). The possibility of meiotic nondisjunction in the oocytes of women with Turner’s syndrome cannot be ignored, because chromosomal aberrations have been shown to be more common in children born after spontaneous pregnancies in these women (22, 23). This must be taken into account when counseling such women. The oocytes that are left in the follicles may well be those with normal chromosomes, however it will be critical to study the chromosomal constitution of the oocytes in cryopreserved tissue. We plan to perform such an analysis using fluorescent in situ hybridization in the biopsy samples. Until the normality of the oocytes has been established, we cannot recommend this procedure for clinical use, and the technique must, for the time being, be regarded as experimental.
The miscarriage rate is also high in pregnancies achieved as a result of oocyte donation (24). Hence, there may be causes other than chromosomal abnormalities as regards the miscarriages. Low blood flow in a hypoplastic uterus might be such a cause. Appropriate estrogen replacement therapy before the planned pregnancy to increase the size and blood flow in the uterus may help to reduce miscarriages among these women (24).
Pregnancies among women with Turner’s syndrome are high risk pregnancies (3). These women have a high incidence of cardiac abnormalities, and two cases of fatal aortic dissection have been reported in pregnant women with Turner’s syndrome (25). Echocardiography and possibly magnetic resonance imaging to diagnose possible aortic root dilation has to be performed to overcome such risks. A careful health check-up is recommended before planned pregnancies among women with Turner’s syndrome (26).
The cause of premature atresia of the follicles among girls with Turner’s syndrome is not known. It is tempting to assume that a double dose of some gene located in the X chromosome is needed to prevent apoptosis and atresia of the follicles.
Our finding that adolescent girls with Turner’s syndrome still have follicles in their ovarian cortical tissue gives hope for future infertility treatment of these girls. It also offers a model for understanding the physiological processes involved in follicular atresia.
Acknowledgments
We thank Dr. Nicholas Bolton for language revision of this manuscript.
Footnotes
Abbreviations: CV, Coefficient(s) of variation; HRT, hormone replacement therapy.
Received February 13, 2002.
Accepted April 23, 2002.
References
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