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Karolinska Institutet, Departments of Pediatrics (B.B.) and Clinical Science, Intervention, and Technology (R.C., S.M., F.G., K.V., F.M., H.O.), Karolinska University Hospital Huddinge, SE 141 86 Stockholm, Sweden; and Department of Women’s and Children’s Health (H.J.), Uppsala University Hospital, SE 751 85 Uppsala, Sweden
Address all correspondence and requests for reprints to: Birgit Borgström, Department of Pediatrics, Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden. E-mail: Birgit.borgstrom{at}ki.se.
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Abstract |
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Objective: Our objective was to study which girls might benefit from ovarian tissue freezing for fertility preservation.
Design: Clinical and laboratory parameters and ovarian follicle counts were analyzed among girls referred by 25 pediatric endocrinologists.
Subjects and Setting: Fifty-seven girls with Turner syndrome, aged 8–19.8 yr, were studied at a university hospital.
Interventions: Ovarian tissue was biopsied laparoscopically, studied for the presence of follicles, and cryopreserved. Blood samples were drawn for hormone measurements.
Main Outcome Measures: Presence of follicles in the biopsied tissue related to age, signs of spontaneous puberty, karyotype, and serum concentrations of gonadotropins and anti-Müllerian hormone were assessed.
Results: Ovarian biopsy was feasible in 47 of the 57 girls. In 15 of the 57 girls (26%), there were follicles in the tissue piece analyzed histologically. Six of seven girls (86%) with mosaicism, six of 22 (27%) with structural chromosomal abnormalities, and three of 28 with karyotype 45X (10.7%) had follicles. Eight of the 13 girls (62%) with spontaneous menarche had follicles, and 11 of the 19 girls (58%) who had signs of spontaneous puberty had follicles. The age group 12–16 yr had the highest proportion of girls with follicles. Normal FSH and anti-Müllerian hormone concentrations for age and pubertal stage were more frequent in girls with follicles.
Conclusions: Signs of spontaneous puberty, mosaicism, and normal hormone concentrations were positive and statistically significant but not exclusive prognostic factors as regards finding follicles.
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Introduction |
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Ovarian failure is one of the characteristic symptoms of this disorder. The new options in infertility treatment, such as oocyte donation (6, 7, 8) and storing remaining oocytes, have become of great interest in this group of subjects.
Cryopreservation of ovarian cortical tissue among young women undergoing cancer treatment is now considered an option for preserving primordial follicles for infertility treatment in the future (9, 10, 11, 12, 13, 14). Healthy children have been born after retransplantation of frozen-thawed tissue (15, 16).
A spontaneous start of puberty occurs in 15–30% of girls with TS, but only 2–5% reach menarche with the possibility of achieving pregnancy (17, 18, 19). This indicates that young teenage girls with TS have ovarian follicles that secrete estrogen but that they disappear prematurely (20, 21, 22). The dynamics of the disappearance have not been completely elucidated. In an earlier study, where we took biopsy specimens from ovarian cortical tissue from girls with TS for cryopreservation and analysis of ovarian follicles, we found that eight of 10 adolescent girls had follicles remaining (23).
The aim of the present study was to seek parameters that could indicate which girls with TS might benefit from ovarian tissue preservation by relating the presence of ovarian follicles to clinical and hormonal data including age, karyotype, spontaneous menarche and onset of puberty, and circulating concentrations of FSH, LH, and the granulosa cell-derived glycoprotein anti-Müllerian hormone (AMH) (24, 25, 26).
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Subjects and Methods |
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The girls were clinically taken care of in their hometowns by pediatric endocrinologists and treated with human GH, T4, and estrogen hormone replacement therapy (HRT) according to Swedish recommendations. Most families accepted the offer to participate in the study, and most of them expressed the opinion that they were pleased to have this opportunity. In the center referring the highest number of girls, only one of 15 refused to participate. Chromosome analysis in lymphocytes, giving the TS diagnosis, showed 45X in 28 girls, mosaicism (M) with complete chromosomes (45X/46XX/47XXX) in seven girls, and structural anomalies (SAs) in one of the X chromosomes (deletions in the p or q arm, isochromosomes, ring chromosome, or y-fragment) in the remaining 22 girls. The chromosome analysis was performed in the laboratory used by the referring clinic at the time of diagnosis. The number of cells analyzed varies between 15 and 105 depending on where and when the test was done (Table 1
). The amount of detail in the results from the genetic laboratories also shows some variation. In a few cases, information was not available regarding which arm of the X chromosome that was deleted or doubled. All presented results are from cultures of lymphocytes even if a fluorescence in situ hybridization analysis (FISH) from buccal cells or some other tissue was also performed.
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Methods
Laparoscopy for obtaining ovarian cortical tissue by biopsy for cryopreservation was carried out under general anesthesia. In 10 girls, the ovaries were streaks and too small for biopsy or even to identify. In 47 girls, 25–50% of the ovarian cortical tissue from one of the ovaries was biopsied. A small piece of this tissue was fixed for histological analysis, and the major part was cryopreserved for possible infertility treatment in the future.
Cryopreservation
The biopsied tissue was frozen using slow programmed freezing, with propanediol-sucrose as the cryoprotectant, as described earlier (13, 14, 23). The ovarian tissue was collected into preequilibrated HEPES-buffered in vitro fertilization culture medium containing human serum albumin and immediately transported to the laboratory. After removing a 0.5- to 3-mm3 piece of the ovarian cortex for histological analysis, the remaining cortical tissue was cut into small strips, 0.5 x 3–5 mm, equilibrated with cryoprotectants containing propanediol and sucrose, and cryopreserved in a programmable freezer (Cryologic PL, Mulgrave, Australia).
Histology and follicle counting
In each case, one piece of fresh ovarian cortex was fixed in Bouin’s solution, embedded in paraffin, cut into 4-µm sections, and stained with hematoxylin and eosin. The total number of follicles and follicular density in the ovarian biopsy sample were evaluated using a digital image analysis system (Easy Image Mätning, Tekno Optik, Stockholm, Sweden) connected to an inverted microscope (Nikon, Tekno Optik), as described by Hreinsson et al. (23). Follicular density was expressed as the number of follicles per cubic millimeter of the analyzed sample. Only normal follicles were counted and included. Healthy teenage girls usually have more than 500 follicles/mm3.
Hormone assays
Blood samples for assay of LH, FSH, and AMH were drawn the day before laparoscopy in 28 girls or were sent to our unit within 1 yr after biopsy in 19 cases and were not available in the remaining 10 girls.
Serum concentrations of FSH and LH were analyzed in 47 girls. In 43 cases, the analyses were performed at the Research Laboratory for Women’s Health, Karolinska Institute, by solid-phase, two-site chemiluminescent immunometric assays (Immulite) according to the instructions of the manufacturer (Diagnostic Products Corp., Los Angeles, CA). For FSH, the analytical sensitivity was 0.1 mIU/ml, and specificity was very high, with no detectable cross-reactivity with human chorionic gonadotropin, TSH, LH, prolactin, human GH, or human placental lactogen.
For LH, the analytical sensitivity was 0.1 mIU/ml, and specificity was very high, with no detectable cross-reactivity with human chorionic gonadotropin, FSH, or TSH.
In the remaining four girls, serum concentrations of FSH were assayed at the Central Laboratory for Clinical Chemistry, Karolinska University Hospital. The assay is standardized against both the World Health Organization (WHO) Second International Reference Preparation (IRP) of FSH for bioassay, number 78/549, and the WHO Second IRP of human menopausal gonadotropin. The sensitivity of the FSH assay was 0.7 mIU/ml. The cutoff level of the assay is widely used in Europe and corresponds to a concentration of 17 IU/liter, if the assay is calibrated against the Second IRP standard 78/549, which is widely used in the United States.
Serum concentrations of AMH were analyzed in serum samples from 43 girls at the Research Laboratory for Women’s Health, Karolinska Institute, by means of a two-step AMH enzyme immunoassay kit according to the instructions of the manufacturer (Immunotech/Beckman-Coulter, Marseille, France). The protocol for an ultrasensitive procedure was used, with a dilution series, as suggested by the manufacturer. The sensitivity, defined as the lowest AMH concentration significantly different from the zero standard with a probability of 95%, was 0.7 pmol/liter. The assay has a very high specificity for AMH, with no cross-reaction with TGF-β.
Statistics
Diagnostic tests for sensitivity and specificity were used to calculate positive and negative predictive values. The positive predictive value is the proportion of patients with positive test results who are correctly diagnosed, referred to as the sensitivity of the test. The negative predictive value is the proportion of patients with negative test results who are correctly diagnosed and gives the specificity of the test. The results are graphically presented in ROC (receiver operating characteristic) curves. Statistical significance was tested with X2 tests. A p-value of <0.05 was considered statistically significant.
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Results |
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All 57 laparoscopies were performed as day surgery, meaning that the girl was admitted in the morning and could leave the hospital in the afternoon or early evening. None of the girls had any complications with anesthesia or surgery.
LH, FSH, and AMH
There were 36 girls without HRT at the time of biopsy. The 21 girls on HRT were excluded regarding presentation of gonadotropin measurements. In 30 cases, measurements of LH and FSH were performed. In 17 girls, serum LH and/or FSH concentrations were higher than normal for age; in 13 of them, no follicles were found in the ovarian biopsy sample, and the remaining four had them. In 13 girls, LH and FSH levels were within normal limits for age and stage of puberty; nine of them showed follicles in the biopsy sample, and four did not.
Concentrations of AMH were analyzed in 43 girls. Healthy teenagers are expected to have AMH higher than 2.0 pmol/liter in all phases of the menstrual cycle. Levels above 2.0 pmol/liter were seen in 11 girls, of whom seven had follicles in the biopsy sample and four did not. Levels of AMH were less than 2.0 pmol/liter in 32 girls. In 28 cases, no follicles were found in the biopsy sample, but in four cases, there were follicles present. These four girls were exactly the same individuals who had high serum concentrations of LH and FSH despite the presence of follicles: girls 16, 37, 59, and 65 in Table 1
. In Fig. 1, serum concentrations of AMH vs. FSH are plotted in the girls not on HRT, and in Table 1, all individual data and results are presented.
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Of the 57 girls in the study (Table 1), an ovarian biopsy sample could be taken in 47, and follicles were identified in 15 cases (26% of 57). The number of follicles varied from 0.7–1200/mm3. In six of seven (86%) girls with mosaicism, follicles were found. The 22 girls with SAs had follicles in six cases (27%). The 28 girls with 45X had follicles in three cases (10.7%). Spontaneous onset of puberty had occurred in 19 girls, and 11 (58%) of them had follicles in the tissue. In 13 girls, menarche had been reached spontaneously, and eight (62%) had follicles. In 30 girls above the age of 12 yr, no signs of spontaneous puberty were seen, but three (10%) had follicles. One 11-yr-old girl did not yet have visible signs of puberty, but follicles were found.
When investigating which factors had the highest positive predictive value (sensitivity) regarding finding follicles, the top five were karyotype M (0.86), normal level of serum FSH, less than 11 mIU/ml (0.69), AMH higher than 2 pmol/liter (0.64), spontaneous menarche (0.62), and spontaneous onset of puberty (0.58). The highest negative predictive value (specificity) regarding finding the girls without follicles were karyotype 45X (0.89), serum AMH less than 2 pmol/liter (0.88), no signs of spontaneous puberty (0.87), age less than 12 yr (0.82), no spontaneous menarche (0.81), high level of serum FSH above 15 mIU/ml (0.77), age over 16 yr (0.76), and karyotype SA (0.73). Five of six factors showed statistical significance in discriminating the girls with follicles. The age factor was not significant despite a high negative predictive value regarding the youngest age group. The results are presented in Table 2 and graphically in Fig. 2.
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The results from this study could discriminate five factors as important for finding remaining follicles in the ovaries of girls with TS: karyotype, low FSH, high AMH, spontaneous menarche, and spontaneous onset of puberty. In summary, the following categories of girls would have the highest chances of having follicles and could be considered for laparoscopy with biopsy for cryopreservation at the age of 13–17 yr: 1) girls with mosaicism (45X/46XX/47XXX); 2) girls with spontaneous onset of puberty and 45X or 45X/46X+SAs; and 3) girls with normal serum FSH and/or normal AMH levels with or without spontaneous onset of puberty.
If these three criteria had been set up for our study girls, 19 laparoscopies would have been performed at the time. Of these 19 girls, follicles would have been found in 11, and not in the remaining eight, resulting in a sensitivity of 0.58 (11 of 19). In the 15 youngest girls, the decision about laparoscopy would have been postponed. It is likely that the two girls in this group who had follicles would have been found later, because both had signs of puberty. In 23 girls, no laparoscopy would have been carried out. Two girls with follicles would have been missed, girls 59 and 16 in Table 1. Two girls would have been included only in connection with the third group of criteria. The specificity regarding excluding the girls without follicles would have been 0.91 (21 of 23).
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Discussion |
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In this study, we did not require ultrasonography or pelvic MRI to visualize the existence of ovaries before laparoscopy. A few girls had undergone an abdominal ultrasonographic scan in their home hospital, but there was low concordance with the result seen in laparoscopy. There are studies (28, 29) in which abdominal ultrasonography results seem to be reliable; investigators with special interest and training and the best possible equipment are likely to be the keys to success. For clinical routine purposes, the use of MRI could be a better method to visualize the existence of ovarian tissue. If a girl does not qualify for laparoscopy according to the criteria described above, but has a very strong desire to have a biopsy performed, MRI may be useful. The two girls in the present study, who had follicles that would have been missed using the criteria, might possibly have been identified by means of MRI.
It is an advantage to discuss biopsy when a girl has reached the age of 13 or 14, even though there was no statistical significance regarding age and the finding of follicles. At this age, most girls are able to understand the possibilities and limitations connected with preserving ovarian tissue for the future. Because experience in using preserved cells in fertility treatment is still limited, it is important to inform the girls that there is no guarantee for biological children in the future (14, 15, 16). The fact that a pregnancy in a woman with TS needs to be very carefully surveyed by a specialist obstetrician, as a result of the increased risks (30), should also be discussed with the girl and her parents. It is possible that the risk of fatal aortic dissection is increased predominantly in women with anomalies in the heart (bicuspid aortic valve and coarctation of the aorta) or hypertension, but until convincing data are available, repeated investigations are recommended (1, 8, 31, 32, 33).
The question of whether or not there is a high risk of chromosomal abnormalities in biological children of TS women cannot be neglected. Published cases are few, and calculation of the risk is unreliable (19, 34). Possibly all women with TS should be offered preimplantation diagnosis, chorion villous sampling, or amniocentesis if fertilization with their own oocytes is successful.
Despite careful information, both oral and in writing, about the limited chances of biological children and the known and suspected risks connected to a future pregnancy, recruitment of the girls for this study was easy. There seems to be a strong desire to do all that is technically possible to have the opportunity to undergo fertility treatment in the future. Only a few families who had the chance to participate in the study refused. From the age of 13–14 yr, the girls themselves took an active part in deciding to participate. The youngest girls were informed and gave their consent, but the parents made the decision. The experience from this study made us convinced that the possibility to perform ovarian biopsy in young girls with TS is an important step in possibly overcoming infertility in this group of girls.
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Footnotes |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online October 28, 2008
1 F.M. and H.O. contributed equally. 
Abbreviations: AMH, Anti-Müllerian hormone; HRT, hormone replacement therapy; IRP, International Reference Preparation; M, mosaicism; MRI, magnetic resonance imaging; SA, structural anomaly; TS, Turner syndrome.
Received March 31, 2008.
Accepted October 21, 2008.
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