Infertility and short stature are two major problems in patients with Turner’s syndrome (TS). In the last 20 years, recombinant growth hormone (Groeth Hormorn, GH) has been used to treat failure to thrive, allowing most TS patients to reach adult height at or slightly below the lower limit of the normal population. Since premature ovarian failure is a classic symptom of the disease, receiving egg donation and preserving the remaining eggs has become one of the effective treatments for infertility.
15-30% of TS patients have spontaneous puberty, but only 2-5% of those with menarche may become pregnant, suggesting that follicles in TS patients are estrogenic but disappear prematurely and that the dynamic process of disappearance has not been fully elucidated. In an earlier study, ovarian cortical tissue biopsy, freezing and analysis of follicles in girls with TS revealed the presence of follicles in 8 out of 10 adolescent girls. Currently, ovarian cortical tissue cryopreservation prior to anticancer treatment in young female oncology patients with fertility requirements has been used as an option to preserve the primordial follicles for future treatment of infertility, and healthy offspring have been obtained by transplantation of frozen-thawed ovarian tissue.
The aim of this study was to predict fertility by freezing ovarian tissue from patients with TS and to predict fertility by a comprehensive assessment of clinical and hormonal aspects including age, karyotype, spontaneous menstruation, pubertal follicle stimulating hormone (FSH), luteinizing hormone (LH), and granulosa cell-derived anti-millerian hormone (AMH) levels.
Clinical data
This study included 57 TS patients, aged 8-19.8 years, referred by 25 pediatric endocrinologists in Sweden (mainly Stockholm and Skåne in the south), and the patients and their parents were informed of the study details and signed an informed consent form. Patients and families were informed in detail that it was not possible to predict the availability of eggs in the obtained ovarian tissue for future treatment of infertility, and that other options for starting a family such as egg donation and adoption were available, and most families accepted the proposal and entered the study.
Only 15 people at the medical center with the highest number of patients refused to enroll. We believe that patients with signs of premature ovarian failure are also likely to find follicles, so no patient was denied participation. The study was approved by the Ethics Committee of the Karolinska Institute and was conducted in strict accordance with the Declaration of Helsinki and the Swedish law on medical research ethics.
Excluding 7 girls younger than 12 years, 19 of 50 girls had signs of spontaneous puberty onset, 13 (26%) showed menarche, and 21 were undergoing HRT. no studies have confirmed the minimum ovarian volume that can detect follicles, so no ultrasound or magnetic resonance imaging (MRI) was performed before conducting the study. Patients received human growth hormone (GH), thyroxine (T4), and estrogen replacement therapy (HRT) according to Swedish treatment norms.
Lymphocyte chromosome analysis determined a TS diagnosis of 45X in 28 cases, chimerism in 7 cases (45X/46XX/47XXX), and structural abnormalities (SAs) on one of the X chromosomes in the remaining 22 cases, including long- or short-arm deletions, heterochromosomes, ring chromosomes, or Y fragments. Chromosome analysis results were provided by the medical center where the patient was seen, and the number of cells analyzed ranged from 15 to 105 depending on the time and place of testing.
The level of detail of the genetic laboratory reports also varies, and a few reports even lack detailed information about the deletion or duplication of the X chromosome arm. All chromosome analysis results were obtained from lymphocyte cultures, and some fluorescence in situ hybridization (FISH) analysis of buccal mucosal cells or other tissues was also reported.
Methods
Laparoscopy
Patients underwent laparoscopic biopsy under general anesthesia to obtain ovarian tissue for freezing and storage. 10 girls had ovaries that were too striated or too small to be biopsied or even difficult to identify. In the remaining 47 patients, 25-50% cortical tissue from one ovary was biopsied.
Cryopreservation
Excised ovarian tissue was immediately placed in an in vitro fertilization culture containing HEPES buffer and human serum albumin. The 0.5-3 mm3 ovarian cortex was excised for histological biopsy, and the remaining tissue was cut into small strips, approximately 0.5 × 3-5 mm, and frozen by slow programming with the cryoprotectant propylene glycol sucrose.
Histologic biopsy and follicle counting
A small piece of fresh ovarian tissue from each patient was cut and fixed in Bouin’s solution, paraffin-embedded, sectioned, and stained with hematoxylin and eosin. The total number of follicles and follicle density of the biopsy specimens were evaluated using a data imaging analysis system connected to an inverted microscope. Follicle density was expressed as the number of follicles per cubic millimeter in the analyzed sample, and only normal follicles were counted. Typically, healthy girls have follicle densities greater than 500/mm3.
Hormone measurements
Blood samples for the detection of FSH, LH and AMH were taken the day before the laparoscopic biopsy in 28 cases, 19 blood samples were sent within one year after the biopsy, and the remaining 10 cases had no blood samples.
Serum FSH and LH were measured in 47 patients, and 43 were measured by solid-phase dual-site chemiluminescence immunoassay (Immulite) in the Women’s Health Research Laboratory at the Karolinska Institute. the sensitivity of the FSH analysis was 0.1 mIU/ml, and the specificity was high, and it was correlated with human chorionic gonadotropin (HCG), thyroid stimulating hormone (TSH) LH, prolactin, human GH or human placental lactogen (HPL) without detectable cross-reactivity.
The sensitivity of LH analysis was 0.1 mIU/ml, and the specificity was also very high, with no detectable cross-reactivity with hCG, FSH or TSH. The remaining four cases of blood FSH were tested in the laboratory of the Clinical Chemistry Center of Karolinsha University Hospital, which was calibrated according to the WHO Second International Reference Preparation (IRP) for biological assays of FSH and hCG. the sensitivity of the FSH assay was 0.7 mIU/ml and its critical level was widely used in Europe, with a corresponding concentration of 17 IU/L, and the widely used standard in the USA is calibrated according to the second IRP standard.
AMH concentrations were determined in 43 children’s sera at the Women’s Health Research Laboratory, Karolinska Institute, Sweden, using a two-step AMH ELISA kit. The ultra-sensitive protocol recommended by the manufacturer was used. This method has a very high specificity for AMH detection and no cross-reactivity with TGF-β. Sensitivity was defined as the lowest AMH concentration with a 95% probability of being significantly different from the zero standard, which was 0.7 pmol/L.
Statistics
The sensitivity and specificity of the diagnostic test were used to calculate positive and negative predictive values, respectively. The positive predictive value is the proportion of patients with a positive test result and reflects the sensitivity of the test. The negative predictive value refers to the proportion of non-patients with a negative test result, reflecting the specificity of the test. The results were expressed as ROC (receiver operating characteristic curve) curves, and the χ2 test was used for statistical treatment, and P<0.05 indicated that the difference was statistically significant.
Results
Laparoscopy
All 57 laparoscopies were performed as day surgery, i.e., patients were admitted in the morning and could be discharged in the afternoon or evening, and there were no anesthetic or surgical complications in this study.
LH, FSH and AMH
All patients were biopsied without HRT in 36 cases and HRT was given in 21 cases. Hormone measurement was not performed considering the artifact of hCG measurement. LH and FSH were measured in a total of 30 cases. 17 cases had serum LH and/or FSH values higher than normal for their age, of which 13 biopsy specimens did not reveal follicles and the remaining 4 had follicles; 13 cases had hormone values within the normal range for their age, of which 9 found follicles and 4 had no follicles.
Ovaries with eggs
Of the 57 patients, 47 could be biopsied on one ovary and 15 (26% of the total) had follicles, ranging from 0.7-1200/mm3. 6 (86%) of the 7 chimeric cases had visible follicles, 6 (27%) of the 22 SAs had follicles, and 3 (10.7%) of the 28 karyotype 45X had follicles. 19 patients with spontaneous pubertal features Eleven (58%) of the 19 patients with spontaneous puberty had follicles, and 8 (62%) of the 13 patients with spontaneous menstruation had follicles.
Of the 30 patients older than 12 years without spontaneous pubertal features, 3 (10%) were found to have follicles. One 11-year-old girl was found to have follicles present despite the absence of obvious pubertal signs.
The top five positive predictive values (sensitivity) for predicting the presence of follicles were: karyotype as chimeric (0.86), FSH value of 11 mIU/ml (0.69), AMH2 pmol/L (0.64), spontaneous menarche (0.62), and spontaneous onset of puberty (0.58).
The negative predictive values (specificity) for absence of follicles were: karyotype 45X (0.89), AMH2 pmol/L (0.88), absence of spontaneous puberty onset (0.87), younger than 12 years (0.82), absence of spontaneous menarche (0.81), FSH value 15 mIU/ml (0.77), older than 16 years (0.76), karyotype SAs (0.73 ). Five of the above six factors were statistically significant, with the age factor being insignificant, although the negative predictive value was higher in the youngest age group.
Conclusion
The results of this study suggest 5 important factors for the ability to detect follicles in the ovaries of TS girls: karyotype, low FSH, high AMH, spontaneous menarche, and spontaneous puberty. In conclusion, the following groups of girls with TS have the highest odds of having follicles and may be considered for laparoscopic biopsy ovarian tissue freezing during 13-17 years of age: 1) chimerism (45X/46XX/47XXX); 2) having spontaneous puberty and 45X or 45X/46X+ SAs; and 3) normal serum FSH and/or AMH with or without spontaneous puberty.
Assuming that the above 3 criteria were used to predict the patients in this study, 19 cases should have undergone laparoscopy at the same age, of which 11 cases could find follicles and 8 cases without follicles, with a sensitivity of 0.58 (9/11). 15 patients at a younger age, delaying laparoscopy meant that 2 cases could still find follicles later, as both had pubertal features. 23 patients who did not undergo laparoscopy , 2 cases with follicles may have been missed according to the third criterion. The specificity to exclude girls with follicle-free TS should be 0.91 (21/23).
Discussion
It is not surprising that follicles can be found in most TS patients with spontaneous puberty and chimerism. Unexpectedly, follicles were also present in 3 patients without spontaneous puberty and 4 patients with high FSH, LH and low AMH. The process of follicular atresia and increased gonadotropin concentrations in the ovaries seems to occur during an overlapping period, at least in some individuals. 3 of the follicles with follicles had at least once had ovarian function but did not show any signs of puberty, implying that their small follicles were unable to secrete sufficient amounts of estrogen to trigger the onset of puberty.
On the other hand, two patients with normal serum gonadotropins and AMH >2 pmol/liter did not show follicles, so it is possible that they did not have follicles in the small pieces of specimen used for histological analysis, while follicles were present in the frozen ovarian tissue or in other parts of the ovarian tissue, which are known to be inconsistently distributed within the ovary. Similarly no follicles were seen in the four cases with normal gonadotropin levels and in the four cases with high AMH levels.
Ultrasound or pelvic MRI was not required to confirm the presence or absence of ovaries before laparoscopy was performed in this study. Some of the girls had undergone transabdominal ultrasound exploration at the local hospital, but the results and the concordance with the laparoscopic findings were extremely low.
Previous studies have also suggested that transabdominal ultrasound findings are reliable, related to the special interest of the investigator, professional skills training and good equipment. MRI may be a better method to detect ovarian tissue in clinical routine and may be an option if laparoscopic ovarian biopsy is not appropriate in patients according to the above criteria. 2 cases in the study who had follicles missed because they did not meet the above criteria may be able to be diagnosed by MRI.
It is necessary to consider whether to perform a biopsy when TS girls reach 13 or 14 years of age, although there is no statistical difference between age and the ability to detect follicles. Most patients who reach this age already understand the possibilities and limitations of preserving ovarian tissue for the future, and because of the limited experience with stored cells for fertility treatment, it is necessary to inform patients that there is no guarantee that they will obtain biological offspring in the future.
Because of the increased risk of complications associated with pregnancy in patients with TS, it should be discussed with the patient and her parents and they should be informed that her pregnancy will require special observation by an obstetrician. Women with cardiac abnormalities (aortic valve insufficiency or aortic stenosis) or hypertension have a significantly increased risk of fatal aortic coarctation aneurysms, but further studies are needed.
The literature on whether the risk of chromosomal abnormalities in the offspring of TS patients is increased is scarce, so the assessment of this risk is not yet reliable. Although TS patients have been informed in detail, both verbally and in writing, of their limited access to offspring and the various known and unknown risks of future pregnancies, it is easy to recruit such girls to participate in the study, as patients seem willing to do whatever they can as long as fertility treatment is technically possible later. From the age of 13-14 years, these patients themselves can become active participants, while for younger girls, the final decision must be made by their parents.
After successful in vitro fertilization of frozen follicles, all women with TS should undergo preimplantation genetic diagnosis (PGD) when conditions permit, and karyotyping of the offspring by chorionic villus sampling or amniocentesis during pregnancy. The experience of this study reinforces our belief that ovarian biopsy and storage at a young age in patients with TS is a very important step forward in the treatment of infertility in this population.