Should we perform oocyte accumulation to preserve fertility in women with Turner syndrome? A multicenter study and systematic review of the literature

Abstract

STUDY QUESTION

Should we perform oocyte accumulation to preserve fertility in women with Turner syndrome (TS)?

SUMMARY ANSWER

The oocyte cryopreservation strategy is not well adapted for all TS women as their combination of high basal FSH with low basal AMH and low percentage of 46,XX cells in the karyotype significantly reduces the chances of freezing sufficient mature oocytes for fertility preservation.

WHAT IS KNOWN ALREADY

An oocyte cryopreservation strategy requiring numerous stimulation cycles is needed to preserve fertility in TS women, to compensate for the low ovarian response, the possible oocyte genetic alterations, the reduced endometrial receptivity, and the increased rate of miscarriage, observed in this specific population. The validation of reliable predictive biomarkers of ovarian response to hormonal stimulation in TS patients is necessary to help practitioners and patients choose the best-personalized fertility preservation strategy.

STUDY DESIGN, SIZE, DURATION

A retrospective bicentric study was performed from 1 January 2011 to 1 January 2023. Clinical and biological data from all TS women who have received from ovarian stimulation for fertility preservation were collected. A systematic review of the current literature on oocyte retrieval outcomes after ovarian stimulation in TS women was also performed (PROSPERO registration number: CRD42022362352).

PARTICIPANTS/MATERIALS, SETTING, METHODS

A total of 14 TS women who had undergone ovarian stimulation for fertility preservation were included, representing the largest cohort of TS patients published to date (n = 14 patients, 24 cycles). The systematic review of the literature identified 34 additional TS patients with 47 oocyte retrieval outcomes after ovarian stimulation in 14 publications (n = 48 patients, n = 71 cycles in total).

MAIN RESULTS AND THE ROLE OF CHANCE

The number of cryopreserved mature oocytes on the first cycle for TS patients was low (4.0 ± 3.7). Oocyte accumulation was systematically proposed to increase fertility potential and was accepted by 50% (7/14) of patients (2.4 ± 0.5 cycles), leading to an improved total number of 10.9 ± 7.2 cryopreserved mature oocytes per patient. In the group who refused the oocyte accumulation strategy, only one patient exceeded the threshold of 10 mature cryopreserved oocytes. In contrast, 57.1% (4/7) and 42.9% (3/7) of patients who have underwent the oocyte accumulation strategy reached the threshold of 10 and 15 mature cryopreserved oocytes, respectively (OR = 8 (0.6; 107.0), P = 0.12; OR= 11 (0.5; 282.1), P = 0.13). By analyzing all the data published to date and combining it with our data (n = 48 patients, n = 71 cycles), low basal FSH and high AMH concentrations as well as a higher percentage of 46,XX cells in the karyotype were significantly associated with a higher number of cryopreserved oocytes after the first cycle. Moreover, the combination of low basal FSH concentration (<5.9 IU/l), high AMH concentration (>1.13 ng/ml), and the presence of 46,XX cells (>1%) was significantly predictive of obtaining at least six cryopreserved oocytes in the first cycle, representing objective criteria for identifying patients with real chances of preserving an adequate fertility potential by oocyte cryopreservation.

LIMITATIONS, REASONS FOR CAUTION

Our results should be analyzed with caution, as the optimal oocyte number needed for successful live birth in TS patients is still unknown due to the low number of reports their oocyte use in the literature to date.

WIDER IMPLICATIONS OF THE FINDINGS

TS patients should benefit from relevant clinical evaluation, genetic counseling and psychological support to make an informed choice regarding their fertility preservation technique, as numerous stimulation cycles would be necessary to preserve a high number of oocytes.

STUDY FUNDING/COMPETING INTEREST(S)

This research received no external funding. The authors declare no conflict of interest.

TRIAL REGISTRATION NUMBER

N/A.

Introduction

Turner syndrome (TS) is a chromosomal disorder affecting ∼1 in 2500 newborn girls (Berglund et al., 2020). It is estimated that 50% of females with TS have a monosomy X karyotype (45,X) while the majority of the remainder have a mosaic karyotype with varying degrees of 45,X/46,XX/47,XXX in their cell lines (Sybert and McCauley, 2004). A minority will have other X-chromosomal structural abnormalities, including deletions, duplications, rings, isodicentric chromosomes, inversions, or translocations (Sybert and McCauley, 2004). The sex chromosome defect in TS women leads to haplo insufficiency of genes that are normally expressed by both X chromosomes, causing a failure during meiosis and prolonging cell cycle (Jeve et al., 2019; Alvarez-Nava and Soto-Quintana, 2022; Fukami, 2023). According to Fukami’s recent review (Fukami, 2023), there are three potential reasons why the loss of germ cells is accelerated in 45,X ovaries. First, a chromosomal pairing failure linked to X-chromosomal aneuploidy causes meiotic arrest, as identified in other dysgonosomias. The X chromosome dosage seems to be more important for the survival of the oocytes than for other ovarian cells. Second, improper coupling between granulosa cells and oocytes may be a factor in causing apoptosis of germ cells. Finally, the lower dosage of multiple genes on the X chromosome may contribute to ovarian dysfunction in women with TS. Hence, monosomic X chromosomes, translocation involving the X chromosomes, or deletions of portions of the X chromosome may lead to genomic instability, decreased mitotic proliferation of germinal cells, arrest meiotic progression in germinal cells, and reduced proliferation of somatic follicular cells, disrupting the survival of ovarian follicles (Modi et al., 2003; Balen et al., 2010; Lundgaard Riis et al., 2021). The phenotype inmosaic TS ovaries seems less severe but can also lead to premature ovarian insufficiency (Lundgaard Riis et al., 2021). Therefore, TS is frequently associated with infertility (Berglund et al., 2020; Lundgaard Riis et al., 2021).

The risk of infertility is a major concern for patients with TS (Sutton et al., 2005) who may be offered fertility preservation through oocyte or ovarian tissue cryopreservation (Huang et al., 2008; Grynberg et al., 2016; Brouillet et al., 2020; Schleedoorn et al., 2020). However, some TS patients poorly respond to hormonal stimulation, leading to a low number of cryopreserved oocytes (El-Shawarby et al., 2010; Oktay et al., 2010; Talaulikar et al., 2019; Vergier et al., 2019; Azem et al., 2020; Ito et al., 2020; Grin et al., 2022). Moreover, there are current investigations regarding the nuclear and cytoplasmic competences of frozen oocytes from TS patients (Bernard et al., 2016; Borini and Coticchio, 2019), suggesting that more oocytes are required for sufficient fertility. In addition, endometrial receptivity seems also to be reduced in TS patients, leading to an increased risk of implantation failure (Li et al., 1991; Rogers et al., 1992; Biljan et al., 1995; Yaron et al., 1996; Cohen et al., 1999; Bodri et al., 2009). In this context, multiple rounds of ovarian stimulation may be necessary to achieve oocyte accumulation and compensate for the diminished ovarian response, the possible genetic alteration of the oocytes, the reduced endometrial receptivity, and the increased risk of miscarriage reported in TS women (Yaron et al., 1996; Kuo and Guo, 2004; Bodri et al., 2009; Bryman et al., 2011; Bernard et al., 2016; Calanchini et al., 2020). However, oocyte accumulation of an adequate number of oocytes does not appear to be achievable in all TS patients.

In this bicentric retrospective study, we report the fertility preservation outcomes in TS patients to whom we proposed oocyte accumulation to preserve their fertility. We also performed a systematic review of the current literature to have a synthesis of existing knowledge and investigate whether specific clinical parameters of TS women are predictive of the preserved fertility potential.

Materials and methods

Ethical approval

The local research ethics committee reviewed and approved the retrospective collection of clinical and biological data required for the clinical study (Institutional Review Board of Montpellier University Hospital, IRB-MTP_2021_07_202100834). All patients included in the clinical study signed an informed consent for the collection of their clinical and biological data. Non-identifying study numbers were assigned to all data.

Patients

This retrospective bicentric cohort study was conducted in the Department of Reproductive Medicine, CHU and University of Montpellier and the Department of Reproductive Medicine and Fertility Preservation, Antoine Beclere Hospital, Clamart, France. Data were collected from the patients’ medical records from 1 January 2011 (corresponding to the authorization of oocyte vitrification in France) to 1 January 2023. Women with TS were referred to the reproductive medicine departments to receive information on fertility preservation.

The inclusion criteria selected women with a karyotype showing at least 10% of cells (or 5% if younger than 25 years) with X chromosome monosomy and/or an abnormal X chromosome (Russell et al., 2007), and documented spontaneous or induced menarche. Exclusion criteria were: women with a Y-chromosome, women with another known genetic abnormality, prepubertal girls or women electing ovarian tissue cryopreservation, women with a condition that prevents them from giving full informed consent, or women at high risk of complications from anesthesia or transvaginal oocyte retrieval.

From 2011 to 2023, 34 patients with TS came for consultation with a fertility preservation specialist. Among them, 15 decided not to cryopreserve their oocytes, and 5 were excluded due to the presence of other genetic alterations. The other 14 women requested oocyte cryopreservation and underwent at least one ovarian stimulation cycle (Fig. 1).

None of them had medical contraindications for pregnancy and they all received genetic counseling before undergoing ovarian stimulation. All women (and their parents if they were minors) signed an informed consent form concerning fertility preservation.

Genetic counseling

Genetic counseling was given to all women, including those who decided against oocyte cryopreservation. During this consultation, patients were informed about the potential genetic risks for the fetus and the higher risk of miscarriage (about two times as higher) compared with the general population (Bernard et al., 2016; Calanchini et al., 2020). The risk of maternal complications in case of pregnancy were discussed with the gynecologist. A pre-conceptional cardiovascular evaluation was carried out according to the French recommendations (Fiot et al., 2022). It included a consultation with a cardiologist and a cardio-aortic imaging.

Ovarian stimulation and oocyte cryopreservation

Ovarian stimulation was performed as previously described (Ranisavljevic et al., 2020) using a GnRH antagonist (ganirelix—Orgalutran 0.25 mg, MSD, France) protocol with recombinant FSH (follitropin alpha—Gonal F, Merck Serono, Geneva, Switzerland; Bemfola, Gedeon Richter, France; follitropin beta—Puregon, MSD Organon, OSS The Netherlands) or human menopausal gonadotropin (hMG; menotropin—Menopur, Ferring Pharmaceuticals, Copenhagen, Denmark; Fertistartkit, Genevrier, France; urofollitropin—Fostimon, Genevrier, France). In all cycles, the daily dose was ≤300 international units (IU) (Montpellier) and ≤450 IU (Paris) of FSH or hMG. The gonadotropin starting dose was high (300–450 IU daily) except in patients with an AMH above 2 ng/ml. Ovulation was triggered either with human chorionic gonadotropin (hCG) (Ovitrelle^® 250 μg Merck Serono), or a GnRH agonist (Decapeptyl^® 0.2 mg, Ipsen, France), or both. The type of trigger was decided by the clinician according to the team prescribing habits in fertility preservation, according to the possible risk of ovarian hyperstimulation (preference for agonist trigger), and according to the patient’s pituitary status (i.e. there was preference for hCG in cases of central hypogonadism). Transvaginal oocyte retrieval was performed under local or general anesthesia. Mature and immature oocytes were cryopreserved by vitrification following the manufacturer’s recommendations (Vit Kit-Freeze, FUJIFILM Irvine Scientific—BioCare Europe^TM).

Systematic review

The systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) guidelines (Liberati et al., 2009).

Registration

The protocol was registered in the International prospective register of systematic reviews (PROSPERO); the registration number is CRD42022362352.

Search

A systematic literature review was performed to identify studies that reported oocyte retrieval outcomes after ovarian stimulation in mosaic TS or TS women, using the MEDLINE database from inception to 1 September 2022. The search terms were (Turner) AND (oocyte).

Study selection

Two reviewers (S.B. and N.R.) independently searched and reviewed the retrieved articles to exclude studies deemed irrelevant by both observers. Studies were first screened for eligibility based on their titles. Abstracts and full texts of potentially relevant articles were retrieved. The retrieved articles were included if they reported the number of retrieved oocytes per patient after ovarian retrieval and ovarian stimulation. Studies mentioning ‘mosaic Turner’ without specifying the karyotype of the patients were included but the absence of information on the chromosomal formula was notified in the results part. Exclusion criteria were women with a karyotype with <10% of cells (or 5% if younger than 25 years) having X chromosome monosomy or no abnormal structure of X chromosome (Russell et al., 2007). Any disagreement or uncertainty was solved by discussion with a third reviewer (M.W.). The final decision was taken by the senior investigator (T.A.).

Data extraction

The following data were extracted to characterize the included studies: study authors, publication year, peripheral blood karyotype, age at ovarian stimulation, baseline FSH (IU/l), AMH (ng/ml), AFC count, number of retrieved oocytes, number of cryopreserved oocytes, use of oocytes (fresh/cryopreserved) if existing, IVF outcomes if existing. Data were extracted independently by two authors (S.B. and N.R.). Any disagreement or uncertainty was solved by discussion with a third reviewer (M.W.). The final decision was taken by the senior investigator (T.A.).

Statistical analyses

Quantitative data were reported as mean and standard deviation, and qualitative data were reported as number/percentage. Statistical analyses were performed with GraphPad Prism (GraphPad Prism 5.0, GraphPad Software Inc). The non-parametric Mann–Whitney test was used for continuous data, and the Chi-square test was used for categorical data. A P-value <0.05 was considered significant. The level of relation between variables was assessed by calculating the Pearson correlation coefficient. This coefficient can range from +1 to −1 (for positive or negative linear correlations). A value of 0 indicates the absence of an association between variables.

ROC curves were performed on basal FSH concentration, basal AMH concentration, and the percentage of 46,XX cells in the karyotype to predict a minimal potential of fertility preservation defined as the cryopreservation of at least six oocytes (cut-off at six oocytes was defined as median value). The ROC curve was the plot of the true positive rate (=sensitivity) versus the false positive rate (=1 − specificity) for all possible threshold values.

The AUC and its 95% confidence interval was also estimated (non-parametric estimator). The AUC indicates the discrimination accuracy as follows: 0.5–0.6 = no discrimination; 0.6–0.7 = poor discrimination; 0.7–0.8 = fair discrimination; 0.8–0.9 = good discrimination; and 0.9–1 = excellent discrimination.

Finally, a combination of basal FSH concentration, basal AMH concentration and the percentage of 46,XX cells in the karyotype based on a logistic regression model was also obtained and evaluated.

Results

The baseline characteristics of the 14 TS patients are summarized in Table 1. The mean age of TS patients was 21.1 ± 5.8 years. The youngest patient was 15 years old while the oldest was 35. Thirteen patients reported spontaneous thelarche and menarche. One patient (Patient #5) beneficiated from puberty induction at the age of 12 by sex steroid treatment. They all had regular menstrual cycles (24–35 days), except one woman who had irregular cycles (Patient #3). All patients were nulliparous. Their ovarian reserves were low, as expected. Indeed, the mean anti-Müllerian hormone (AMH) level was 1.12 ± 1.01 ng/ml (median: 0.85 ng/ml) and the mean antral follicle number was 11.08 ± 9.97 (median: 8).

The ovarian stimulation outcomes are presented in Table 2. A total of 24 cycles of ovarian stimulation were initiated among 14 TS patients. One cycle was interrupted due to an ovarian hyporesponse (Patient #8). An hCG trigger was used in most cycles (11/21, 52.4%). Three cycles of three patients were associated with an absence of cryopreserved oocytes (Patients #3, #8, #9). The mean duration of ovarian stimulation was 10.3 ± 2.01 days. No complication related to ovarian stimulation or oocyte retrieval was reported. At the end of the first cycle, the number of cryopreserved mature oocytes ranged from 0 (Patients #3 and #9) to 11 (Patient #4), with a mean number of 4.0 ± 3.7. Oocyte accumulation was proposed to increase the preserved fertility potential and accepted by 50% (7/14) of patients (Patients #1, #2, #5, #6, #7, #8, and #13). The mean number of cycles among patients who accepted oocyte accumulation was 2.4 ± 0.5 cycles. In total, two and three cycles were performed in four and three patients, respectively. A high heterogeneity between cycles was found for 28.6% (2/7) of patients. Indeed, Patient #1 produced 3, 10, and 4 matures oocytes in Cycles #1, #2, and #3, respectively. Patient #6 produced 1, 3, and 12 matures oocytes in Cycles #1, #2, and #3, respectively. Immature oocytes were retrieved and vitrified (without in vitro maturation) from 4 patients (Patient #1: 1 Metaphase I (MI) oocyte; Patient #5: 1 MI oocyte, Patient #8: 1 MI oocyte, Patient #11: 1 germinal vesicle stage oocyte).

In the group who refused the oocyte accumulation strategy, only one patient exceeded the threshold of 10 mature cryopreserved oocytes. In contrast, 57.1% (4/7) of patients who underwent the oocyte accumulation strategy reached the threshold of 10 mature cryopreserved oocytes (OR= 8 (0.6; 107.0), P = 0.12). Moreover, 42.9% (3/7) of patients exceeded the threshold of 15 oocytes with oocyte accumulation strategy (OR= 11 (0.5; 282.1), P = 0.13). (Patient #3 who then had puberty induction produced 20 mature oocytes in two cycles among the 23 oocytes collected.) Oocyte accumulation was associated with a non-significant improved total number of cryopreserved mature oocytes per patient compared to the patients who declined this strategy (10.9 ± 7.2 versus 4.0 ± 4.3, P = 0.1).

Concerning their female parameters, there was a significant correlation between the number of cryopreserved mature oocytes after the first cycle and basal FSH concentration (negative correlation, r = −0.6, P = 0.04) as well as with the percentage of 46,XX cells in the karyotype (positive correlation, r = 0.77, P = 0.001). In contrast, there was no correlation between the number of cryopreserved mature oocytes after the first ovarian cycle and woman’s age (r = −0.01, P = 0.96), basal AMH concentration (r = 0.52, P = 0.07), AFC count (r = 0.43, P = 0.12), percentage of cells with X chromosome monosomy (r = −0.42, P = 0.14), nor percentage of cells with an abnormal X (r = −0.23, P = 0.42).

The systematic review of the current literature on oocyte retrieval outcomes after ovarian stimulation in TS women identified 259 potentially relevant articles in the initial search (Fig. 2). After screening the titles, 99 abstracts and their corresponding full-text articles were assessed for eligibility. After exclusion of irrelevant articles, 14 studies reporting the outcomes of ovarian retrieval following hormonal stimulation in 34 TS women were included (Table 3). Among them, only 29.4% (10/34) beneficiated from oocyte accumulation (#4, #6, #16, #19, #20, #24, #25, #30, #31, and #33 in Table 3).

When analyzing all the data from Table 3 and our data, significant correlations between the number of cryopreserved mature oocytes after the first cycle of ovarian stimulation and basal FSH concentration (negative correlation, r = −0.33, P = 0.04, n = 39), basal AMH concentration (positive correlation, r = 0.62, P = 4.7 × 10⁻⁵, n = 36), and the percentage of 46,XX cells in the karyotype (positive correlation, r = 0.42, P = 0.008, n = 37) were observed (Fig. 3A). In contrast, there was no correlation between the number of cryopreserved mature oocytes after the first cycle and woman’s age (n = 44, r = −0.07, P = 0.61), the percentage of cells with X chromosome monosomy (n = 37, r = −0.06, P = 0.7), nor the percentage of cells with an abnormal X (n = 38, r = −0.17, P = 0.29).

The basal FSH concentration, basal AMH concentration, and the percentage of 46,XX cells were significantly predictive of obtaining at least six cryopreserved oocytes after the first cycle, with optimal thresholds, according to ROC curves, of FSH < 5.9 IU/l (OR = 6.96 (1.66, 29.26), P = 0.0081), AMH >1.13 ng/ml (OR = 12.47 (2.47, 62.99), P = 0.0023), and 46,XX cells >1% (OR = 9.75 (2.15, 44.14), P = 0.0031), respectively. The AUC_ROC evaluating the individual performance of basal FSH concentration, basal AMH concentration and the percentage of 46,XX cells in predicting cryopreserved oocytes reached 0.68 (95% CI (0.52, 0.83)) (poor discrimination), 0.84 (95% CI (0.67, 0.94)) (good discrimination), and 0.77 (95% CI (0.62, 0.90)) (fair discrimination), respectively (Fig. 3B). The combination of these three parameters [FSH + AMH + 46,XX cells] allowed an improvement in the AUC_ROC to 0.91 (95% CI (0.73, 0.98)) (excellent discrimination).

To date, IVF outcomes using autologous oocytes from only six TS patients have been reported in literature (Table 3, Patients #1, #4, #21, #22, #24, #25), with only one attempt from one patient using autologous cryopreserved oocytes (patient #25; Strypstein et al., 2022)). Nine embryo transfers were performed in only four patients (#1, #22, #24, #25), leading to the birth of three children (#1, #22, #25). We report the IVF outcomes using fresh autologous oocytes from an additional patient with TS mosaicism (Table 4). The couple already had two children from two spontaneous pregnancies and wished for a third child. The investigation of the causes of their secondary infertility highlighted a diminished ovarian reserve and a Turner mosaic syndrome for the female partner. A first ovarian stimulation cycle was performed, resulting in the retrieval of 7 oocytes of which only 3 were mature after denudation. After intracytoplasmic sperm injection, a top embryo was transferred on D3 leading to only a biochemical pregnancy. The supernumerary embryo frozen on D3 was thawed and transferred in a subsequent cycle, unfortunately resulting in no pregnancy. A second ovarian stimulation cycle was performed, resulting in the retrieval of 13 oocytes of which only 7 were mature after denudation. After intracytoplasmic sperm injection, two embryos were cryopreserved on D3 because the patient was at risk of hyperstimulation syndrome. The two embryos were thawed, with one embryo was lysed after thawing. The other thawed embryo was transferred, unfortunately resulting in no pregnancy.

Discussion

Oocyte cryopreservation after ovarian stimulation is one of the options for fertility preservation in patients with TS. To our knowledge, we report here the largest cohort of TS patients who have undergone oocyte cryopreservation and oocyte accumulation. More importantly, we performed the first systematic review on ovarian stimulation outcomes in TS patients (Table 3). This review allowed us to identify that basal FSH concentration, basal AMH concentration and the percentage of 46,XX cells in the karyotype were associated with the number of mature cryopreserved oocytes and that the corresponding combination of these three parameters was highly predictive of the ovarian response in this specific population (Fig. 3). This point is relevant because it could help practitioners to identify TS patients at risk of poor ovarian response, providing concrete elements for an informed choice regarding the best-personalized option for fertility preservation technique by TS patients. Our study also has other strengths. The bicentric design allows us to report the largest cohort of patients with TS who underwent oocyte cryopreservation and oocyte accumulation with a wide age range from 15 to 35 years. Our results enrich the current literature as the previous best-documented publication presented oocyte cryopreservation outcomes in only seven TS women with a narrower age range from 18 to 26 years, with only one patient benefiting from oocyte accumulation (Talaulikar et al., 2019). We also have added the result of IVF outcomes from an additional mosaic TS patient to the six patients with TS who had already been reported in the literature, increasing the data on the use of autologous oocytes of TS patients.

The choice of the best fertility preservation strategy in TS patients requires the use of predictive markers specifically validated within this population to predict the potential for fertility preservation. Our systematic review with the data from the literature and our data together represent 71 cycles from 48 TS patients in total. This first systematic review allowed us to identify that the combination of low basal FSH concentration (<5.9 IU/l), high AMH concentration (>1.13 ng/ml) and the presence of 46,XX cells (>1%) in the karyotype was significantly predictive of obtaining at least six cryopreserved oocytes in the first cycle (Fig. 3). The identification of these objective parameters and their validation in further studies could open up interesting perspectives on the identification of TS patients who can hope to preserve a sufficient fertility potential with oocyte accumulation. To date, several predictive biomarkers of ovarian response to hormonal stimulation have been explored during the last decades but none has been validated in TS population. Low AMH concentrations have demonstrated a high accuracy for diagnosis of early premature ovarian insufficiency in various etiologies (Jiao et al., 2021) and for predicting poor ovarian response (Wang et al., 2021). Increased basal FSH has also been reported to be associated with low ovarian response to hormonal stimulation (Reichman et al., 2014). Recently, some authors have suggested that the basal FSH level may display a higher predictive value in younger women whereas AMH measurement could be more appropriate in women aged ≥35 years (Salama et al., 2021). Due to the lack of data specific to TS patients in the literature, further studies are highly necessary in TS patients to validate a combination of predictive markers to help practitioners and patients choose the best option(s) for fertility preservation between oocyte and ovarian tissue cryopreservation.

We have demonstrated in this study that the oocyte accumulation strategy requires repeated ovarian stimulations in the hope of preserving a sufficient fertility potential in TS patients. Indeed, a high number of mature oocytes seems necessary to compensate for the low ovarian response, the potential oocyte genetic alterations, the reduced endometrial receptivity and the higher miscarriage rates observed in this specific population (Yaron et al., 1996; Kuo and Guo, 2004; Bodri et al., 2009; Bryman et al., 2011; Bernard et al., 2016; Calanchini et al., 2020). These adverse effects are assumed to be the consequence of the absence or alteration of X chromosome genes. Hence, TS patients should presumably undergo more stimulation cycles to cryopreserve a sufficient number of oocytes to allow them to hope for the birth of at least one live child. In addition, we noted a high heterogeneity in ovarian response between stimulated cycles among some TS patients, highlighting major variations between consecutive ovarian stimulation cycles in mature oocyte yield despite similar stimulation protocols. This illustrates that the magnitude of ovarian response to stimulation may exhibit significant variations from cycle to cycle in TS patients. This heterogeneity is certainly due to karyotype mosaicism between ovarian follicles, with some oocyte cohorts being composed of more or less follicles presenting a normal karyotype (Jeve et al., 2019; Nadesapillai et al., 2021; Alvarez-Nava and Soto-Quintana, 2022). Hence, low oocyte yield in one cycle should not discourage the medical team nor the TS patient from performing another cycle. In consequence, these results highlight the need to perform several cycles of ovarian stimulation (possibly at least three cycles) before deciding on the medical interest of continuing oocyte accumulation or changing the fertility preservation strategy. Hence, practitioners should clearly state how cumbersome the process is for TS women. The proposition of dual stimulation (Ito et al., 2020) may help to reduce the period dedicated to fertility preservation. To date, publications reporting the use of TS cryopreserved oocytes are still rare in the current literature, which raises questions about the ideal number of oocytes to preserve. The first live birth after fertility preservation using autologous cryopreserved oocytes in a woman with mosaic TS was recently reported (Strypstein et al., 2022), but the optimal number of oocytes needed for at least one successful live birth is still unknown in the TS population. However, a retrospective study in young patients with various etiologies undergoing elective cryopreservation reported a live birth rate of 15.4%, 60.5%, and 85.2% when 5, 10, and 15 mature oocytes could be defrosted during the IVF procedure, respectively (Cobo et al., 2016). These results suggest that far more than 15 frozen oocytes would be needed to preserve sufficient fertility potential in TS patients to compensate for the fertility alterations associated with this condition. Unfortunately, this threshold is rarely reached in this population, supporting that oocyte cryopreservation is not an adequate strategy for all TS patients.

The genetic status of TS patients seems also highly associated with TS fertility potential. In our study, we demonstrated that a threshold below 1% of 46,XX cells in the karyotype was a negative predictive parameter for oocyte cryopreservation strategy (Fig. 3). The presence of an abnormal X chromosome could also represent a negative factor for oocyte accumulation. Indeed, theoretically ∼50% of oocytes will contain the der(X), leading to different risks for the pregnancy outcome according to the gender of the fetus. A female fetus will present with TS whereas male fetus may not be viable or may lead to different syndromes, according to the size of the der(X). Thus, it would be relevant to provision a much larger number of oocytes in TS patients with an abnormal X (between +25% to +50%, which might be difficult to achieve), and to discuss preimplantation genetic diagnosis when possible according to the local legislation. If spontaneous pregnancy occurs, prenatal diagnosis is recommended for male pregnancies according to the configuration of the der(X) (strongly recommended for small Xp deletion). In contrast, women without X-chromosomal structural abnormalities would have a relatively low risk (i.e. below 10%) of giving birth a daughter with X-chromosomal aneuploidy (Peek et al., 2019; Liao et al., 2021). This could be explained by the lack of correlation observed between the level of mosaicism in lymphocytes, granulosa cells and that of ovarian cells (Peek et al., 2019; Nadesapillai et al., 2021) or by the theory of a cryptic mosaicism with a rescue cell line in 45,X individuals (Hook and Warburton, 2014). However, the observed rate of X chromosome aneuploidy rate remains higher in TS women than in non-TS women and a higher risk of other chromosomal abnormalities has been described in autologous TS women’s pregnancies (Tarani et al., 1998; Bryman et al., 2011; Bernard et al., 2016; Nadesapillai et al., 2021). Hence, genetic counseling should be given to all TS women whatever their karyotype, including those who do not choose cryopreservation, with a systematic discussion on preimplantation genetic testing for aneuploidies (PGT-A) and prenatal diagnosis according to the local legislation (Giles et al., 2020). In France, TS women are systematically informed about current and future management as well as screening for potential associated comorbidities, including especially cardiovascular diseases according to French recommendations (Fiot et al., 2022). The cardiologist’s agreement is required before the implementation of medically assisted procreation. Patients are monitored by a specialized cardiologist throughout the pregnancy according to PNDS recommendations to receive adequate information about their increased risks of eclampsia, gestational diabetes, cholestatic hepatitis or aortic dissection during pregnancy, ensuring ethical and safe care of these patients (Fiot et al., 2022).

Our study had some limitations. Although this is the largest published cohort, its sample size remains limited but this is expected due to the study population. Moreover, our data should be interpreted with caution as all steps of fertility preservation are financially supported by the national health insurance in France. It can be assumed that patients in other countries will be less inclined to carry out multiple stimulation cycles to preserve fertility because of the financial burden that these numerous cycles represent. On the other hand, we did not assess the competence of the cryopreserved oocytes in our study as no patient has yet asked for the use of their cryopreserved oocytes.

Fertility preservation in TS patients has been recently recommended as soon as spontaneous menarche is achieved (La Marca and Mastellari, 2021). Intriguingly, recent data have demonstrated that ovarian stimulation in a TS prepubertal girl could lead to the freezing of several mature oocytes (Azem et al., 2020), questioning the ideal timing of fertility preservation. However, women before the age of 20 display a high level of oocyte aneuploidy caused by numerous meiotic nondisjunction events (Gruhn et al., 2019). Hence, a larger number of oocytes would be required in young TS patients to compensate the higher aneuploidy rate. Moreover, very young patients do not necessarily have the psychological resources to withstand multiple cycles of stimulation. Indeed, the oocyte retrieval procedure might be unacceptable for such young patients or their parents, due to personal representations, cultural and/or religious beliefs. Moreover, the necessity to multiply the stimulation cycles for oocyte accumulation might discourage them, threatening the chances of achieving a large number of cryopreserved oocytes. Appropriate psychological support must be offered to all TS women, especially young patients, and their families who may experience fertility-related distress and may need supportive care to assist them in decision-making (Oktay et al., 2016; Logan and Anazodo, 2019).

Conclusions

Accumulation of cryopreserved oocytes for women with TS should be proposed after appropriate medical and genetic counseling with the objective of obtaining a high number of oocytes. The optimal oocyte yield in patients with TS might be much higher than in other fertility preservation settings. Future studies on the use of vitrified oocytes will allow us to confirm whether oocyte accumulation increases the chances of a live birth.

Data availability

The main data underlying this article are available in the article. Other data underlying this article will be shared on reasonable request to the corresponding author.

Acknowledgements

The authors thank Dr Claire Jeandel for her dedicated care of young patients with Turner syndrome and her collaboration with our department to preserve their fertility. The authors are grateful to Dr Florence Perron for her help in data collection.

Authors’ roles

S.B., N.R., M.W., and T.A. participated in the study design. S.B., N.R., C.S., E.H., S.B.D., V.L.C., and S.H. participated in the study execution. S.B. and N.R. participated in the analysis. S.B., N.R., C.S., M.W., E.H., and T.A. participated in the manuscript drafting. All authors participated in the critical discussion.

Funding

This research received no external funding.

Conflict of interest

The authors declare no conflict of interest.

References

Author notes