Browse Subject Areas

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Effect of Previous Chemotherapy on the Quality of Cryopreserved Human Ovarian Tissue In Vitro

  • Babak Asadi Azarbaijani ,

    Affiliations Women and Children’s Division, Oslo University Hospital, Rikshospitalet, Oslo, Norway, University of Oslo, Oslo, Norway

  • Mona Sheikhi,

    Affiliations Division of Obstetrics and Gynecology, Karolinska Institute, Karolinska University Hospital, Huddinge, Stockholm, Sweden, Stockholm IVF, Stockholm, Sweden

  • Irma C. Oskam,

    Affiliation Faculty of Veterinary Medicine and Bioscience, University of Oslo, Oslo, Norway

  • Mirja Nurmio,

    Affiliation Department of Physiology and Pediatrics, University of Turku, Turku, Finland

  • Tiina Laine,

    Affiliations Department of Pediatrics, Helsinki University Central Hospital, Helsinki, Finland, Division of Obstetrics and Gynecology, University of Helsinki, Helsinki, Finland

  • Helena Tinkanen,

    Affiliation Tampere University Central Hospital, Tampere, Finland

  • Sirpa Mäkinen,

    Affiliation The Family Federation of Finland, Fertility Clinic, Helsinki, Finland

  • Tom G. Tanbo,

    Affiliations Women and Children’s Division, Oslo University Hospital, Rikshospitalet, Oslo, Norway, University of Oslo, Oslo, Norway

  • Outi Hovatta,

    Affiliation Division of Obstetrics and Gynecology, Karolinska Institute, Karolinska University Hospital, Huddinge, Stockholm, Sweden

  • Kirsi Jahnukainen

    Affiliations Department of Pediatrics, Helsinki University Central Hospital, Helsinki, Finland, Division of Obstetrics and Gynecology, University of Helsinki, Helsinki, Finland, Department of Women’s and Children´s Health, Karolinska Institute and University Hospital, Stockholm, Sweden

Effect of Previous Chemotherapy on the Quality of Cryopreserved Human Ovarian Tissue In Vitro

  • Babak Asadi Azarbaijani, 
  • Mona Sheikhi, 
  • Irma C. Oskam, 
  • Mirja Nurmio, 
  • Tiina Laine, 
  • Helena Tinkanen, 
  • Sirpa Mäkinen, 
  • Tom G. Tanbo, 
  • Outi Hovatta, 
  • Kirsi Jahnukainen



Cryopreservation of ovarian tissue has been widely accepted as an option for fertility preservation among cancer patients. Some patients are exposed to chemotherapy prior to ovarian tissue cryopreservation. Consequently, assessment of the developmental capacity of human ovarian tissue after chemotherapy is of primary importance.


In order to study the impact of previous chemotherapy on in vitro development and viability of ovarian follicles, quality control samples from 34 female cancer patients at median age of 15 years (range 1‒35), cryopreserved for fertility preservation before (n = 14) or after (n = 20) initiation of chemotherapy, were thawed and cultured for 7 days. The morphology and developmental stages of ovarian follicles were studied by light microscopy before and after culture. Possible associations between follicular densities, age and exposure to alkylating agents, expressed as cyclophosphamide equivalent dose (CED) were tested.


Exposure to chemotherapy significantly impaired the survival and development of ovarian follicles in culture. After seven days, significantly higher densities of intermediary, primary and secondary follicles and lower densities of atretic follicles was detected in the samples collected before chemotherapy. Increasing dose of alkylating agents was identified by multivariate linear regression analysis as an independent predictor of a higher density of atretic follicles, whereas increasing age of the patient predicted a better outcome with less follicle atresia and a higher density of maturing follicles.


This study provides quantitative in vitro evidence of the impact of chemotherapy on developmental capacity of cryopreserved human ovarian tissue. The results indicate that fertility preservation should be carried out, if possible, before initiation of alkylating agents in order to guarantee better in vitro survival of ovarian follicles. In addition, ovarian samples from younger girls show lower viability and fewer developing follicles in culture.


Infertility is one of the late effects of cancer treatment among female survivors. Cryopreservation of ovarian tissue has been widely accepted as an option for fertility preservation [1]. It is the only option available for pre-pubertal girls and women who cannot delay the start of chemotherapy [1;2]. To achieve fertility, the follicles in the cryopreserved ovarian tissue ought to undergo full maturation from primordial follicles to antral follicles containing fully mature oocytes. At present, only patients with cancers associated with a low risk of ovarian metastasis are considered for auto-transplantation of ovarian tissue [35]. For the time being, it is not regarded safe to perform auto-transplantation on patients with high risk of ovarian metastasis, such as those with hematological cancers, because of possibility to reseeding malignant cells into cured patients [35].

Maturation of ovarian follicles and oocytes in vitro is a promising but challenging strategy to overcome the problems of cancer contamination. Even though there is progress in the procedures [69], full maturity with fertilizable oocytes has so far not been feasible in humans. In experimental animals, such as rodents, the procedure is shorter. The first live mouse was born from in vitro matured oocytes in 1996 [10], followed by large numbers of healthy offspring after merely improving the culture conditions [11]. New culture methods with using inhibitor of phosphatase and tensin homologue (PTEN) have been successfully used in activation of the primordial follicles in vitro and generation of fertilized egg in mouse [12]. Now, after the encouraging data on the mouse oocyte maturation, the in vitro methods need to be refined so that they can be applied to human follicles. We and others have successfully used PTEN inhibitor in cultures of human ovarian follicles during the first day of culture to promote follicle activation and development to the secondary stage [13;14].

The sterilizing effect of chemotherapy on the ovary is well known [15;16] and most oncologists recommend cryopreservation of ovarian tissue before initiation of chemotherapy [3]. Childhood cancers and hematological malignancies require prompt initiation of cancer therapy and the patients are therefore often exposed to chemotherapy prior to ovarian biopsy [1719]. Up to date, there is no data regarding the effect of chemotherapy on in vitro development and survival of ovarian follicles in cryopreserved human ovarian tissue, despite its high importance for the feasibility of obtaining oocytes from the cryopreserved tissue.

In the present study, we used in vitro tissue culture to evaluate developmental capacity of cryopreserved human ovarian tissue taken as quality control samples for fertility preservation of children and young women with cancer. The study was conducted with special emphasis on the effects of chemotherapy with alkylating agents on the in vitro development and viability of ovarian follicles.

Materials and Methods

Tissue donors

Patients for fertility preservation were recruited at the Children's Hospital, University Central Hospital of Helsinki, Finland, the Department of Gynecology, Oslo University Hospital, Norway and the Department of Gynecology, University Hospital of Tampere, Finland [17].

Adult patients in the Oslo University Hospital and the University Hospital of Tampere were offered cryopreservation of ovarian tissue as a part of the fertility preservation program, which provided them with a full range of fertility-saving options. The participants signed an informed consent form for quality control of ovarian tissue including morphological analysis and in vitro culture. Ethical approval was therefore not necessary. All samples were anonymized before access and start of analysis.

In the Children's Hospital, Helsinki fertility preservation was performed as a part of research protocol approved by the Ethics Committee of Helsinki University Central Hospital. All age-appropriate patients or guardians provided their written informed consent for participation in the study.

Parental written consent was obtained from all patients under 18 years old.

Ovarian tissues used for control of the culture method were donated by four healthy women undergoing Cesarean section at Karolinska University Hospital, Stockholm, Sweden. They had signed an informed consent form for donation of a small piece of tissue for this research. The study and the consent form had been approved by The Regional Ethics Board in Stockholm.

The study samples were consecutive quality-control material for fertility preservation from patients with hematological and solid cancers (Helsinki, n = 19; Oslo, n = 13; Tampere n = 2) (Table 1) at median age of 15 years (range 1–35) at the time of biopsy. It was not possible to select tissue donors on the basis of age. Of the total of 34 samples obtained, 14 were collected at the time of diagnosis before chemotherapy and 20 after initiation of chemotherapy. The median age of healthy controls was 33 years (range 31–36).

Table 1. Controls and cancer patients who underwent biopsy before or after chemotherapy.

The hospitals’ medical records were used to collect information regarding the patients; i.e. age, diagnosis, type of treatment, timing and cumulative doses of chemotherapeutic agents. Cumulative exposure to alkylating agents was assessed by calculating the cyclophosphamide equivalent dose (CED), as described before [20]. Except for one, all patients treated with chemotherapeutic drugs received alkylating agents, cyclophosphamide being a major treatment element (Table 1). The interval between last dose of chemotherapy and ovarian biopsy was less than 56 days for all patients treated with chemotherapy (Table 1).

Ovarian tissue cryopreservation and thawing

Ovarian tissue was biopsied by laparotomy or laparoscopy and transported on ice to the laboratory in cold phosphate-buffered saline prior to cryopreservation. Ovarian tissue from each patient was prepared in the laboratory by cutting the ovarian cortex into small pieces. Cryopreservation of ovarian tissue was carried out by using a slow freezing method with either propanediol (PrOH) or ethylene glycol (EG) as cryoprotectant agent depending on the cryopreservation protocol used in the respective hospital. The protocols for freezing of human ovarian tissue have been described earlier [21;22]. A programmable freezer was used and the tissue specimens were stored in liquid nitrogen. The cryopreserved pieces were thawed according to the reverse cryopreservation procedure [21;23;24]. The procedure took place at room temperature under a laminar flow hood in sterile conditions before transfer to culture medium.

Ovarian tissue culture

Ovarian tissue culture was based on an established method as previously described [6;13;25;26]. The thawed ovarian cortical tissue was cut into pieces of 2–3 × 3–4 × 1.5 mm3. One piece was fixed in Bouin’s solution for histology. The remaining pieces were immediately cultured at 37°C in a humidified atmosphere containing 5% CO2, in 0.5 ml Dulbecco’s Modified Eagle’s Medium with Glutamax (Gibco, Invitrogen Inc.) supplemented with human serum albumin (10%; Vitrolife, Goteborg, Sweden), glutamine (3mM), follicle-stimulating hormone (0.5IU/ml; Gonal-F Serono Nordic Inc.), insulin-transferrin-selenium (1%; Invitrogen Inc.) and antibiotic/antimycotic (50IU/ml; Invitrogen Inc.). All samples were treated with phosphatase and tensin inhibitor (1 μM; Calbiochem, Merck Chemicals Ltd.) for the first 24 hours of the culture period to activate the growth of ovarian follicles. The culture medium was changed daily. The cultured ovarian tissue pieces were fixed in Bouin’s solution after seven days.


The fixed ovarian tissue samples were placed in 70% alcohol after 24 h and stored at 4°C. To analyze the development of ovarian follicles by light microscopy, the dehydrated ovarian tissue strips were embedded in paraffin, sectioned (4 μm), and stained with hematoxylin and eosin (HE). Two persons counted the number of follicles at each developmental stage in all sections and controlled the results by use of an inter-observer variation method. To avoid double counting, each follicle was followed through neighboring sections.

Follicles were classified as primordial, intermediary, primary and secondary (Fig 1) [26;27]. They were further classified as intact, influenced and atretic in order to evaluate the quality of the ovarian cortical pieces (Fig 1). Intact follicles were defined as those with an intact basement membrane attached to granulosa cells and without contraction of the cytoplasm or any pyknotic nuclei. The oocytes were in close contact with the surrounding granulosa cells, and the granulosa cells were without pyknotic nuclei or any signs of shrinkage or swelling. Influenced follicles were defined as those having intact nuclei and membranes of the oocyte, with less than 50% detachment of the oocyte from surrounding granulosa cells and/or less than 10% vacuolization in cytoplasm, and less than 50% atretic granulosa cells. The follicles were defined as atretic if the nucleus or more than 50% of any of the follicle structures described above were pyknotic. All intact and influenced primordial, intermediary, primary and secondary follicles were evaluated separately in each category.

Fig 1. Representative images of ovarian cortex before and after seven days of culture (magnification ×40), from a 15-year-old girl with lymphoma and no chemotherapy (A,B), and from a 2-year-old girl with neuroblastoma exposed to CED of 7200 mg/m2 (C,D).

a) Intact primordial follicle, b) intact secondary follicle, c) influenced primordial follicle, d) atretic follicle.

The volume (V) of ovarian cortices was calculated by summing the number (n) of sections, of area Amm2 and thickness 0.004 mm: V (mm3) = (A1+A2+…+An) × 0.004. The areas were measured by using Nikon's NIS-Elements with ×4 magnification. The densities of each type of follicle in the ovarian cortex were then calculated as the total number of follicles divided by the total volume and expressed as the number of follicles/mm3 of ovarian tissue [28].

Statistical analysis

SPSS statistical software version 21 was used to analyze the data. All data are presented as median and interquartile range (IQR). The Mann–Whitney U test was used to compare follicle densities in tissue samples removed before chemotherapy vs. those removed after initiation of chemotherapy. The entire study material was included in Spearman’s rank correlation analysis to assess univariate correlations between follicular density and age, and follicular density and CED. Follicular densities of the entire study material were further entered as dependent variables, and age, CED and cryoprotectant agents (PrOH or EG) as independent variables in multiple linear regression analysis. All tests of significance were two-tailed and p-values ≤ 0.05 indicated statistical significance.


The viability and developmental stages of a total of 17,212 non-cultured follicles and 41,231 follicles after seven days of culture were analyzed in tissues from 34 cancer patients and four healthy donors. Of a total of 34 samples, 33 were evaluated after thawing and 34 after seven days of culture. The results are based on ovarian follicular density (Table 2). Developmental stages from primordial to secondary follicles were observed after culture (Fig 1).

Table 2. Median and interquartile range (IQR) of follicle densities in cryopreserved human ovarian tissue before and after seven days of culture.

Viability and development of ovarian follicles in control tissue

The samples from the four healthy donors were evaluated independently as controls for the culture method. Median density (IQR) of total, intact and atretic follicles were before culture 20 (31), 20 (31), 17 (10) and after culture 23 (17), 11 (5), 7 (8) per mm3, respectively. Median density of primordial, intermediary, primary and secondary follicles were before culture 6 (21), 6 (11), 6 (2), 0 (4) and after culture 4 (4), 4 (7), 6 (6), 1 (2) per mm3, respectively. The proportion of atretic follicles increased from 0% to 30% (P = 0.014) while the proportion of intact follicles decreased from 100% to 47% (P = 0.021) after the seven days of culture. No changes in the proportion of more developing follicles-primary and secondary follicles- were observed in culture (35% before and 40% after seven days of culture).

Viability and development of follicles in ovarian tissue collected before and after the initiation of chemotherapy

Median follicular densities before and after culture are shown in Table 2. Before culture, the density of primordial follicles was similar in ovarian samples collected before and after the initiation of chemotherapy, however, more atretic follicles and fewer follicles in primary or secondary stages were seen in samples exposed to chemotherapy (Table 2). After seven days of culture the majority of follicles in samples collected after the initiation of chemotherapy had entered atresia (Table 2). Significantly higher densities of intermediary, primary and secondary follicles were detected in the samples collected before chemotherapy (Table 2).

The proportion of atretic follicles increased in culture. This was observed both in the samples not exposed (8% before and 53% after seven days of culture, P = 0.004) and exposed to chemotherapy (before 41%, after 86%, P = 0.070). The decrease in the proportion of intact follicles was more notable in samples exposed to chemotherapy (before 46%, after 6%, P<0.001) than in samples that were not exposed (before 82%, after 28%, P = 0.001). No changes in the proportions of more developing follicles-primary and secondary follicles- were detected in the samples that were not exposed to chemotherapy (before 17%, after 17%), while proportions of more developing follicles were decreased in the samples collected after exposure to chemotherapy (before 8%, after 5%, P = 0.014).

Effects of age and ovarian exposure to alkylating agents on viability and development of ovarian follicles

Patients who had not received chemotherapy before ovarian biopsy were significantly older than those who had received chemotherapy (Tables 1 and 2, Fig 2). Exposure to alkylating agents, expressed as CED, varied significantly between the patients (Tables 1 and 2). Spearman’s rank correlation analysis was performed to identify if age or CED correlated to follicle densities using 33 samples before and 34 samples after seven days of culture (Table 3). Increasing age correlated significantly with an increasing density of developing follicles and with a lower density of total and atretic follicles both before and after seven days of culture (Table 3). Increasing exposure to CED correlated with an increased density and proportion of atretic follicles before and after culture (Table 3). Increased CED also correlated with a decreased density of developing follicles after culture (Table 3).

Fig 2. Relationship between follicle density and age before and after seven days of culture in controls (circles) and cancer patients with (triangles) and without (squares) chemotherapy.

Table 3. Spearman’s rank correlation analysis of age and cumulative Cyclophosphamide Equivalent Dose (CED) and their association with follicle densities after seven days of culture.

Multivariate linear regressions were performed to adjust for differences in age, cryoprotectant agents (PrOH or EG) and exposure to CED between the groups. Multivariate linear regression analysis identified increasing age of the patient as the only independent predictor of lower total density of follicles in ovarian samples before culture (Table 4). After culture for seven days, both age and exposure to alkylating agents independently predicted viability of ovarian follicles. Increasing age predicted a lower density of total and atretic follicles, while exposure to increasing CED predicted higher density of total and atretic follicles. Increasing age was the only independent predictor of higher density of the differentiating primary and secondary follicles after seven days of culture (Table 4). The cryoprotectants PrOH or EG were not significantly associated with follicle densities when adjusted to other factors in the regression model (Table 4).

Table 4. Multivariate linear regression analysis of follicle densities using age, cumulative Cyclophosphamide Equivalent Dose (CED) and propanediol (PrOH) as predictors after 7 days of culture.


The aim of ovarian tissue cryopreservation is to increase the chance of fertility in cured cancer patients, either via transplantation or in vitro culture. To achieve the goal, the evaluation of the developmental capacity of cryopreserved tissue is of primary importance. This study provides the first quantitative evidence of the impact of alkylating agents on in vitro viability and developmental capacity of ovarian tissue that was cryopreserved for fertility preservation. We demonstrated that previous chemotherapy significantly impaired the survival and development of the ovarian follicles in culture. The cyclophosphamide equivalent dose was an independent predictor for the density of atretic follicles. The study demonstrates that the youngest patients, exposed to the highest cumulative doses of alkylating agents, had the highest probability of increased follicular atresia and the lowest probability of developing follicles after culture.

Previous analyses of clinical histological samples and the results of xenotransplantation studies show that chemotherapy, in particular with alkylating agents, induces atresia of ovarian follicles [2931]. Patients who received chemotherapy presented with significantly lower primordial follicle counts in morphological analysis and decreased estrogen production in vitro [32]. Cyclophosphamide is one of the most widely used alkylating agents. It is known to have an adverse effect on rapidly dividing cells and it damages DNA repair mechanisms [33]. In the ovary, granulosa cells surrounding the oocytes are an important target of alkylating agents [15;34]. DNA damage can lead to apoptosis of proliferating granulosa cells and decrease intercellular communication between them and the oocyte [35]. Alkylating agents may also directly cause DNA and RNA damage and therefore affect even non-dividing ovarian follicles [35;36]. It has also been suggested that ovarian exposure to cyclophosphamide may trigger dormant follicle activation, resulting in burnout of the follicular reserve [15;37].

As pre-pubertal girls have more primordial follicles than adults, it has been assumed that their follicle cohort has a higher maturation potential and better survival capacity after cancer treatment [38;39]. However, this hypothesis has been questioned after a recent in vitro study suggesting that there may be key differences in follicular recruitment and development between the ovaries of young girls and adults. The capacity of primordial follicles to be activated and reach the secondary stage of development may be age-dependent [40]. The present results confirm decreased in vitro viability and development of ovarian follicles from very young girls. Increased age correlated with fewer follicles, more developing and fewer atretic follicles after culture. A study on mouse ovaries revealed two classes of primordial follicles; the first wave, which are activated immediately after they are formed, and adult primordial follicles, which are activated gradually during reproductive life [41]. These two primordial follicle pools have been shown to differ in their developmental dynamics and location in the maturing ovary [42]. Our results may indicate that a similar situation exists in humans. A decreased rate of activation and development of follicles in ovaries from young girls may reflect compromised developmental competence in vitro. Presently no international consensus exists on the age at which reproductive potential is actually reached making it unclear how recommendations for fertility preservation can be effectively applied to cancer patients younger than 18 years old [1].

The present study population was small and heterogeneous with a significant difference in the median age of patients who underwent chemotherapy versus those who did not. Ideally, the effect of chemotherapy should have been studied in groups of comparable age and compared to age-matched control tissue. This was not possible because of the clinical nature of the samples available. The fact that first-line therapy seldom associates with subfertility among pre-pubertal girls delays the decision of fertility preservation. Childhood cancers also require prompt initiation of cancer therapy often before ovarian biopsy. For ethical reasons, it was not possible to collect age-matched tissue from healthy young girls. The healthy control ovarian tissue cultures were from few adult samples. Their performance in in vitro conditions was comparable with those in previous [6;13;25]. In order to decrease the effect of skewed study populations, the entire study material was included in multivariate analysis to adjust for differences in age, use of cryoprotectant agents and exposure to CED. The analysis identified CED and age as independent predictors of follicle survival. No independent effect on viability of the follicles was found to be associated with the use of either PrOH or EG as cryoprotectants during slow freezing. This is in agreement with the results of previous studies [26;43].

In conclusion, the results of this study demonstrate that exposure to an increased cumulative dose of alkylating agents prior to ovarian cryopreservation decreases survival of cultured human ovarian follicles. Therefore, if possible, fertility preservation should be carried out before initiation of chemotherapy. The findings further confirm that the capacity of ovarian follicles to survive and develop in culture may be reduced among young girls. The results of the present study can have implications for future implementation, timing and quality control of fertility-preservation methods.


We would like to thank Prof. Leo Dunkel and Dr. Anne Wikström for their contribution in initiating the fertility preservation study at Helsinki University Central Hospital, Taija Poikkipuoli and Erica Nyman for skillful technical assistance and Björn Jonsson for help with statistical analysis.

Author Contributions

Conceived and designed the experiments: KJ ICO OH. Performed the experiments: BA MS. Analyzed the data: BA ICO KJ. Contributed reagents/materials/analysis tools: MN TL HT SM TGT. Wrote the paper: BA ICO OH KJ.


  1. 1. Donnez J, Dolmans MM: Fertility preservation in women. Nat Rev Endocrinol 2013;9:735–749. pmid:24166000
  2. 2. Oktay K, Oktem O: Fertility preservation medicine: a new field in the care of young cancer survivors. Pediatr Blood Cancer 2009;53:267–273. pmid:19301406
  3. 3. Kim SS, Donnez J, Barri P, Pellicer A, Patrizio P, Rosenwaks Z, et al. Recommendations for fertility preservation in patients with lymphoma, leukemia, and breast cancer. J Assist Reprod Genet 2012;29:465–468. pmid:22648282
  4. 4. Rosendahl M, Greve T, Andersen CY: The safety of transplanting cryopreserved ovarian tissue in cancer patients: a review of the literature. J Assist Reprod Genet 2013;30:11–24. pmid:23263841
  5. 5. Bastings L, Beerendonk CC, Westphal JR, Massuger LF, Kaal SE, van Leeuwen FE, et al. Autotransplantation of cryopreserved ovarian tissue in cancer survivors and the risk of reintroducing malignancy: a systematic review. Hum Reprod Update 2013;19:483–506. pmid:23817363
  6. 6. Carlsson IB, Scott JE, Visser JA, Ritvos O, Themmen AP, Hovatta O: Anti-Mullerian hormone inhibits initiation of growth of human primordial ovarian follicles in vitro. Hum Reprod 2006;21:2223–2227. pmid:16720622
  7. 7. Carlsson IB, Laitinen MP, Scott JE, Louhio H, Velentzis L, Tuuri T, et al. Kit ligand and c-Kit are expressed during early human ovarian follicular development and their interaction is required for the survival of follicles in long-term culture. Reproduction 2006;131:641–649. pmid:16595715
  8. 8. Xu M, Barrett SL, West-Farrell E, Kondapalli LA, Kiesewetter SE, Shea LD, et al. In vitro grown human ovarian follicles from cancer patients support oocyte growth. Hum Reprod 2009;24:2531–2540. pmid:19597190
  9. 9. Telfer EE, McLaughlin M, Ding C, Thong KJ: A two-step serum-free culture system supports development of human oocytes from primordial follicles in the presence of activin. Hum Reprod 2008;23:1151–1158. pmid:18326514
  10. 10. Eppig JJ, O'Brien MJ: Development in vitro of mouse oocytes from primordial follicles. Biol Reprod 1996;54:197–207. pmid:8838017
  11. 11. O'Brien MJ, Pendola JK, Eppig JJ: A revised protocol for in vitro development of mouse oocytes from primordial follicles dramatically improves their developmental competence. Biol Reprod 2003;68:1682–1686. pmid:12606400
  12. 12. Adhikari D, Gorre N, Risal S, Zhao Z, Zhang H, Shen Y, et al. The safe use of a PTEN inhibitor for the activation of dormant mouse primordial follicles and generation of fertilizable eggs. PLoS One 2012;7:e39034. pmid:22761722
  13. 13. Sheikhi M, Hultenby K, Niklasson B, Lundqvist M, Hovatta O: Preservation of human ovarian follicles within tissue frozen by vitrification in a xeno-free closed system using only ethylene glycol as a permeating cryoprotectant. Fertil Steril 2013;100:170–177. pmid:23566598
  14. 14. McLaughlin M, Kinnell HL, Anderson RA, Telfer EE: Inhibition of phosphatase and tensin homologue (PTEN) in human ovary in vitro results in increased activation of primordial follicles but compromises development of growing follicles. Mol Hum Reprod 2014;20:736–744. pmid:24830779
  15. 15. Meirow D, Biederman H, Anderson RA, Wallace WH: Toxicity of chemotherapy and radiation on female reproduction. Clin Obstet Gynecol 2010;53:727–739. pmid:21048440
  16. 16. Sklar CA, Mertens AC, Mitby P, Whitton J, Stovall M, Kasper C, et al. Premature menopause in survivors of childhood cancer: a report from the childhood cancer survivor study. J Natl Cancer Inst 2006;98:890–896. pmid:16818852
  17. 17. Jahnukainen K, Tinkanen H, Wikstrom A, Dunkel L, Saarinen-Pihkala UM, Makinen S, et al. Bone marrow remission status predicts leukemia contamination in ovarian biopsies collected for fertility preservation. Leukemia 2013;27:1183–1185. pmid:23079961
  18. 18. Meirow D, Hardan I, Dor J, Fridman E, Elizur S, Ra'anani H, Slyusarevsky E, et al. Searching for evidence of disease and malignant cell contamination in ovarian tissue stored from hematologic cancer patients. Hum Reprod 2008;23:1007–1013. pmid:18344563
  19. 19. Schmidt KT, Nyboe AA, Greve T, Ernst E, Loft A, Yding AC: Fertility in cancer patients after cryopreservation of one ovary. Reprod Biomed Online 2013;26:272–279. pmid:23352102
  20. 20. Green DM, Nolan VG, Goodman PJ, Whitton JA, Srivastava D, Leisenring WM, et al. The cyclophosphamide equivalent dose as an approach for quantifying alkylating agent exposure: A report from the childhood cancer survivor study. Pediatr Blood Cancer 2014;61:53–67. pmid:23940101
  21. 21. Hovatta O, Silye R, Krausz T, Abir R, Margara R, Trew G, et al. Cryopreservation of human ovarian tissue using dimethylsulphoxide and propanediol-sucrose as cryoprotectants. Hum Reprod 1996;11:1268–1272. pmid:8671438
  22. 22. Schmidt KL, Byskov AG, Nyboe AA, Muller J, Yding AC: Density and distribution of primordial follicles in single pieces of cortex from 21 patients and in individual pieces of cortex from three entire human ovaries. Hum Reprod 2003;18:1158–1164. pmid:12773440
  23. 23. Tao T, Del VA: Human oocyte and ovarian tissue cryopreservation and its application. J Assist Reprod Genet 2008;25:287–296. pmid:18670872
  24. 24. Oskam IC, Asadi BA, Santos RR: Histologic and ultrastructural features of cryopreserved ovine ovarian tissue: deleterious effect of 1,2-propanediol applying different thawing protocols. Fertil Steril 2010;93:2764–2766. pmid:20236634
  25. 25. Scott JE, Carlsson IB, Bavister BD, Hovatta O: Human ovarian tissue cultures: extracellular matrix composition, coating density and tissue dimensions. Reprod Biomed Online 2004;9:287–293. pmid:15353079
  26. 26. Keros V, Xella S, Hultenby K, Pettersson K, Sheikhi M, Volpe A, et al. Vitrification versus controlled-rate freezing in cryopreservation of human ovarian tissue. Hum Reprod 2009;24:1670–1683. pmid:19359339
  27. 27. Gougeon A: Some aspects of the dynamics of ovarian follicular growth in the human. Acta Eur Fertil 1989;20:185–192. pmid:2519570
  28. 28. Lass A, Skull J, McVeigh E, Margara R, Winston RM: Measurement of ovarian volume by transvaginal sonography before ovulation induction with human menopausal gonadotrophin for in-vitro fertilization can predict poor response. Hum Reprod 1997;12:294–297. pmid:9070714
  29. 29. Meirow D, Nugent D: The effects of radiotherapy and chemotherapy on female reproduction. Hum Reprod Update 2001;7:535–543. pmid:11727861
  30. 30. McDermott EM, Powell RJ: Incidence of ovarian failure in systemic lupus erythematosus after treatment with pulse cyclophosphamide. Ann Rheum Dis 1996;55:224–229. pmid:8733438
  31. 31. Oktem O, Oktay K: A novel ovarian xenografting model to characterize the impact of chemotherapy agents on human primordial follicle reserve. Cancer Res 2007;67:10159–10162. pmid:17974956
  32. 32. Oktem O, Oktay K: Quantitative assessment of the impact of chemotherapy on ovarian follicle reserve and stromal function. Cancer 2007;110:2222–2229. pmid:17932880
  33. 33. Hales BF: Comparison of the mutagenicity and teratogenicity of cyclophosphamide and its active metabolites, 4-hydroxycyclophosphamide, phosphoramide mustard, and acrolein. Cancer Res 1982;42:3016–3021. pmid:7046914
  34. 34. Rosendahl M, Andersen CY, la Cour FN, Juul A, Lossl K, Andersen AN: Dynamics and mechanisms of chemotherapy-induced ovarian follicular depletion in women of fertile age. Fertil Steril 2010;94:156–166. pmid:19342041
  35. 35. Perez GI, Maravei DV, Trbovich AM, Cidlowski JA, Tilly JL, Hughes FM Jr.: Identification of potassium-dependent and-independent components of the apoptotic machinery in mouse ovarian germ cells and granulosa cells. Biol Reprod 2000;63:1358–1369. pmid:11058539
  36. 36. Kerr JB, Hutt KJ, Michalak EM, Cook M, Vandenberg CJ, Liew SH, et al. DNA damage-induced primordial follicle oocyte apoptosis and loss of fertility require TAp63-mediated induction of Puma and Noxa. Mol Cell 2012;48:343–352. pmid:23000175
  37. 37. Haie-Meder C, Mlika-Cabanne N, Michel G, Briot E, Gerbaulet A, Lhomme C, et al. Radiotherapy after ovarian transposition: ovarian function and fertility preservation. Int J Radiat Oncol Biol Phys 1993;25:419–424. pmid:8436519
  38. 38. Sanders JE, Hawley J, Levy W, Gooley T, Buckner CD, Deeg HJ, et al. Pregnancies following high-dose cyclophosphamide with or without high-dose busulfan or total-body irradiation and bone marrow transplantation. Blood 1996;87:3045–3052. pmid:8639928
  39. 39. Jadoul P, Dolmans MM, Donnez J: Fertility preservation in girls during childhood: is it feasible, efficient and safe and to whom should it be proposed? Hum Reprod Update 2010;16:617–630. pmid:20462941
  40. 40. Anderson RA, McLaughlin M, Wallace WH, Albertini DF, Telfer EE: The immature human ovary shows loss of abnormal follicles and increasing follicle developmental competence through childhood and adolescence. Hum Reprod 2014;29:97–106. pmid:24135076
  41. 41. Zheng W, Zhang H, Liu K: The two classes of primordial follicles in the mouse ovary: their development, physiological functions and implications for future research. Mol Hum Reprod 2014;20:286–292. pmid:24448914
  42. 42. Zheng W, Zhang H, Gorre N, Risal S, Shen Y, Liu K: Two classes of ovarian primordial follicles exhibit distinct developmental dynamics and physiological functions. Hum Mol Genet 2014;23:920–928. pmid:24087793
  43. 43. Newton H, Aubard Y, Rutherford A, Sharma V, Gosden R: Low temperature storage and grafting of human ovarian tissue. Hum Reprod 1996;11:1487–1491. pmid:8671490