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ORIGINAL ARTICLE
Year : 2014  |  Volume : 1  |  Issue : 3  |  Page : 148-152

Ovarian tissue vitrification as a method for fertility preservation: A study of follicle number and morphology after vitrification


1 Department of Obstetrics and Gynecology, University of Indonesia, Cipto Mangunkusumo National Hospital, Jakarta, Indonesia
2 Department of Histology, University of Indonesia, Cipto Mangunkusumo National Hospital, Jakarta, Indonesia
3 Department of Anatomy and Physiology, Institute of Veterinary and Agricultural, Bogor, Indonesia

Date of Web Publication7-Oct-2014

Correspondence Address:
Budi Wiweko
Research Manager, Faculty of Medicine Universitas Indonesia, Jakarta
Indonesia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2348-2907.142328

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  Abstract 

Purpose: The aim was to evaluate the effects of ovarian cortex vitrification on follicle number and morphology. Materials and Methods: We excised approximately 5 mm of ovarian tissue from one ovary of each of 10 reproductive aged women who underwent surgery for the removal of benign ovarian tumors. A cryoprotectant agent containing 7.5% dimethylsulphoxide and 7.5% ethylene glycol was added to the ovarian cortex. Post-vitrification ovarian cortex was placed in a Cryotissue device and frozen in liquid nitrogen. We evaluated the shape and number of the follicles as well as the granulosa cell layers. The quality of the follicles was evaluated based on the morphological integrity of the oocytes, granulosa cells, and basal membranes as determined by hematoxylin and eosin staining. Results: The median age of the patients was 33 years (range: 28-40 years). The mean number of follicles per patient was 17 ± 7. After thawing, 59 primordial follicles (35.3%), 45 (26.9%) primary follicles and 6 (3.5%) secondary follicles were retrieved. The remaining follicles (34.1%) were atretic. There was a significant relationship between number of follicles and age (P = 0.012, r = −0751). No differences were found in the morphology of the granulosa cells, stroma or collagen in both groups. The number of primordial follicles, primary follicles, secondary follicles, and follicular atresia were not significantly different before and after vitrification. Conclusion: In our study, vitrification does not cause significant changes to the morphology and number of follicles.

Keywords: Follicle, granulosa cells, morphology, ovarian follicle, vitrification.


How to cite this article:
Wiweko B, Maidarti M, Mansyur E, Yuningsih T, Ahmad A, Boediono A, Soebijanto S, Affandi B. Ovarian tissue vitrification as a method for fertility preservation: A study of follicle number and morphology after vitrification. IVF Lite 2014;1:148-52

How to cite this URL:
Wiweko B, Maidarti M, Mansyur E, Yuningsih T, Ahmad A, Boediono A, Soebijanto S, Affandi B. Ovarian tissue vitrification as a method for fertility preservation: A study of follicle number and morphology after vitrification. IVF Lite [serial online] 2014 [cited 2021 Jan 21];1:148-52. Available from: http://www.ivflite.org/text.asp?2014/1/3/148/142328


  Introduction Top


With advances in oncologic treatment, the 5-year survival rate of cancer patients is currently approximately 90%.[1] However, these treatments are associated with premature ovarian failure.[2] Attempts to preserve fertility and ovarian function are, therefore, important, especially in women of reproductive age.[3],[4] One of the most commonly used chemotherapeutic agents is cyclophosphamide. Depending on the age of the patient, and the total dose of cyclophosphamide delivered, ovarian failure is observed in 12-83% of cyclophosphamide-treated cases.[5]

To preserve ovarian function, oocytes, embryos or ovarian tissue can be cryopreserved. [6] In general, oocyte cryopreservation is limited by the number of mature oocytes that can be obtained after one cycle of ovarian stimulation, thus limiting its efficacy (Stoop, 2012). [21] The time required for stimulation could also delay the initiation of the main oncologic treatment, especially for chemotherapy procedures.

The cryopreservation of ovarian tissue has several potential advantages over other preservation techniques, including the presence of a large number of follicles in the cortex. In addition, this tissue will provide ovarian hormones after a successful ovarian transplantation. [19] Ovarian tissue freezing can be performed readily because it is a single surgical procedure that does not require a pretreatment period, and this tissue can be obtained anytime, independent of the phase of the menstrual cycle. In addition, primordial follicles are less cryosensitive than mature oocytes. [7]

There are two types of cryopreservation, the slow cooling method and vitrification. [8] Vitrification is superior to slow cooling because it is not associated with the formation of intracellular ice crystals. [9] In contrast to the increased apoptosis observed in response to the slow cooling method, [10] several investigators found that ovarian vitrification had no effect on the follicle structure or apoptosis. [11],[12] However, Mazoochi et al. demonstrated differences in fatty acid synthetase and survivin gene expression between vitrified tissues and controls. [11] Apoptosis can be assessed by evaluating the morphology and histology of the follicles, gene expression/DNA, and cell counting. [13]

Until date, (at least in our country) no standard technique of ovarian cryopreservation has been developed. [8] The purpose of our study was therefore to evaluate the effects of ovarian cortex vitrification on follicle number and morphology.


  Materials and Methods Top


The study was conducted at the Department of Obstetrics and Gynecology, Dr. Cipto Mangunkusumo Hospital, Faculty of Medicine, University of Indonesia, from January 2011 to May 2011. We excised approximately 5 mm of ovarian tissue from the ovaries of 10 reproductive aged women who underwent surgery for the removal of benign ovarian tumors. The Ethics Committee of the University of Indonesia and Cipto Mangunkusumo Hospital approved the study. Patients with ovarian cancer or a history of chemotherapy or radiotherapy were excluded.

Vitrification and thawing

Immediately after ovarian excision, the ovarian tissues were soaked in normal saline solution and transported to the laboratory, where the tissues were washed with phosphate-buffered saline solution. [13] The vitrification procedure was performed as previously described by Kagawa et al. [13],[14] Briefly, tissues were incubated in medium containing 7.5% ethylene glycol (EG) and 7.5% dimethylsulfoxide (DMSO) (Kitazato, Japan) for 25 min at room temperature, and then incubation in medium containing 20% EG, 20% DMSO and 0.5 mol/l sucrose (Kitazato, Japan) for 15 min. The ovarian cortex was inserted into a special device (Cryotissue, Kitazato, Japan) for freezing in liquid nitrogen. [14]

The tissues were kept frozen for 1-3 months before thawing. After removal from liquid nitrogen, the tissues were submerged in 40 ml of medium containing 1.0 mol/l of sucrose (Kitazato, Japan) for 1 min at 37°C, followed by incubation in 15 ml of medium containing 0.5 mol/l of sucrose for 5 min at room temperature. They were then rinsed twice in HEPES-buffered TCM-199 solution supplemented with 20% synthetic serum substitute (SSS: Irvine Scientific, Santa Anna, USA). [13]

Histological analysis

The tissues were fixed in 10% buffered formalin, embedded in paraffin wax, serially sectioned at 5 mm, stained with hematoxylin and eosin, and analyzed under a light microscope (Olympus, Tokyo, Japan) at × 100 and × 400 magnification. We evaluated the shape and number of the follicles and the granulosa cell layers. Follicle quality was evaluated based on the morphological integrity of the oocyte, granulosa cells, and basal membrane.

We defined histologically normal follicles as follicles containing an oocyte with intact granulosa cells. Degenerated follicles contained an oocyte with a pycnotic nucleus, reduced ooplasm or irregular granulosa cells, including large follicles or follicles that had come loose from the basal membrane).

Statistical analysis

For continuous variables, inferential testing was conducted using the Student's t-test for normally distributed data. For non-normally distributed data, the Mann-Whitney test was used. Proportions were compared using the Chi-Square or Fisher's exact test, when appropriate. The relationship between the number of follicles and patient age was evaluated using Spearman's correlation test. Differences were considered as statistically significant if alpha was <0.05. Statistical analysis was per  formed in SPSS 17.0 software (Chicago, SPSS Inc.).


  Results Top


The median patient age was 33 years (range: 28-40 years). We obtained and analyzed 167 follicles. The mean number of follicles per patient was 17 ± 7. [Table 1] shows the number of follicles based on the patient characteristics before and after vitrification. A total of 59 primordial follicles (35.3%), 45 (26.9%) primary follicles and 6 (3.5%) secondary follicles were retrieved after thawing. The remaining follicles (34.1%) were atretic. There was a significant relationship between follicle number and age (P = 0.012 r = -0751; [Figure 1]). No differences were found in the morphology of the granulosa cells, stroma and collagen before and after vitrification [Figure 2], [Figure 3], [Figure 4]. Similarly, the number of primordial follicles, primary follicles, secondary follicles and follicular atresia before and after vitrification were comparable (P ≥ 0.05) [Table 2].
Figure 1: The relationship between follicle number and age

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Figure 2: The morphology of ovarian tissue before and after vitrification. (a-c) The primordial follicle (red arrowhead) and primary follicles (blue arrow heads) in fresh ovarian tissue. (d-f) Primary follicles in vitrified ovarian tissue. (e) Ovarian tissue post-vitrification. The secondary follicle (black arrow) contains an intact oocyte and neatly arranged granulosa cells (H and E, ×100)

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Figure 3: Examples of ovarian cortex (H and E, ×100). (a) Before vitrification, a healthy primary follicle (white arrow) and an abnormal follicle with irregularly arranged granulose cells or reduced cytoplasm (black arrow). (b) A primary follicle in vitrified ovarian tissue

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Figure 4: Ovarian tissue before (a) and after vitrification (b), demonstrating a primordial follicle with intact oocytes surrounded by a layer of flattened cells (granulosa cells)

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Table 1: Number of follicles before and after vitrification based on patient characteristics

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Table 2: Comparision of the number of follicles before and after vitrification

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  Discussion Top


In this study, we found that there were no statistically significant differences in the morphology of the granulosa cells, stroma and collagen of ovarian tissue before and after vitrification. The number of primordial follicles, primary follicles, secondary follicles and follicular atresia was also comparable in the before and after vitrification groups (P ≥ 0.05).

As expected, we found a decrease in the number of follicles with advancing age. Fabbri [15] previously demonstrated that the number of follicles per female varies and is related to patient age and to the geometry of ovarian follicular distribution in the ovarian cortex. [15] The number of follicles also depends on the sampling technique and the selection of the ovarian cortical surface. [16]

Ovarian tissue can be collected by laparoscopy at any time, independent of the stage of the menstrual cycle. [7] We found that the morphology of ovarian tissue was similar before and after vitrification. Donnez et al. [3] previously demonstrated that primordial follicles in the ovarian cortex were resistant to the vitrification process (2010). Indeed, primordial follicles are less cryosensitive than mature oocytes. This may be due to their smaller size, underdevelopment, and metabolic arrest during development and differentiation. [3]

In our study, we did not evaluate the occurrence of apoptosis. However, morphological changes in the mitochondria of the primary follicles after vitrification have been reported. [16] Mitochondrial volume is associated with the integrity of organelles and cells, and mitochondrial swelling is also one of the key events in cytochrome c release, which is associated with apoptotic cell death (Kaasik, 2007). [22] It also appears that mitochondrial volume is related to increased oxidative stress. [16] On the other hand, a few authors have reported that apoptosis was not a predominant factor in cell death related to cryopreservation, either in primordial follicles or primary follicles. [3] The majority of our patients had endometriosis. However, as previously reported, the presence of ovarian endometrioma is not associated with a reduced number of oocytes retrieved from the affected ovary during in vitro fertilization treatment. [17]

Navarro-Costa et al. [18] previously reported that the most severe damage to frozen ovarian tissue was observed in the stromal cells while the follicles were more resistant to the freeze-thawing process. Stromal cells are not only smaller in size but also subjected to faster warming and dehydration compared with granulosa cells and oocytes. Furthermore, high concentrations of cryoprotectant could cause excessive dehydration, leading to cell membrane damage during the rehydration phase. [18]

Our study has several limitations, namely the small number of cases included and that we did not evaluate the viability or the apoptotic activity of the follicle. Although apoptosis plays a role in maintaining tissue homeostasis, some studies observed no significant increase in apoptotic activity in cultured ovarian tissue. [20] Further research into this issue is still needed.

The results of our study suggest that vitrification does not cause significant changes to follicle morphology or number. Further studies to examine the function of the follicle and the levels of apoptosis after cryopreservation are needed.

 
  References Top

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Chang HJ, Suh CS. Fertility preservation for women with malignancies: Current developments of cryopreservation. J Gynecol Oncol 2008;19:99-107.  Back to cited text no. 1
    
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Donnez J, Jadoul P, Donnez O, Eyck AS, Squifflet J, Dolmans MM. Cryopreservation of ovarian tissue: An overview. In: Chian RC, Quinn P, editors. Fertility Cryopreservation. 1 st ed. Philadephia: Cambridge University Press; 2010. p. 189-99.  Back to cited text no. 3
    
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Vajta G, Nagy ZP. Are programmable freezers still needed in the embryo laboratory? Review on vitrification. Reprod Biomed Online 2006;12:779-96.  Back to cited text no. 9
    
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Mazoochi T, Salehnia M, Valojerdi MR, Mowla SJ. Morphologic, ultrastructural, and biochemical identification of apoptosis in vitrified-warmed mouse ovarian tissue. Fertil Steril 2008;90:1480-6.  Back to cited text no. 11
    
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Mazoochi T, Salehnia M, Pourbeiranvand S, Forouzandeh M, Mowla SJ, Hajizadeh E. Analysis of apoptosis and expression of genes related to apoptosis in cultures of follicles derived from vitrified and non-vitrified ovaries. Mol Hum Reprod 2009;15:155-64.  Back to cited text no. 12
    
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Demirci B, Salle B, Frappart L, Franck M, Guerin JF, Lornage J. Morphological alterations and DNA fragmentation in oocytes from primordial and primary follicles after freezing-thawing of ovarian cortex in sheep. Fertil Steril 2002;77:595-600.  Back to cited text no. 13
    
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Kagawa N, Silber S, Kuwayama M. Successful vitrification of bovine and human ovarian tissue. Reprod Biomed Online 2009;18:568-77.  Back to cited text no. 14
    
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Fabbri R. Cryopreservation of human oocytes and ovarian tissue. Cell Tissue Bank 2006;7:113-22.  Back to cited text no. 15
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Wang LH, Mullen SF, Li Y, Zhong JQ, Crister JK, Chen ZJ. Morphological and apoptotic comparison of primordial and primary follicles in cryopreserved human ovarian tissue. Reprod Domest Anim 2009;44:879-83.  Back to cited text no. 16
    
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Navarro-Costa P, Correia SC, Gouveia-Oliveira A, Negreiro F, Jorge S, Cidadão AJ, et al. Effects of mouse ovarian tissue cryopreservation on granulosa cell-oocyte interaction. Hum Reprod 2005;20:1607-14.  Back to cited text no. 18
    
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Siebzehnrubl E, Kohl J, Dittrich R, Wildt L. Freezing of human ovarian tissue-not the oocytes but the granulosa is the problem. Mol Cell Endocrinol 2000;169:109-111.  Back to cited text no. 19
    
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Blumenfeld Z. Preservation of fertility and ovarian function and minimalization of chemotherapy associated gonadotoxicity and premature ovarian failure: The role of inhibin-A and -B as markers. Mol Cell Endocrinol 2002;187:93-105.  Back to cited text no. 20
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21.
Stoop D, De Vos M, Tournaye H, Devroey P. Fertility preservation utilizing controlled ovarian hyperstimulation and oocyte cryopreservation in a premenarcheal female with myelodysplastic syndrome. Fertil Steril 2012;98:1121-2.  Back to cited text no. 21
    
22.
Kaasik A, Safiulina D, Zharkovsky A, Veksler V. Regulation of mitochondrial matrix volume. Am J Physiol Cell Physiol 2007;292.  Back to cited text no. 22
    

 
  Authors Top


Dr. Budi Wiweko, MD, OG (REI) is a Research Manager of Faculty of Medicine Universitas Indonesia and also a Supporting Manager for Yasmin IVF Clinic - Jakarta, Indonesia. After graduated from Universitas Indonesia in 2005, he spent his time as a research fellow on ovarian tissue vitrification and in vitro culture of follicles at Hyogo College of Medicine Japan. He continued his research on basic laboratory on ART and had some clinical IVF training in Osaka, Barcelona, Thailand and Vietnam. He defensed his PhD thesis on "Pre-antral follicle vitrification" in Faculty of Medicine Universitas Indonesia in 2014. Besides as a General Secretary, now he is The President Elect of Indonesian Association for IVF (IA-IVF) for 2016-2020 and a member of advisory board of Merck Serono Asia Pacific. He is also a secretary of Indonesian Society for Reproductive Endocrinology and Infertility (HIFERI) and Indonesian Society for Obstetrics and Gynecology (POGI-JAYA). He is very active in a lot of regional organization such as Asia Pasific Iniative on Reproduction (ASPIRE) as a member of executive board, Pacific Rim Fertility Society (PRFS), European Society for Human Reproduction and Embryology (ESHRE), International Federation of Fertility Society (IFFS), American Society for Reproductive Medicine (ASRM), International Society for Fertility Preservation (ISFP) and American Association for Gynecology Laparoscopy (AAGL). Now he is trying to develop ovarian tissue cryopreservation in Indonesia.


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2]


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