DNA damage and primordial follicle activation after in vitro culture of sheep ovarian cortex in Morus nigra leaf extract

ABSTRACT: This study evaluated the effect of Morus nigra leaf extract, with or without supplementation, on morphology, activation and DNA damage of preantral follicles cultured within sheep ovarian tissue. Ovaries were collected and divided into fragments, being one fixed for histological and Terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labeling (TUNEL) analysis (fresh control). The remaining fragments were cultured for 7 days in alpha minimum essential media (α-MEM) supplemented with bovine serum albumin (BSA), insulin, transferrin, selenium, glutamine, hypoxanthine and ascorbic acid (α-MEM+; control medium) or into medium composed of M. nigra extract without supplements (0.1; 0.2 or 0.4mg/mL) or supplemented with the same substances described above for α-MEM+ (MN 0.1+; 0.2+ or 0.4+mg/mL). Then, tissues were destined to histological and TUNEL analysis. The α-MEM+ treatment had more morphologically normal follicles than all M. nigra extract treatments. However, α-MEM+ treatment also showed signs of atresia because the percentage of TUNEL positive cells was similar in α-MEM+ and in 0.1mg/mL M. nigra without and with supplements. Moreover, a reduction in the primordial follicles and an increase in the growing ones were observed in all treatments, except 0.2mg/mL M. nigra. In conclusion, the follicles cultured at 0.1mg/mL M. nigra extract were in good condition and able to continue their development, as demonstrated by the same rates of DNA damage and follicular activation as the control medium.

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The activin-follistatin system and in vitro early follicle development in goats

Journal of Endocrinology ◽

10.1677/joe.1.06487 ◽

2006 ◽

Vol 189 (1) ◽

pp. 113-125 ◽

Cited By ~ 35

Author(s):

J R V Silva ◽

T Tharasanit ◽

M A M Taverne ◽

G C van der Weijden ◽

R R Santos ◽

...

Keyword(s):

Ovarian Tissue ◽

Primordial Follicle ◽

Activin A ◽

Terminal Deoxynucleotidyl Transferase ◽

Follicle Development ◽

Cortical Tissue ◽

Primary Follicle ◽

Growth And Survival ◽

Primordial Follicles

The aim of the present study was to investigate the effects of activin-A and follistatin on in vitro primordial and primary follicle development in goats. To study primordial follicle development (experiment 1), pieces of ovarian cortex were cultured in vitro for 5 days in minimal essential medium (MEM) supplemented with activin-A (0, 10 or 100 ng/ml), follistatin (0, 10 or 100 ng/ml) or combinations of the two. After culture, the numbers of primordial follicles and more advanced follicle stages were calculated and compared with those in non-cultured tissue. Protein and mRNA expression of activin-A, follistatin, Kit ligand (KL), growth differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15) in non-cultured and cultured follicles were studied by immunohistochemistry and PCR. To evaluate primary follicle growth (experiment 2), freshly isolated follicles were cultured for 6 days in MEM plus 100 ng/ml activin-A, 100 ng/ml follistatin or 100 ng/ml activin-A plus 200 ng/ml follistatin. Morphology, follicle and oocyte diameters in cultured tissue and isolated follicles before and after culture were assessed. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) reactions were performed to study DNA fragmentation in follicles. In experiment 1, it was found that goat primordial follicles were activated to develop into more advanced stages, i.e. intermediate and primary follicles, during in vitro culture, but neither activin-A nor follistatin affected the number of primordial follicles that entered the growth phase. Activin-A treatment enhanced the number of morphologically normal follicles and stimulated their growth during cortical tissue culture. The effects were, however, not counteracted by follistatin. The follicles in cultured goat tissue maintained their expression of proteins and mRNA for activin-A, follistatin, KL, GDF-9 and BMP-15. Fewer than 30% of the atretic follicles in cultured cortical tissue had TUNEL-positive (oocyte or granulosa) cells. Activin-A did not affect the occurrence of TUNEL-positive cells in follicles within cortical tissue. In experiment 2, addition of activin-A to cultured isolated primary follicles significantly stimulated their growth, the effect being counteracted by follistatin. Absence of such a neutralizing effect of follistatin in the cultures with ovarian cortical tissue can be due to lower dose of follistatin used and incomplete blockage of activin in these experiments. In contrast to cortical enclosed atretic follicles, all atretic follicles that had arisen in cultures with isolated primary follicles had TUNEL-positive cells, which points to differences between isolated and ovarian tissue-enclosed follicles with regard to the followed pathways leading to their degeneration. In summary, this in vitro study has demonstrated that cultured goat primordial follicles are activated to grow and develop into intermediate and primary follicles. During in vitro culture, the follicles maintain their ability to express activin-A, follistatin, KL, GDF-9 and BMP-15. The in vitro growth and survival of activated follicles enclosed in cortical tissue and the in vitro growth of isolated primary follicles are stimulated by activin-A.

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In-vitro fragmentation of ovarian tissue activates primordial follicles through the Hippo pathway

Human Reproduction Open ◽

10.1093/hropen/hoaa048 ◽

2020 ◽

Vol 2020 (4) ◽

Author(s):

C De Roo ◽

S Lierman ◽

K Tilleman ◽

P De Sutter

Keyword(s):

Tissue Culture ◽

Ovarian Tissue ◽

Hippo Pathway ◽

Primordial Follicle ◽

Akt Pathway ◽

Primordial Follicles ◽

Primordial Follicle Activation ◽

The Hippo Pathway ◽

Follicle Activation

Abstract STUDY QUESTION What is the role of the Hippo and PI3K/Akt pathway in follicles during ovarian tissue culture in tissue derived from oncological patients and transgender men? SUMMARY ANSWER Results highlight a Hippo pathway driven primordial follicle activation in vitro, predominantly from Day 0 to Day 4. WHAT IS KNOWN ALREADY In-vitro ovarian tissue culture aims at activating and maturing primordial follicles for fertility restoration in patients with a threatened ovarian reserve. Not all patients are eligible for ovarian cortex transplantation and therefore several groups are attempting to culture ovarian tissue in-vitro. Cortex fragmentation disrupts the Hippo pathway, leading to increased expression of downstream growth factors and follicle growth. The PI3K/Akt pathway is considered the intracellular pathway to where different extracellular factors involved in primordial follicle activation in-vivo converge. In order to optimise current ovarian tissue culture models, information on progression of these pathways during tissue culture is mandatory. STUDY DESIGN, SIZE, DURATION The first step of a multistep cortex culture system was performed using 144 ovarian cortex pieces from a total of six patients. Per patient, 24 cortical strips were cultured for 6 days and six pieces per patient were collected for downstream analysis of follicle development and Hippo and PI3K/Akt pathway targets every second day. PARTICIPANTS/MATERIALS, SETTING, METHODS Ovarian tissue was obtained from oncological (N = 3; 28.67 ± 4.51 years) and transgender (N = 3; 23.33 ± 1.53 years) patients. Follicles were analysed using haematoxylin-eosin staining and pathways were studied using immunohistochemistry and precise follicle excision by laser capture micro-dissection for RT-qPCR analysis. MIQE guidelines for RT-qPCR were pursued. Reference gene selection (GAPDH, RPL3A, 18s rRNA) was performed using GeNorm Reference Gene Selection Kit. Statistical analysis was conducted with IBM SPSS Statistics 23 (Poisson regression, negative binomial regression, ANOVA and paired t-test). MAIN RESULTS AND THE ROLE OF CHANCE Immunohistochemical analysis confirmed a Hippo pathway driven primordial follicle activation due to mechanical manipulation of the cortical strips. Ovarian tissue preparation and culture induced the inhibitory phosphorylated Yes-associated protein (pYAP) to disappear in granulosa cells of primordial follicles on Day 2. The stimulatory YAP on the contrary appeared in primordial granulosa cells over increasing culture days. Looking at the YAP target connective tissue growth factor (CTGF), a significantly up-regulated CTGF was noted in primordial follicles when comparing Day 2 and Day 4 (ratio Day 2/4 = 0.082; P < 0.05), clearly showing an effect on the Hippo pathway in primordial follicles during tissue culture. Follicle classification showed a significant drop in estimated primordial follicle counts in the oncological cohort (−78%; P = 0.021) on Day 2 and in the transgender cohort on Day 4 (−634%; P = 0.008). Intermediate follicle counts showed a non-significant increasing trend to during culture and this follicle recruitment and growth resulted in a significant rise in estimated primary follicle counts on Day 6 in oncological patients (170%; P = 0.025) and, although limited in absolute numbers, a significant increase in secondary follicles on Day 4 (367%; P = 0.021) in the transgender cohort. Subsequent antral follicle development could not be observed. LIMITATIONS, REASONS FOR CAUTION A limitation is the small sample size, inherent to this study subject, especially as a large amount of tissue was needed per patient to reduce inter-patient variation in different downstream analysis techniques. A particular and specific weakness of this study is the inability to include an age-matched control group. WIDER IMPLICATIONS OF THE FINDINGS These findings support an adapted tissue preparation for Hippo pathway disruption and a shorter first phase of tissue culture. This work may also have implications for transplantation of cryopreserved tissue as larger strips (and thus slower burnout due to less Hippo pathway disruption) could be a benefit. STUDY FUNDING/COMPETING INTEREST(S) This research was financially supported by the Foundation Against Cancer (Stichting tegen Kanker, TBMT001816N), the Flemish Foundation of Scientific Research (FWO Vlaanderen, FWO G0.065.11N10) and the Gender Identity Research and Education Society (GIRES) foundation. The authors declare no competing interests. TRIAL REGISTRATION NUMBER N/A.

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In vitro follicle culture in the context of IVF

Reproduction ◽

10.1530/rep-18-0173 ◽

2018 ◽

Vol 156 (1) ◽

pp. F59-F73 ◽

Cited By ~ 10

Author(s):

Anamaria C Herta ◽

Francesca Lolicato ◽

Johan E J Smitz

Keyword(s):

Fertility Preservation ◽

Polycystic Ovary ◽

Ovarian Tissue ◽

Primordial Follicle ◽

Assisted Reproduction Techniques ◽

Realistic Potential ◽

Primordial Follicles ◽

Primordial Follicle Activation ◽

Extracellular Matrix Components

The currently available assisted reproduction techniques for fertility preservation (i.e.in vitromaturation (IVM) andin vitrofertilization) are insufficient as stand-alone procedures as only few reproductive cells can be conserved with these techniques. Oocytes in primordial follicles are well suited to survive the cryopreservation procedure and of use as valuable starting material for fertilization, on the condition that these could be grown up to fully matured oocytes. Our understanding of the biological mechanisms directing primordial follicle activation has increased over the last years and this knowledge has paved the way toward clinical applications. New multistepin vitrosystems are making use of purified precursor cells and extracellular matrix components and by applying bio-printing technologies, an adequate follicular niche can be built. IVM of human oocytes is clinically applied in patients with polycystic ovary/polycystic ovary syndrome; related knowhow could become useful for fertility preservation and for patients with maturation failure and follicle-stimulating hormone resistance. The expectations from the research on human ovarian tissue and immature oocytes cultures, in combination with the improved vitrification methods, are high as these technologies can offer realistic potential for fertility preservation.

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Effects of jacalin and follicle-stimulating hormone on in vitro goat primordial follicle activation, survival and gene expression

Zygote ◽

10.1017/s0967199414000173 ◽

2014 ◽

Vol 23 (4) ◽

pp. 537-549 ◽

Cited By ~ 3

Author(s):

Regislane P. Ribeiro ◽

Antonia M.L.R. Portela ◽

Anderson W.B. Silva ◽

José J.N. Costa ◽

José R.S. Passos ◽

...

Keyword(s):

Gene Expression ◽

Ovarian Tissue ◽

Follicle Stimulating Hormone ◽

Primordial Follicle ◽

Nuclear Antigen ◽

Primordial Follicles ◽

Primordial Follicle Activation ◽

Follicle Activation ◽

Stimulating Hormone

SummaryThis study aims to investigate the effects of jacalin and follicle-stimulating hormone (FSH) on activation and survival of goat primordial follicles, as well as on gene expression in cultured ovarian tissue. Ovarian fragments were cultured for 6 days in minimum essential medium (MEM) supplemented with jacalin (10, 25, 50 or 100 μg/ml – Experiment 1) or in MEM supplemented with jacalin (50 μg/ml), FSH (50 ng/ml) or both (Experiment 2). Non-cultured and cultured tissues were processed for histological and ultrastructural analysis. Cultured tissues from Experiment 2 were also stored to evaluate the expression of BMP-15, KL (Kit ligand), c-kit, GDF-9 and proliferating cell nuclear antigen (PCNA) by real-time polymerase chain reaction (PCR). The results of Experiment 1 showed that, compared with tissue that was cultured in control medium, the presence of 50 μg/ml of jacalin increased both the percentages of developing follicles and viability. In Experiment 2, after 6 days, higher percentages of normal follicles were observed in tissue cultured in presence of FSH, jacalin or both, but no synergistic interaction between FSH and jacalin was observed. These substances had no significant effect on the levels of mRNA for BMP-15 and KL, but FSH increased significantly the levels of mRNA for PCNA and c-kit. On the other hand, jacalin reduced the levels of mRNA for GDF-9. In conclusion, jacalin and FSH are able to improve primordial follicle activation and survival after 6 days of culture. Furthermore, presence of FSH increases the expression of mRNA for PCNA and c-kit, but jacalin resulted in lower GDF-9 mRNA expression.

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Essential role of follicle stimulating hormone in the maintenance of caprine preantral follicle viability in vitro

Zygote ◽

10.1017/s0967199407004169 ◽

2007 ◽

Vol 15 (2) ◽

pp. 173-182 ◽

Cited By ~ 76

Author(s):

M.H.T. Matos ◽

I.B. Lima-Verde ◽

M.C.A. Luque ◽

J.E. Maia Jr ◽

J.R.V. Silva ◽

...

Keyword(s):

Ovarian Tissue ◽

Follicle Stimulating Hormone ◽

Control Medium ◽

Primordial Follicles ◽

Ultrastructural Studies ◽

Follicle Diameter ◽

Transmission Electron ◽

Stimulating Hormone

SummaryThe aims of the present study were to investigate the effects of follicle-stimulating hormone (FSH) on survival, activation and growth of caprine primordial follicles using histological and ultrastructural studies. Pieces of caprine ovarian cortex were cultured for 1 or 7 days in minimum essential medium (MEM – control medium) supplemented with different concentrations of FSH (0, 10, 50 or 100 ng/ml). Small fragments from non-cultured ovarian tissue and from those cultured for 1 or 7 days in a specific medium were processed for classical histology and transmission electron microscopy (TEM). Additionally, effects of FSH on oocyte and follicle diameter of cultured follicles were evaluated. The results showed that the lowest percentage of normal follicles was observed after 7 days of culture in control medium. After 1 day of culture, a higher percentage of growing follicles was observed in the medium supplemented with 50 ng/ml of FSH. In the presence of 10 and 50 ng/ml of FSH, an increase in diameter of both oocyte and follicle on day 7 of culture was observed. TEM showed ultrastructural integrity of follicles after 1 day of culture in MEM and after 7 days in MEM plus 50 ng/ml FSH, but did not confirm the integrity of those follicles cultured for 7 days in MEM. In conclusion, this study demonstrated that FSH at concentration of 50 ng/ml not only maintains the morphological integrity of 7 days cultured caprine preantral follicles, but also stimulate the activation of primordial follicles and the growth of activated follicles.

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Supplementation of granulosa cells conditioned medium with pyruvate and testosterone could improve early follicular development in cultured 1-day-old mouse ovaries

Zygote ◽

10.1017/s0967199421000174 ◽

2021 ◽

pp. 1-8

Author(s):

Mohammad Jafari Atrabi ◽

Parimah Alborzi ◽

Vahid Akbarinejad ◽

Rouhollah Fathi

Keyword(s):

Conditioned Medium ◽

Ovarian Tissue ◽

Follicular Development ◽

Primordial Follicle ◽

Ovarian Tissue Cryopreservation ◽

Primordial Follicles ◽

Patients With Cancer ◽

Primordial Follicle Activation ◽

Follicle Activation

Summary In vitro activation of primordial follicles could serve as a safe method to preserve fertility in patients with cancer subjected to ovarian tissue cryopreservation during oncotherapy, however the culture medium for this purpose requires to be optimized. Granulosa cell conditioned medium (GCCM) has been recognized to enhance primordial follicle activation and the present study was conducted to understand whether addition of pyruvate, a combination of insulin, transferrin and selenium (ITS) or testosterone to GCCM could improve its efficiency in this regard. To this end, 1-day-old mouse ovaries were cultured in four different media including CON (control; containing GGCM only), PYR (containing GCCM plus pyruvate), ITS (containing GCCM plus ITS) or TES (containing GCCM plus testosterone) for 11 days. Furthermore, follicular dynamics and gene expression of factors involved in follicular development were assessed using histological examination and RT-PCR, respectively, on days 5 and 11 of culture. Pyruvate decreased follicular activation, but it enhanced the progression of follicles to the primary stage. Moreover, it upregulated Bmp15 and Cx37 (P < 0.05). In the ITS group, activation of follicles was not affected and total number of follicles was reduced by day 11 of culture. Additionally, ITS downregulated Pi3k, Gdf9, Bmp15 and Cx37 (P < 0.05). Although testosterone did not affect primordial follicle activation, it enhanced the development of follicles up to the preantral stage (P < 0.05). Furthermore, testosterone inhibited the expression of Pten but stimulated the expression of Gdf9 and Cx37 (P < 0.05). In conclusion, the present study revealed that inclusion of pyruvate and testosterone into GCCM could enhance the early development of follicles in cultured 1-day-old mouse ovaries.

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Leptin decreases apoptosis and promotes the activation of primordial follicles through the phosphatidylinositol-3-kinase/protein kinase B pathway in cultured ovine ovarian tissue

Zygote ◽

10.1017/s0967199421000034 ◽

2021 ◽

pp. 1-7

Author(s):

T.J.S. Macedo ◽

V.G. Menezes ◽

R.S. Barberino ◽

R.L.S. Silva ◽

B.B. Gouveia ◽

...

Keyword(s):

Protein Kinase ◽

Protein Kinase B ◽

Ovarian Tissue ◽

Pi3k Inhibitor ◽

Akt Pathway ◽

Control Medium ◽

Phosphatidylinositol 3 Kinase ◽

Primordial Follicles ◽

Follicle Activation

Summary This study evaluated the effects of leptin on primordial follicle survival and activation after in vitro culture of ovine ovarian tissue and if leptin acts through the phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) pathway. Ovarian fragments were fixed for histology (fresh control) or cultured for 7 days in control medium (α-MEM+) alone or supplemented with leptin (1, 5, 10, 25 or 50 ng/ml). Follicle morphology, activation and apoptosis were analyzed. Next, the fragments were cultured in the medium that showed the best results in the absence or the presence of the PI3K inhibitor (LY294002), and immunohistostaining of p-Akt protein was assessed. After culture, the percentage of normal follicles decreased (P < 0.05) in all treatments compared with the fresh control. Moreover, control medium and 1 ng/ml leptin had similar (P > 0.05) percentages of normal follicles, which were significantly higher than those in other treatments. However, culture with 1 ng/ml leptin maintained apoptosis similarly (P > 0.05) to that of the fresh control and lower (P < 0.05) than that in α-MEM+. Leptin did not influence follicle activation (P > 0.05) compared with the control medium (α-MEM+). Culture in 1 ng/ml leptin with LY294002 decreased the normal follicles and increased apoptosis, inhibited follicle activation (P < 0.05), and reduced p-Akt immunostaining, compared with the medium containing 1 ng/ml leptin without PI3K inhibitor. In conclusion, leptin at 1 ng/ml reduces apoptosis and promotes the activation of primordial follicles compared with the fresh control after in vitro culture of ovine ovarian tissue possibly through the PI3K/Akt pathway.

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The in vitro growth and maturation of follicles

Reproduction ◽

10.1530/rep-08-0290 ◽

2008 ◽

Vol 136 (6) ◽

pp. 703-715 ◽

Cited By ~ 150

Author(s):

H M Picton ◽

S E Harris ◽

W Muruvi ◽

E L Chambers

Keyword(s):

Culture Media ◽

Ovarian Tissue ◽

Cell Communication ◽

Primordial Follicle ◽

Primordial Follicles ◽

Follicle Culture ◽

Preantral Follicles ◽

Large Animals

The development of technologies to grow oocytes from the most abundant primordial follicles to maturity in vitro holds many attractions for clinical practice, animal production technology and research. The production of fertile oocytes and live offspring has been achieved in mice following the long-term culture of oocytes in primordial follicles from both fresh and cryopreserved ovarian tissue. In contrast, in non-rodent species advances in follicle culture are centred on the growth of isolated preantral follicles. As a functional unit, mammalian preantral follicles are well-suited to culture but primordial and primary follicles do not grow well after isolation from the ovarian stroma. The current challenges for follicle culture are numerous and include: optimisation of culture media and the tailoring of culture environments to match the physiological needs of the cell in vivo; the maintenance of cell–cell communication and signalling during culture; and the evaluation of the epigenetic status, genetic health and fertility of in vitro derived mature oocytes. In large animals and humans, the complete in vitro growth and maturation of oocytes is only likely to be achieved following the development of a multistage strategy that closely mimics the ovary in vivo. In this approach, primordial follicle growth will be initiated in situ by the culture of ovarian cortex. Isolated preantral follicles will then be grown to antral stages before steroidogenic function is induced in the somatic cells. Finally, cytoplasmic and nuclear maturation will be induced in the in vitro derived oocytes with the production of fertile metaphase II gametes.

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Cyclophosphamide Exposure Causes Long-Term Detrimental Effect of Oocytes Developmental Competence Through Affecting the Epigenetic Modification and Maternal Factors’ Transcription During Oocyte Growth

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.682060 ◽

2021 ◽

Vol 9 ◽

Author(s):

Weijie Yang ◽

Yerong Ma ◽

Jiamin Jin ◽

Peipei Ren ◽

Hanjing Zhou ◽

...

Keyword(s):

Dna Damage ◽

Culture Media ◽

Epigenetic Modification ◽

Primordial Follicle ◽

Oocyte Quality ◽

Mature Oocyte ◽

Developmental Competence ◽

Oocyte Growth ◽

Primordial Follicles

Cyclophosphamide (CTX) is widely used in various cancer therapies and in immunosuppression, and patients can still have babies after CTX chemotherapy. CTX directly causes primordial follicle loss with overactivation and DNA damage-induced apoptosis. Previous studies have shown that maternal exposure to CTX before conception increases the incidence of birth abnormalities and alters the methylation of genes in the oocytes of offspring. Mice were treated with a single dose of CTX (100 mg/kg) at post-natal day 21 and sacrificed 47 days later when primordial follicles surviving chemotherapy developed to the antral stage. Acute DNA damage and acceleration of the activation of primordial follicles after CTX treatment were repaired within several days, but the remaining follicle numbers remarkably decrease. Although partial surviving primordial follicle were developed to mature oocyte, oocyte quality hemostasis was impaired exhibiting aberrant meiosis progression, abnormal spindle and aneuploidy, mitochondrial dysfunction and increased endoplasmic reticulum stress. Thereafter, embryo development competency significantly decreased with fewer blastocyst formation after CTX exposure. CTX treatment resulted in alteration of DNA methylations and histone modifications in fully grown GV oocytes. Single-cell RNA-seq revealed CTX treatment suppressed multiple maternal genes’ transcription including many methyltransferases and maternal factor YAP1, which probably accounts for low quality of CTX-repaired oocyte. In vitro addition of lysophosphatidic acid (LPA) to embryo culture media to promote YAP1 nuclear localization improved CTX-repaired embryo developmental competence. This study provides evidence for the consistent toxic effect of CTX exposure during follicle development, and provide a new mechanism and new insights into future clinical interventions for fertility preservation.

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Primordial follicle survival of goat ovarian tissue after vitrification and transplantation on chorioallanthoic membrane

Middle East Fertility Society Journal ◽

10.1186/s43043-020-00044-1 ◽

2020 ◽

Vol 25 (1) ◽

Author(s):

Shofwal Widad ◽

Detty Siti Nurdiati ◽

Sarrah Ayuandari ◽

Kuky Cahya Hamurajib ◽

Muhammad Dimas Reza Rahmana ◽

...

Keyword(s):

Dna Damage ◽

Dna Fragmentation ◽

Ovarian Tissue ◽

Primordial Follicle ◽

Young Female ◽

Primordial Follicles ◽

Significant Difference ◽

Transplantation Procedures ◽

Hematoxylin Eosin ◽

Potential Issue

Abstract Background Ovarian tissue vitrification is an alternative fertility preservation procedure for young female patients prior to gonadotoxic treatment. Primordial follicle loss might be a potential issue for vitrification and transplantation procedures. This study aimed to evaluate primordial follicle density and deoxyribonucleic acid (DNA) fragmentation in each stage of the preservation procedure of goat ovarian tissue. Follicle density and DNA fragmentation were examined microscopically after staining with hematoxylin eosin and TUNEL assay, respectively. Both parameters were compared between fresh, fresh-transplanted, vitrification, and vitrification-transplanted groups. Results A significant decrease was observed in the primordial follicle proportion after vitrification and transplantation compared to the primordial follicle proportion in the fresh group (88.09% vs 52.42%, p < 0.05, 95% CI 11.54, 66.94). There was no significant difference in DNA fragmentations of primordial follicles between each group (p > 0.05). Conclusions The vitrification and transplantation process of goat ovarian strips could cause the primordial follicles loss and DNA damage of the follicles. However, primordial follicles loss and DNA damage were not significantly different in each procedure.

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