Oosit Kriyoprezervasyonunda Ultra Hızlı Dondurma-Çözme Yönteminin Morfolojik ve Mitokondriyal Avantajları

Abstract

Giris: Oositlerin kriyoprezervasyonu, yardımcı üreme teknolojileri (ART) ve fertilite korumasında merkezi bir rol oynamaktadır. Yaygın olarak kullanılan konvansiyonel vitrifikasyon (CVT), yüksek konsantrasyonlarda kriyoprotektan ajanlara (CPA) uzun süreli maruziyeti içerdiğinden hücresel stres oluşturma potansiyeline sahiptir. Ultra hızlı dondurma-çözme (UFFT), CPA maruziyetini en aza indirmeyi ve aşırı soğutma/ısıtma hızlarına ulaşarak kriyohasarı azaltmayı hedeflemektedir. Amaç: UFFT ve CVT yöntemlerinin metafaz II evresindeki fare oositleri üzerindeki etkilerinin karşılaştırılması; odak noktaları olarak iğ ipliği/kutuplu cisim morfolojisi, PI⁺ ekstraselüler veziküller (EV) aracılığıyla nekroz yüzdesi ve mitokondriyal yanlış katlanmış protein yanıtı (mtUPR) aktivasyonu değerlendirilmiştir. Yöntemler: Metafaz II oositleri (n = 100/grup) taze kontrol, CVT ve UFFT gruplarına ayrıldı. Çözme sonrası morfoloji ışık/kutuplaşmalı mikroskopi ile değerlendirildi; nekroz, akım sitometrisi ile PI⁺ EV fraksiyonu olarak kantitatif olarak belirlendi; mtUPR aktivasyonu (Hsp60, Lonp1, Atf5, ClpP genleri) qRT-PCR ile ölçüldü. Veriler ANOVA ve Tukey'nin post-hoc testi ile analiz edildi. Sonuçlar: UFFT uygulanan oositlerde iğ ipliği bütünlüğü (%91 ± 3) ve kutuplu cisim korunumu (%94 ± 3), CVT grubuna kıyasla (%65 ± 5 ve %70 ± 4) anlamlı olarak daha yüksekti (p < 0.001). PI⁺ EV yüzdesi UFFT grubunda (%18.0 ± 3.0), CVT’ye (%34.0 ± 4.0) kıyasla anlamlı derecede düşüktü (p < 0.001). mtUPR gen ekspresyonu en yüksek CVT grubunda, orta düzeyde UFFT grubunda ve en düşük taze kontrolde gözlendi. UFFT ile kontrol grubu arasındaki fark istatistiksel olarak anlamlı değildi (p > 0.05). Sonuç: UFFT, oosit morfolojisinin korunmasında ve membran hasarının azaltılmasında CVT’ye kıyasla üstünlük göstermiştir; ayrıca mitokondriyal stres belirteçlerinde kontrol grubunun üzerine çıkmadan etkili olmuştur. Bu bulgular, UFFT’nin ART uygulamalarında umut vaat eden bir alternatif olduğunu ve daha geniş ölçekli klinik çalışmalarla desteklenmesi gerektiğini göstermektedir.

Keywords

• oosit
• vitrifikasyon
• mtUPR

Morphological and Mitochondrial Advantages of Ultrafast Freeze–Thaw in Oocyte Cryopreservation

Abstract

Background: Cryopreservation of oocytes is central to assisted reproductive technologies (ART) and fertility preservation. Conventional vitrification (CVT), though widely used, involves prolonged exposure to high concentrations of cryoprotective agents (CPAs), potentially inducing cellular stress. Ultrafast freeze–thaw (UFFT) aims to minimize CPA exposure and achieve extreme cooling/warming rates, reducing cryodamage. Objective: To compare UFFT and CVT in mouse oocytes, focusing on spindle/polar body morphology, necrosis percentage via PI⁺ extracellular vesicles (EVs), and mitochondrial unfolded protein response (mtUPR) activation. Methods: Metaphase II oocytes (n = 100/group) were assigned to fresh control, CVT, or UFFT groups. Post-thaw morphology was assessed via light/polarized microscopy; necrosis was quantified as PI⁺ EV fraction by flow cytometry; mtUPR activation (Hsp60, Lonp1, Atf5, ClpP) was measured by qRT-PCR. Data were analyzed by ANOVA with Tukey’s post-hoc test. Results: UFFT oocytes exhibited significantly higher spindle integrity (91% ± 3%) and polar body retention (94% ± 3%) than CVT (65% ± 5% and 70% ± 4%, respectively; p < 0.001). PI⁺ EV percentage was reduced in UFFT (18.0% ± 3.0%) compared to CVT (34.0% ± 4.0%; p < 0.001). mtUPR gene expression was highest in CVT, intermediate in UFFT, and lowest in the fresh control. Differences between UFFT and control were not statistically significant (p > 0.05). Conclusion: UFFT preserved oocyte morphology and reduced membrane damage compared to CVT, without significantly elevating mitochondrial stress markers above control. These findings support UFFT as a promising alternative in ART, warranting further large-scale and clinical studies.

Keywords

• oocyte
• vitrification
• mtUPR

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