Cryopreservation time does not decrease follicular viability in ovarian tissue frozen for fertility preservation

Abstract

OBJECTIVE:To determine the effect of storage duration on cryopreserved ovarian tissue using fresh and frozen-thawed samples.METHODS:Seventeen fertile patients underwent an ovarian biopsy during elective laparoscopic tubal ligation. The tissue sample was divided into three parts: one part was processed fresh (FG), and two were slowly frozen, cryopreserved for 30 (G30) or 180 days (G180), thawed and analyzed. Follicular density, follicular viability, and steroidogenic capacity were assessed.RESULTS:We observed no differences between the groups in follicular density, which was assessed in hematoxylin and eosin–stained tissue sections. A heterogeneous follicular distribution was observed in the parenchyma, with a mean density of 361.3±255.4, 454.9±676.3, and 296.8±269.0 follicles/mm3 for FG, G30 and G180, respectively (p = 0.46). Follicular viability was greater in FG (93.4%) when compared with the cryopreserved tissues (70.8% for G30 (p<0.001) and 78.4% for G180 (p<0.001)), with no difference in viability between the frozen samples (p>0.05). The steroidogenic capacity of the tissue was not significantly reduced following cryopreservation.CONCLUSION:The slow freezing procedures used for ovarian cryopreservation are capable of preserving follicular viability and maintaining the steroidogenic capacity of the tissue despite a roughly 30% decrease in follicular viability. Furthermore, short-term storage of ovarian tissue does not appear to compromise follicle integrity.

Efficacy of ovarian tissue cryopreservation in a major European center

“…The observed percentages of tissue viability impairment as a result of cryopreservation and thawing are roughly consistent with the 70-80 % follicle survival reported earlier for various slowfreezing protocols. [8,27,29,30,35,38,39] In addition, we observed a 24-27 % reduction in overall tissue viability using the glucose uptake assay. This decreased glucose uptake indicates a reduced metabolism of the tissue during culture as a result of cryodamage.…”

“…Although various large centers for cryopreservation of ovarian tissue exist, [12,13,15,17,18,21,24] to the best of our knowledge, cohort studies in which the impact of currently used cryopreservation/ thawing protocols on both follicle and stromal cell survival in young cancer patients is measured have not been published before. Earlier studies considering the impact of slow freezing techniques other than evaluated here, generally focused on follicle viability only [27][28][29][30][31][32][33][34][35][36][37][38] and often described patient populations without an indication for fertility preservation (e.g. patients applying for a sterilization, cystectomy, or caesarean section) [8, 27-29, 32, 34, 35, 38].…”

“…Although there are studies that quantify the impact of slow-freezing on human ovarian tissue viability, these studies do not per definition focus on the protocols that are currently being used by the major centers. [27][28][29][30][31][32][33][34][35][36][37][38] Moreover, most of these studies did not take the viability of the stromal cell compartment into account. [27][28][29][30][31][32][33][34][35][36][37][38] This compartment, however, is essential for post-autotransplantation neovascularization, follicle survival, and life span of the ovarian graft [39] and is considered to be more sensitive to ischemic and cryoinjury than primordial follicles [8,40].…”

Three Dimensional &lt;i&gt;In Vitro&lt;/i&gt; Culture of Murine Secondary Follicles in a Defined Synthetic Matrix

“…gel system derived from the medium (DMEM/F12 with 10% FCS and 1% ITS), the diameters and morphological integrity of follicles grown in SFX-1 were not significantly lower than those grown in Matrigel and were clearly superior to [24]. It was surprising that Matrigel did not maintain significantly higher viable cell numbers than either of the other two culture systems.…”

Ethical, moral and other aspects related to fertility preservation in cancer patients

“…Although uncertainties are still common among oncologists, we can happily say that today they are not endorsed anymore, since the advent of random-start treatment protocols, not only allowing the patient to continue cancer treatment in two to three weeks after the first query (Cakmak et al ., 2013a), but also being carried out in association with aromatase inhibitors (Cakmak & Rosen, 2013b) or selective estrogen receptors modulators (Franco et al ., 2012), which reduces or eliminates the risk of hormonal-dependent tumor stimulation. Current literature provides promising results with random-start protocols and, although studies are still small and with low levels of evidence, many consider the data strong enough to encourage oncologists refer their patients to an oncofertility specialist early on (Cakmak et al ., 2013a; Rashidi et al ., 2014; Checa et al ., 2015; Kim et al ., 2015).…”