Dynamic in vitro culture of cryopreserved-thawed human ovarian cortical tissue using a microfluidics platform does not improve early folliculogenesis

Abstract

Current strategies for fertility preservation include the cryopreservation of embryos, mature oocytes or ovarian cortical tissue for autologous transplantation. However, not all patients that could benefit from fertility preservation can use the currently available technology. In this regard, obtaining functional mature oocytes from ovarian cortical tissue in vitro would represent a major breakthrough in fertility preservation as well as in human medically assisted reproduction. In this study, we have used a microfluidics platform to culture cryopreserved-thawed human cortical tissue for a period of 8 days and evaluated the effect of two different flow rates in follicular activation and growth. The results showed that this dynamic system supported follicular development up to the secondary stage within 8 days, albeit with low efficiency. Surprisingly, the stromal cells in the ovarian cortical tissue were highly sensitive to flow and showed high levels of apoptosis when cultured under high flow rate. Moreover, after 8 days in culture, the stromal compartment showed increase levels of collagen deposition, in particular in static culture. Although microfluidics dynamic platforms have great potential to simulate tissue-level physiology, this system still needs optimization to meet the requirements for an efficient in vitro early follicular growth.

Enhanced solute transport and steady mechanical stimulation in a novel dynamic perifusion bioreactor increase the efficiency of the in vitro culture of ovarian cortical tissue strips

“…However, both transport enhancement and shear stresses are difficult to evaluate quantitatively, non-reproducible, and beyond the operator’s control. Recently, microfluidic bioreactors have been proposed for ovarian tissue culture although with unsatisfactory effects on folliculogenesis ( Del Valle et al, 2022 ). None of the proposed bioreactors permits the application of a direct mechanical challenge on the tissue.…”

“…The compound effect of these resistances is that the dissolved oxygen concentration near the follicles may be much lower than it is at the gas/liquid interface that may cause hypoxic or even anoxic conditions in their culture, thus limiting long-term follicle development and viability. In recent times, dynamic bioreactors of macro- to microscales have been proposed for ovarian tissue culture that mimic the continuous supply of nutrients and oxygen (and waste metabolites removal) enabled by the blood circulation in natural tissues ( da Rocha and Smith, 2014 ; Heise et al, 2009 ; Sadeghzadeh Oskouei et al, 2021 ; Del Valle et al, 2022 ; Mastrorocco et al, 2022 ). Bioreactor designs for ovarian strip culture have been proposed to decrease the liquid resistance to oxygen transport external to the tissue strips in which the medium is mechanically or fluid dynamically mixed, apparently with inconsistent results ( Hovatta et al, 1997 ; Wright et al, 1999 ; Isachenko et al, 2006 ; Liebenthron et al, 2013 ).…”

Bioengineered 3D Ovarian Models as Paramount Technology for Female Health Management and Reproduction

“…This study followed the first culture step described by McLaughlin and colleagues [40], but used cryopreserved ovaries from transmasculine individuals on hormone replacement therapy for several years instead. They showed that neither a low flow rate (0.1 mL/min) nor a high flow rate (0.5 mL/min) were beneficial for the maturation to secondary follicles [51]. The secondary follicles obtained presented a disorganized layer of granulosa cells, often with a peripherally located oocyte.…”

“…This has led to the hypothesis that ECM remodeling during this procedure could trigger follicular activation. Del Valle and colleagues reported a significant increase in collagen IV in the stromal compartment of human ovarian cortex tissue cultured for 8 days, indicating that the ovarian cortex became more fibrotic during culture [51]. By contrast, Groibos and colleagues reported that human ovarian cortex tissue cultured for 6 days showed decreased collagen and increased elastin deposition [65].…”

“…Microfluidics technologies have proven beneficial to create a more physiological microenvironment for the culture of several female reproductive tissues (ovary, fallopian tube, uterus, cervix and liver) with sustained circulation between them for a period of 28 days [50]. Recently, Del Valle and colleagues investigated whether culturing human cryopreserved ovarian cortex fragments (1 mm 3 ) under dynamic flow conditions could improve the maturation rate of unilaminar follicles to secondary follicles after 8 days of culture [51]. This study followed the first culture step described by McLaughlin and colleagues [40], but used cryopreserved ovaries from transmasculine individuals on hormone replacement therapy for several years instead.…”