Effect of cryopreservation on human granulosa cell viability and responsiveness to gonadotropin

In human, the use of freshly recovered granulosa cells for experiments remains difficult. Because of the single use of human cells, the experiments cannot be repeated, and no additional conditions can be tested afterwards with the cells of the same patient. Therefore, granulosa cell cryopreservation could be a good alternative to keep part of these cells for later controls or experiments. The aim of this study is to compare the responsiveness to FSH of fresh and frozen-thawed human primary granulosa-lutein cells (hGLC) and determine if cryopreserved granulosa cells can be used in place of fresh cells. Two cryopreservation methods were also compared: a conventional versus a simplified freezing method. This experimental study was undertaken at Igyxos S.A., Nouzilly, France. Seventy women undergoing oocyte retrieval at the IVF Unit from Bretonneau University Hospital (Tours, France) were recruited in 2016. Fresh and frozen-thawed hGLC were cultured for 7 days and then stimulated by r-FSH for 48 h. To assess r-FSH efficacy and potency, extracellular cAMP accumulated in the supernatant for each stimulation point was measured. We demonstrated that hGLC remain responsive to FSH stimulation after freezing-thawing and 7 days of pre-culture. They are able to secrete cAMP with a similar EC50 value as fresh hGLC, but FSH efficacy is lowered. As our study did not show any significant difference between the two freezing methods concerning the sensitivity of hGLC to FSH, hGLC could be cryopreserved with the simplified freezing method without taking up too much time for IVF laboratories.