Production of endometrial placental protein 14 and prolactin by cultured endometrial explants after collagenase and freeze/thaw treatment, and in response to progesterone.

Abstract

Objective: Investigation of methods for maintaining functional endometrial explants in culture after cryopreservation with or without previous enzymatic dispersion of stromal cells and epithelial glands. Such a standardized culture system is a requirement for the development of a non-invasive bioassay for embryo quality in in vitro fertilization programs, a method that will eventually measure endometrial response to embryo conditioned media.

Method: Culture of mid-luteal phase endometrial biopsies, in the presence of [35S]methionine, with or without prior collagenase treatment and/or storage in liquid nitrogen in the presence of dimethyl sulfoxide. Determination of released de novo synthesized total protein by trichloroacetic acid precipitation of culture media. Measurement, after culture in absence and presence of progesterone, of prolactin and placental protein 14 (PP14) production by sensitive non-isotopic immunoassays.

Results: Production of prolactin, but not PP14, was increased by 200 nmol/l progesterone, 2-8-fold after 4 days and 1.5-700-fold after 7 days in culture. After limited collagenase treatment (but without separation of stromal cells from glands), both marker protein productions were similar compared to untreated explants; however, there was no significant stimulation of prolactin by progesterone. After freezing and thawing, production was markedly reduced, particularly from explants frozen following collagenase treatment.

Conclusions: Both stromal and glandular viability are maintained after collagenase treatment but the response to progesterone is lost. Cryopreservation reduced prolactin and PP14 production in subsequent culture. Therefore, novel freezing protocols should be developed which preserve both endometrial structure and function.