O-239 CD147 expression in human embryos and its role during implantation
Authors: Essahib W , Verheyen G , De Vos A , De Munck N , Tournaye H , Van de Velde H 
Published: June 2022

Abstract

Study question
Do human pre-implantation embryos express CD147, does it play a role during implantation, and can it be used as non-invasive marker for embryo selection?

Summary answer
CD147 is expressed throughout pre-implantation development. Blocking results in implantation failure in vitro, but CD147 secretion has no immediate link with implantation in vivo.

What is known already
Embryo selection remains a subjective process with room for improvement. Researchers focus on alternative techniques to reduce time to pregnancy. While preimplantation genetic testing and time-lapse studies increased our understanding, they failed to efficiently improve embryo selection. CD147, a cluster domain well studied in cancer, has been proven to positively influence implantation in mice. When the protein is blocked, the embryo develops to blastocyst but fails to implant. This study aims to visualize the expression of CD147 throughout human pre-implantation development and to understand its role during implantation in humans, using both an in vitro and in vivo model.

Study design, size, duration
CD147 expression was studied in oocytes (n = 13) and embryos (n = 30) between 3 and 7 days post-fertilisation (dpf) with immunohistochemistry. To investigate its function, 5dpf blastocysts (n = 45) were co-cultured with Ishikawa cells for two days in an in vitro attachment model with two distinct anti-CD147 antibodies (ab666 and ab119114). Attachment models containing embryos (n = 8) were stained using ActinGreen™ probes. Additionally, spent culture medium was collected in the IVF lab after fresh blastocyst transfer (n = 153, sequential medium).

Participants/materials, setting, methods
Fresh immature oocytes and vitrified/warmed embryos were obtained after patients’ informed consent, fixed in 4% paraformaldehyde and stained using ab666. The counterstained samples were visualized with confocal microscopy. For blocking experiments, embryos were incubated in ab666 or ab119114 (Abcam) and co-cultured in the attachment model. Attachment was measured after 24hrs by gentle pipetting of the medium. Attached embryos were stained for F-actin. CD147 concentrations in spent embryo culture medium were determined by ELISA (EH78RB, Thermo-Fisher-Scientific).

Main results and the role of chance
In immature oocytes, CD147 was present on the oolemma only. After fertilization, CD147 protein was restricted to the membrane of all blastomeres but fluorescence intensity decreased gradually during cleavage stages. After embryonic genome activation the expression increased again mainly on the membranes of the cells. After blastulation (5 dpf), the protein was expressed in all cells but mainly concentrated on the membranes. With the expansion of the blastocysts (6-7 dpf) CD147 became more abundant on the membranes of trophectoderm cells (TE), the signal being the strongest on the apical membranes and TE junctions.
Blocking of CD147 binding in blastocysts was achieved with two anti-CD147 antibodies ab666 and ab119114 with respectively 52% and 70 % reduction of attachment on Ishikawa cells compared to non-treated control groups (n = 45, p &lt; 0.05).
Finally, secreted CD147 was found in culture medium of embryos 5 dpf. Most of the blastocysts (150/153) secreted CD147 with concentrations ranging from 40–229 pg/ml. No correlation with pregnancy outcome or morphology was noted, indicating that CD147 cannot be used as non-invasive marker for embryo selection. Although these results clearly highlight the importance of CD147 in human embryo implantation in vitro, more research is needed to investigate its role.

Limitations, reasons for caution
The experiments have been performed using an in vitro model for attachment, the impact of additional interfering factors in vivo cannot be ruled out.

Wider implications of the findings
Protein expression highlights the importance of CD147 in human blastocysts, especially for the attachment function of the apical TE membrane. Blocking CD147protein reduces implantation dramatically, proving its involvement in establishing a successful pregnancy. In addition, secretion of CD147 demonstrates its potential paracrine role during the interaction with the endometrium.

Trial registration number
not applicable