The impact of soluble HLA-G in IVF/ICSI embryo culture medium on implantation success

Abstract

The HLA-G molecule is widely accepted as an important factor for pregnancy success. Its expression has been detected in the extravillous trophoblasts. Soluble HLA-G (sHLA-G) was found in the genital tract, pre-implanted embryos as well as in seminal fluid. In this study, we investigated the concentration of sHLA-G (sHLA-G1 and sHLA-G5) in media from 344 single cultured embryos following in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI). The level of sHLA-G (U/ml) was tested with a sandwich enzyme-linked immunosorbent assay (ELISA) kit. We correlated sHLA-G secretion with ovarian stimulation protocols, the type of embryo transfer (fresh or frozen cycle) and the quality of the embryos. The ovarian stimulation protocol affects the secretion of sHLA-G by the embryo. Embryos obtained from the long agonist protocol secreted more sHLA-G than those originating from the short antagonist protocol (p = 0.0001). Embryos whose transfer resulted in a clinical pregnancy and/or live birth secreted more sHLA-G compared to those whose transfer ended without pregnancy. This was particularly observable in embryos following the long ovarian stimulation protocol and from a frozen embryo cycle. In conclusion, sHLA-G secreted by the embryo has an impact on implantation and live birth and could be a developmental potential marker of the embryo. Its concentration depends on the ovarian stimulation protocol used.

Keywords: SHLA-G; embryo; in vitro fertilization; ovarian stimulation; reproductive success.

Figure 1

Quality of the embryos depending on the day of development. 1) development of embryos over time from the second to the sixth day (height of the colored stripes); 2) comparisons of secretion between embryos of different classes or stages of development (vertical clamps). A, B, C – class of the embryo; M – embryo in the morula stage; no-M – embryo that has not reached the morula stage; BC – embryo in the blastocyst stage; no-BC – embryo that has not reached the blastocyst stage. Median concentration of sHLA-G was presented in brackets. P-values are calculated by Mann-Whitney test.

Figure 2

Secretion of soluble HLA-G (U/ml) by embryos in different ovarian stimulation protocols. Green box represents the level of sHLA-G measured in embryo media in long cycle, blue box – in short cycle. Boxes are drawn from the first quartile (25^th percentile) to the third quartile (75^th percentile). Black lines in boxes are medians. Whiskers represent 10-90 percentiles. P-values are calculated by Mann-Whitney test.

Figure 3

Effect of embryo quality on the concentration of secreted HLA-G in the long and short stimulation protocol. 1) development of embryos over time from the second to the sixth day (height of the colored stripes); 2) secretion comparisons between long and short protocol groups (horizontal arrows); 3) comparisons of secretion between embryos of different classes or stages of development (vertical clamps). A, B, C – class of the embryo, M – embryo in the morula stage; no-M – embryo that has not reached the morula stage; BC – embryo in the blastocyst stage; no-BC – embryo that has not reached the blastocyst stage. Median concentration of sHLA-G was presented in brackets. Dashed line – p-value resulting from analyses with insufficient number of embryos. P-values are calculated by Mann-Whitney test or t-test.

Figure 4

Secretion of soluble HLA-G (U/ml) by embryos in the fresh and frozen cycle. Green box represents the level of sHLA-G measured in embryo media in fresh cycle, blue box – in frozen cycle. Boxes are drawn from the first quartile (25^th percentile) to the third quartile (75^th percentile). Black lines in boxes are medians. Whiskers represent 10-90 percentiles. P-values are calculated by Mann-Whitney test. Analysis do not include deferred transfers.

Figure 5

Effect of embryo quality on the concentration of secreted HLA-G in the fresh and frozen cycle. 1) development of embryos over time from the second to the sixth day (height of the colored stripes); 2) secretion comparisons between fresh and frozen cycle groups (horizontal arrows); 3) comparisons of secretion between embryos of different classes or stages of development (vertical clamps). A, B, C – class of the embryo, M – embryo in the morula stage; no-M – embryo that has not reached the morula stage; BC – embryo in the blastocyst stage; no-BC – embryo that has not reached the blastocyst stage; Dashed line – p-value resulting from analyses with insufficient number of embryos. Median concentration of sHLA-G was presented in brackets. P-values are calculated by Mann-Whitney test or t-test.

Figure 6

Effect of sHLA-G secretion by embryos on the presence of pregnancy markers. Green box represents the level of sHLA-G measured in embryo media resulting in FHR-positivity; blue box – FHR-negativity. Boxes are drawn from the first quartile (25^th percentile) to the third quartile (75^th percentile). Black lines in boxes are medians. Whiskers represent 10-90 percentiles. P-values are calculated by Mann-Whitney test. FHR – fetal heart rate.

Figure 7

Influence of soluble HLA-G (U/ml) secreted by embryos and long/short cycle on the pregnancy outcome. Green boxes represent the level of sHLA-G measured in embryo media in the long cycle, blue boxes – in the short cycle. Boxes are drawn from the first quartile (25^th percentile) to the third quartile (75^th percentile). Black lines in boxes are medians. Whiskers represent 10-90 percentiles. P-values are calculated by Mann-Whitney test. Dashed line – p-value resulting from analyses with insufficient number of embryos. P-values are calculated by Mann-Whitney test. LB means live birth, P – pregnancy, MIS – miscarriage, no-P – no pregnancy.

Figure 8

Influence of soluble HLA-G (U/ml) secreted by embryos and fresh/frozen cycle on the pregnancy outcome. Green boxes represent the level of sHLA-G measured in embryo media in fresh cycles, blue boxes – in frozen cycles. Boxes are drawn from the first quartile (25^th percentile) to the third quartile (75^th percentile). Black lines in boxes are medians. Whiskers represent 10-90 percentiles. P-values are calculated by Mann-Whitney test. LB means live birth, P – pregnancy, MIS – miscarriage, no-P – no pregnancy.