PLANTEAMIENTO DEL PROBLEMA

In order to determine a role for trypsin activity in human implantation, embryo culture media (ECM) drops, in which embryos were cultured for IVF treatment, were routinely collected and stored at -80°C. These culture drops were tested for the presence of protease and trypsin activity in order to identify whether such activity may be as important in human implantation as observed in other species. The ECM either contained a number of co-cultured embryos from a single patient (‘pooled’), or embryos that were cultured individually for time-lapse imaging (‘individual’).

Protease activity was measured using the Enzchek® assay. This assay measures protease catalysed hydrolysis of a casein derivative, which causes release of the quenched fluorescently labelled peptides (BODIPY FL dye). The level of fluorescence measured is proportional to protease activity. Trypsin activity was measured using an assay that employs a trypsin-specific substrate, which upon cleavage by trypsin generates p-nitroaniline (p-NA) detectable at OD = 405. The colour change is thus proportional to p-NA content.

To determine if trypsin activity is generated by human embryos, ECM of 163 pooled embryos from 16 individuals were analysed using the trypsin assay (Figure 3.2.2.1a). As shown, trypsin activity was detectable, although the level of activity varied widely between culture droplets (median: 20.9 mU/ml; range: 0.7 to 45.4 mU/ml).

In order to obtain more precise measurements of protease and trypsin activity, ECM of 87 individually cultured embryos from 21 individuals were analysed using the Enchek® and trypsin assays (Figure 3.2.2.1b,c). Embryos were divided into two groups according to the day of pre-implantation embryo development at the time of ECM collection (day 2 or day 5). Data were normalised to activity in unconditioned

are displayed.

Protease activity was measured in 47 individual ECM droplets and was increased in the ECM of day 5 embryos (median: 247.7 % change from control; range: 0.0 to 806.6 % change from control) compared to day 2 embryos (median: 38.3 % change from control; range: 0.0 to 546.1 % change from control) (Figure 3.2.2.1b).

Trypsin activity was measured in 40 individual ECM droplets and similar to protease activity, was increased in the ECM of day 5 embryos (median: 34.7 % change from control; range: 2.1 to 76.7 % change from control) compared to day 2 embryos (median: 10.3 % change from control; range: 0.0 to 46.4 % change from control) (Figure 3.2.2.1c).

The recorded levels of protease and trypsin activity varied markedly between individual ECM droplets (range: 0 to 806.6 % change from control; range: 0 to 76.7 % change from control, respectively) (Figure 3.2.2.1b,c). This variation is partly, but not entirely, explained by increased activity from embryos at day 5.

A high rate of attrition is observed during in vitro culture of human embryos with a

majority of embryos arresting before the blastocyst stage. Therefore separation of ECM according to the day of development does not accurately reflect the developmental stage of the embryo. In our unit embryo transfers are conducted

Day 5 transferred blastocysts are routinely graded according to the Gardner grading system (Gardner et al., 2000), which assigns values to the level of blastocoel expansion in combination with the morphological quality of two polarised cell compartments that comprise the blastocyst. The outer shell-like TE that will give rise to the extraembryonic membranes and the inner cluster of cells that will give rise to the fetus, the ICM. These compartments are graded independently by an embryologist just prior to embryo transfer and the grades have been shown to reflect embryo implantation potential (Gardner et al., 2000).

Blastocoel expansion is graded as follows:

1, the blastocoel is less than half of the volume of the embryo;

2, the blastocoel is half of or greater than half of the volume of the embryo; 3, the blastocoel completely fills the embryo;

4, the blastocoel volume is larger than that of the early embryo, with a thinning zona; 5, the trophectoderm is starting to herniate though the zona;

6, the blastocyst has completely escaped from the zona.

For fully formed blastocysts (with a blastocoele grading of 3-6) the inner cell mass is graded as follows:

A, tightly packed, with many cells; B, loosely grouped, with several cells; C, very few cells.

For fully formed blastocysts (with a blastocoele grading of 3-6) the trophectoderm is graded as follows:

A, many cells forming a cohesive epithelium; B, few cells forming a loose epithelium; C, very few large cells.

To determine if trypsin activity varies between high and low quality embryos, 21 individual ECM drops from blastocysts transferred on day 5 were measured using the trypsin assay, and separated according to their morphological grading via the Gardner scale (Figure 3.2.2.3). In brief, groups A and B denote good to average quality embryos whereas group C denotes poor quality embryos. Though numbers are limited, this analysis revealed that increased trypsin activity was detectable in ECM drops from embryos with poorer quality trophectoderm. This may be explained by a propensity of trypsin, produced by the embryo, to leak out between the cells within the trophectoderm layer in cases where the cells are more loosely packed. The result may be decreased trypsin levels within the blastocyst itself potentially impairing the ability of the blastocyst to adequately undergo the hatching process, a prerequisite for implantation.

This work provides novel evidence of trypsin activity being produced by human embryos, which complements the wealth of literature designating a role for trypsin

Figure 3.2.2.1 Protease and Trypsin activity in ECM a) Trypsin activity in day

5 pooled ECM (n = 16). b) Protease activity in individual ECM (n = 49, P =

0.006) using the Enzchek® assay. c) Trypsin activity in individual ECM (n = 38,

P = 0.0015). All measurements were performed in duplicate, normalised to

UCM and are displayed as mean values. Mann Whitney statistical analysis was applied. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3.2.2.2 Trypsin activity and pre-implantation embryo development.

Trypsin activity in individual ECM (n = 39). All measurements were performed in

duplicate, normalised to UCM and are displayed as mean values. Median values are

Figure 3.2.2.3 Trypsin activity and morphological grade. Trypsin activity in

individual ECM from blastocysts transferred on day 5 (n = 21) and separated

according to morphological grade (A = good, B = satisfactory, C = poor). All measurements were performed in duplicate, normalised to UCM and are displayed as mean values. Median values are displayed for each group. Mann Whitney