Apoptotic cells were mainly detected in the glandular epithelium of the endometrium. Only very few positive cells were detected in the stroma at any stage of the cycle. In the late proliferative phase of the cycle apoptosis in the glands was scarce (Fig. 16). During the secretory phase, the abundance of apoptotic cells increased and reached a maximum at menstruation. At early proliferation apoptosis was still present but the number of apoptotic cells decreased towards late proliferation (IV: Fig. 3).
DNA fragmentation analysis confirmed the results of in situ 3"-end labelling. Due to the abundance of blood cells present in the curettage samples the quality of analysis was not entirely satisfactory, but it clearly showed that fragmentation was highest in the menstrual phase (IV: Fig. 2, 3, 4).
Figure 16. Presentation of apoptosis in the endometrium during the menstrual cycle in endometrial glands. EP = early proliferative (cycle days 3–9), LP = late proliferative endometrium (10–14), ES = early secretory (15–19), MS = middle secretory (20–26), LS = late secretory endometrium (27–29), ME = menstruating endometrium (1–5)
The intensity of Bcl-2 staining in glandular epithelial cells was highest in the late proliferative phase, although there was considerable variation in staining intensity between the samples. In the mid- and late secretory phase and in the menstruating phase, it was absent or scarce. In the stroma, Bcl-2 immunostaining was negligible throughout the cycle. Only a small decrease in the intensity of staining was detected during the secretory phase. Western analysis of Bcl-2 protein revealed the expected 26 kDa protein in endometrium and a similar expression pattern as seen in immunohistochemistry was observed (IV: Fig. 5, 6, Table 1) (Fig. 17).
Bax was expressed at all phases of the cycle. In glandular epithelium staining was most intense, but the protein was also expressed in the stroma. In the glandular epithelium, the expression of Bax was high during the late proliferative and early secretory phase. In secretory and menstruating endometrium, immunostaining of Bax decreased, but the relative decrease seemed to be less pronounced than that of Bcl-2. In the stroma Bax was also found at all phases of the cycle. In proliferative endometrium, stromal Bax immunostaining was more intense than in the secretory or menstruating phases. In Western analysis of Bax the expected 21 kDa protein was observed, and there
was a decreasing tendency in the amount of protein detected towards menstruation (IV: Fig. 5, 6, Table 1) (Fig. 17).
Cell proliferation in the endometrium was studied by immunostaining the endometrial samples for antigen Ki-67, which is a nuclear protein present in cells at the replicating phase of the cell cycle. As expected, proliferative endometrium revealed intense staining of Ki-67. Positive cells were found mostly in the glandular epithelium, but also in the stroma. The number of immunopositive cells diminished towards the secretory phase, and almost disappeared in the late secretory phase. During menstruation, the expression of Ki-67 was also low. The sections showing high intensity of Ki-67 staining clearly showed very little apoptosis (IV: Fig. 3).