## 4 Discussion  
The postponement of childbearing to the fourth decade of life has made reproductive aging a central topic that entails negative consequences for individual fertility and pregnancy outcome but also for society. Reproductive organs are exposed to metabolites that irreversibly accumulate during the reproductive lifespan, including compounds such as AGEs [25]. The main precursors of AGEs are reactive dicarbonyls, such as MG.MG, which is a ubiquitous reactive dicarbonyl that is eliminated by the glyoxalase system [18; 21]. However, some reactive dicarbonyls can escape detoxification and react to form stable AGEs. The accumulation of reactive dicarbonyl compounds can be caused by decreased elimination or increased formation. In the current study, we focused on the key enzyme of the detoxification system, GLO1, in reproductive tissues and embryos of old and young rabbits. GLO1 represents the frontline defence against dicarbonyl glycation in physiological systems, limiting dicarbonyl stress [21; 22]. We could show that GLO1 is expressed in the ovary, oviduct, uterus and blastocyst of rabbits. We observed lower levels of GLO1 in the endometrium of old rabbits whilst the specific GLO1 activity remained the same, implicating an absolute decrease of GLO1 activity in this tissue. In the ovary of polycystic ovary syndrome (PCOS) rats, AGEs lead to reduced GLO1 [37]. In the immunohistochemical analysis of the uterus we observed an increased accumulation of the AGE argpyrimidine in endometrial cells of old rabbits. The decreased amount of GLO1 in the endometrium may be caused by higher AGE levels in the uterine environment of old rabbits. Our findings indicate an impaired AGE defence and higher exposure to dicarbonyl stress in endometrial cells--the cell layer that directly forms and interacts with the surrounding milieu of the developing blastocyst. The importance of GLO1 has been shown in several knock down studies. The lack of GLO1 in cell culture and animal models results in increased levels of free MG and MG-derived AGEs [38; 39]. GLO1 activity is inversely correlated to elevated levels of AGEs and androgen levels, in consequence leading to hormonal imbalance [37]. Hormonal imbalance can impair fertility and pregnancy establishment, as observed in women with advanced maternal age [40]. Lower levels of GLO1  
Figure 5: Quantification of GLO1 mRNA and protein amount in 6-day old blastocysts from young and old rabbits. (**A**) GLO1 mRNA levels were quantified by qPCR. (\(N=2\), \(n\geq 11\)) (**B**) For GLO1 protein analysis by the Simple Western method pooled embryonic samples with 5–13 blastocysts were used (\(N=2\), \(n\geq 4\)). Results are shown as box plots. Relative mRNA and protein amount are normalised to the levels of vinculin. All values are expressed in per cent, with young = 100%. in the endometrium may indicate higher AGE levels and oxidative stress in the uterine cavity fluid, the surrounding milieu of the developing embryo. We have shown already that AGEs such as argpyrimidine, CML and pentosidine are present in rabbit blastocysts [28]. Further research is needed to analyse whether oxidative stress and the levels of AGEs are increased in the blastocysts of old rabbits. In humans, AGE-modified proteins were detected in follicular cell layers, granulosa and theca cells and in luteinised cells, with a more prominent staining in granulosa cells of women with PCOS [41]. In PCOS patients, AGEs are able to modulate extracellular matrix organisation, leading to deregulation of folliculogenesis [42]. Increased MG and AGE levels, paired with a lower GLO1 expression, resulted in ovarian proteome damage in the mouse model [25]. Accumulation of AGEs in the ovary may, therefore, be one reason for the impaired folliculogenesis in women with an advanced maternal age [43]. A possible correlation between AGEs and follicle aging was first described by an observational study. They could show that the AGE pentosidine was increased in the primordial, primary and atretic follicles of postmenopausal women [44].