## 3 Results
### Increased GPR30 Abundance Levels in Human Spermat Are Associated with Clinical Pregnancies  
From the 81 couples seeking medical reproductive treatments at the beginning of this study, embryo transfer (ET) (of one or two embryos) was performed in 60 women. This decision was made by the medical team working with each couple. The reasons for ET not being performed were: the women's physical condition was not ideal for ET and the embryos were cryopreserved (\(n=15\)); lack of fertilization (\(n=2\)); bad embryo development (\(n=2\)); and lack of good-quality oocytes (\(n=2\)). The following analysis concerns only the 60 couples that were allowed to continue with treatment and performed ET. The fertilization rate and embryo development indicators (embryo cleavage rate, high-quality embryo rate, and blastocyst rate) were assessed before embryo transfer. Oocyte donations were used in 16 cases. We could not find any correlation between the _GPR30_ transcript abundance in human spermatozoa and the fertilization rate or with the embryo development indicators (Figure 3). Women were classified with a biochemical pregnancy when the serum beta-human chorionic gonadotropin (\(\beta\)-HCG) concentration surpassed the value of 20 mIU/mL (positive serum \(\beta\)-HCG levels), 12 days after embryo transfer [24]. Pregnancy was not detected in 32 of the initial 60 women (negative serum \(\beta\)-HCG levels). The remaining 28 cases were classified as biochemical pregnancies (positive serum \(\beta\)-HCG levels). After this point, clinicians consider that the embryo has started the process of implantation. Notwithstanding this fact, the rise of \(\beta\)-HCG can be transient and pregnancy can still fail to progress [24]. We could not identify a direct correlation between _GPR30_ mRNA abundance in human spermatozoa and serum \(\beta\)-HCG concentration (Figure 4A). Nevertheless, our data show that higher levels of _GPR30_ mRNA abundance were present in those spermatozoa that originated biochemical pregnancies (1.63 \(\pm\) 0.27 arbitrary units). Concomitantly, lower levels of _GPR30_ transcript abundance were found in spermatozoa where the resulting embryos failed to implant in the uterus (1.13 \(\pm\) 0.17 arbitrary units), \(p=0.0262\) (Figure 4B). In our cohort, up until April 2019, 4 women who were previously classified as biochemically pregnant suffered an abortion. Furthermore, we lost track of 2 women who  
Figure 2: Association of _GPR30_ mRNA expression with paternal body mass index and age. The figure shows the distribution of _GPR30_ mRNA abundance in spermatozoa according to paternal BMI, (range 19.4–33.8 kg/m\({}^{2}\)), \(n=79\) (**A**); and age, (range 27–58 years), \(n=81\) (**B**). The association between _GPR30_ mRNA abundance, paternal BMI, and age was evaluated by computing the Pearson correlation coefficients (r), assuming a Gaussian distribution (confidence interval of 95%). No correlations between the studied parameters were found to be statistically significant. were previously classified with biochemical pregnancies. The remaining pregnancies evolved into clinical pregnancies. To summarize, from the 60 embryo transfers performed, only 22 cases evolved into clinical pregnancies. As before, our data show that the mean of _GPR30_ transcript abundance in human spermatozoa associated with clinical pregnancies (1.68 \(\pm\) 0.33 arbitrary units) was higher than the mean of _GPR30_ transcript abundance in human spermatozoa that were not associated with clinical pregnancies (1.13 \(\pm\) 0.16 arbitrary units), where \(p\) = 0.0288 (Figure 3C). No correlation between _GPR30_ transcript abundance in spermatozoa and paternal age was found (Figure 2B). It is noteworthy that no statistical difference was found regarding the mean maternal age nor the BMI between the clinical pregnancy and non-pregnancy groups (Figure S2). The same finding is true regarding the age and BMI of the oocyte donors for the same groups.