Semen samples were provided from 113 volunteer patients at the Naresuan Infertility Center, Faculty of Medicine, Naresuan University, Thailand. The experimental protocols were approved by the Institutional Review Board of Naresuan University (IRB No. 0549/60). A signed consent form was obtained from all volunteers. The semen samples were collected by masturbation, following 3–4 days of sexual abstinence, and allowed to liquefy for 30–60 minutes at room temperature. The semen samples were then assessed for liquefaction, appearance, viscosity, pH, volume, and sperm quality (including sperm concentration, motility, and morphology) following the World Health Organization (2010) guidelines [30]. An overview of the sample composition is shown in Figure 1. Ninety-nine semen samples with normal appearance, liquefaction, and pH values along with a sperm concentration ≥5 ×10⁶/mL were included in this study, while 14 semen samples with a sperm concentration <5 ×10⁶/mL were excluded. The included samples were divided into three groups: NOR (n=32), OAT (n=22), and TER (n=45). The volunteer patients in the NOR group had normal sperm concentrations (≥15 ×10⁶/mL), progressive motility (PR; ≥32%) and morphology (normal form ≥4%), while those in the OAT group had abnormal sperm concentrations (<15 ×10⁶/mL), PR (<32%), and morphology (normal form <4%). Volunteer patients with normal sperm concentration and PR, but abnormal sperm morphology, were defined as TER men.

A drop (~10 µL) of the semen sample was loaded into a Makler counting chamber. Sperm concentration and motility were recorded using an Optikam microscope camera and software (OPTIKA Microscopes, Ponteranica, Italy) under a bright-field microscope at ×200 magnification. The average number of sperm per 10 squares was defined as the sperm concentration (×10⁶ sperm/mL). The sperm were also graded into PR, non-progressive motility (NP), and immotility. The total of motile sperm was defined as the sum of PR and NP. For sperm morphology analysis, a drop of semen was smeared on a glass slide, allowed to air-dry, fixed, and stained using the Diff-Quick stain. Approximately 200 sperm were examined for sperm morphology under a bright-field microscope at ×1,000 magnification. The percentage of sperm with normal and abnormal morphology was calculated. The label of each slide for the sperm morphology analysis was blinded. For the study of gene expression, all semen samples were stored at –80°C until used.

Total RNA was isolated from 5–10 ×10⁶ sperm in each sample using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and chloroform. The RNA was converted to complementary DNA (cDNA) using a qScript XLT cDNA Supermix (Quanta Biosciences, Beverly, MA, USA) according to the manufacturer’s protocol. The synthesized cDNA was used as a template for reverse transcription-polymerase chain reaction. Oligonucleotide primers for GABA receptors, GABA A-α1 and GABA B-R2 receptors, and GAPDH (reference gene used as an internal control) genes were used following previous studies [31-33], as shown in Table 1. Complementarity with other sequences in the human genome was searched using BLAST on the NCBI website (freely available at https://blast.ncbi.nlm.nih.gov/Blast.cgi). The oligonucleotide primers were synthesized by Macrogen (Macrogen Inc., Seoul, Korea). Each PCR product was amplified from 2 ng of cDNA template using 2×SensiFAST SYBR No-Rox (BIO-98005, Bioline, London, UK) and performed on the LineGene 9600 Plus QPCR system (Bioer, Hangzhou, China). The expression levels of GABA receptor genes were normalized to the GAPDH gene and represented as relative mRNA expression values [34].

In total, 35 volunteer patients received ICSI treatment at the Naresuan Infertility Centre, Faculty of Medicine, Naresuan University, Thailand, including 10 of the 32 patients in the NOR group, 10 of the 22 patients in the OAT group, and 15 of the 45 patients in the TER group (Figure 1). The female partner of each patient undergoing ICSI underwent controlled ovarian stimulation using a standard protocol (a gonadotropin-releasing hormone antagonist protocol), and oocyte retrieval, as previously described [35]. After incubation for 3 hours, oocyte denudation was performed using a hyaluronidase solution (80 IU/mL hyaluronidase in FertiCult Flushing Medium; FertiPro NV, Beernem, Belgium). Sperm were prepared using two-layer density gradient centrifugation, with 45% and 90% of Sil-Select solutions (FertiPro NV), following the standard swim-up method. After ICSI, the fertilization rate and embryo quality were assessed. The fertilization of the injected oocyte was monitored on day 1 (~16–18 hours after ICSI). An embryo that showed two polar bodies (first and second polar bodies) and two pronuclei was defined as a fertilized oocyte. Embryo quality in each stage of embryo development was then observed from day 2 to 5 after ICSI, including the cleavage stage on day 3; the morula stage on day 4; and the blastocyst stage on day 5. The cell number, symmetry, and fragmentation were used to verify the embryo quality at the cleavage stage. A good-quality embryo was defined as having 6–8 cells and less than 10% cytoplasmic fragmentation [36]. At the morula stage, an embryo with evidence of compaction was defined as a good-quality embryo. Blastocyst formation, the inner cell mass (ICM), and the trophectoderm (TE) were used to assess embryo quality at the blastocyst stage according to the guideline of Gardner et al. [37]. Blastocyst formation was evaluated using 6 grades based on the degree of expansion and hatching status, as follows: grade 1, early blastocyst; grade 2, blastocyst; grade 3, full blastocyst; grade 4, expanded blastocyst; grade 5, hatching blastocyst; and grade 6, hatched blastocyst. The ICM was categorized as grade A, tightly packed with many cells; grade B, loosely grouped with several cells; and grade C, very few cells. The TE scores were grade A, many cells forming a cohesive epithelium; grade B, few cells forming a loose epithelium; and grade C, very few cells [37]. In this study, the embryo quality at the blastocyst stage was divided into good (3–6 with AA, AB, BA, or BB), moderate (3–6 with BC, CB, or CC), and poor (1–2 regardless of ICM and TE grades and arrested embryos; defective at reaching the blastocyst stage). The fertilization rate and number of embryos of good, moderate, and poor quality in each stage of embryo development were recorded.

To investigate the relationship between the mRNA expression of GABA A-α1 and GABA B-R2 receptors and the embryo quality on cleavage stage, all patients undergoing ICSI were divided into two groups based on the percentage of good-quality embryos at the cleavage stage: good (having a >50% proportion of good-quality embryos, n=22) and poor (having a ≤50% proportion of good-quality embryos, n=13).

The normality of the data distribution was determined using the Shapiro-Wilk test. The statistical significance of differences between the two groups was analyzed using the Student t-test (for parametric data) and the Mann-Whitney test (for nonparametric data). The statistical significance of differences among three groups was analyzed using one-way analysis of variance followed by the Dunnett post hoc test (for parametric data) and the Kruskal-Wallis test followed by the Dunn multiple comparison test (for nonparametric data). Moreover, the Pearson correlation coefficient was used to investigate the relationships of the relative mRNA expression of GABA receptors with sperm parameters, fertilization rate, and embryo quality. The chi-square test was used to compare the categorical variables of groups of volunteer patients regarding the rate of fertilization and embryo quality. Statistical significance was considered to be indicated by p-values <0.05.

The results of the semen analysis for all parameters are shown in Table 2. There were no significant differences in the age of the male patients or semen volume in the OAT and TER groups compared to the NOR group. The OAT group had significantly lower sperm concentration, PR, total motility, and normal morphology than the NOR group. A significantly lower percentage of sperm with normal sperm morphology was found in the TER group than in the NOR group.

Significantly higher mRNA expression of the GABA A-α1 and GABA B-R2 receptors was found in the OAT group than in the NOR group, as shown in Figure 2A (GABA A-α1: 1.7±0.2 vs. 1.0±0.1 for OAT vs. NOR) and Figure 2B (GABA B-R2: 2.2±0.3 vs. 1.0±0.2 for OAT vs. NOR); however, the differences between the TER and NOR groups were not significant.

As shown in Table 3, a significantly lower proportion of good-quality embryos at the cleavage stage was found in the OAT and TER groups than in the NOR group. There were no significant differences between groups in the age of the female partner, fertilization rate, or embryo quality in the morula and blastocyst stages.

High expression of both GABA A-α1 and GABA B-R2 receptors was significantly correlated with low sperm concentration, PR, total motility, and normal morphology (Figure 3). Patients in the good group had significantly lower levels of GABA A-α1 receptor expression than patients in the poor group (1.00±0.16 vs. 2.24±0.40), but a significant difference was not found for GABA B-R2 receptor expression (1.32±0.27 vs. 1.72±0.40), as shown in Figure 4. A strong negative correlation was present between the expression of the GABA A-α1 receptor and the percentage of good-quality embryos at the cleavage stage (r=–0.464, p=0.005) (Figure 5A); however, a significant correlation was not found for GABA B-R2 receptor expression (r=–0.227, p=0.191) (Figure 5B). Moreover, the percentage of good-quality embryos at the cleavage stage exhibited a positive correlation with normal sperm morphology (r=0.468, p=0.005) (Figure 6). There were no significant correlations between the expression of GABA receptors and other outcomes, including fertilization rate and embryo quality at the morula and blastocyst stages (data not shown).