Administration of N-acetylcysteine influence the expression of apoptotic genes in the granulosa cells of infertile women diagnosed with endometriosis

In Table1 , we present a comparison of the demographic and clinical characteristics of patients in the two groups. The analysis of background variables revealed no statistically significant differences between the groups. Consequently, these variables were evenly distributed across both groups before the intervention. Table1 Demographic and clinical characteristics of the patients. Variable NAC-treated group Placebo-treated group P value Infertility period (year) 6.43 ± 4.19 3.45 ± 3.50 0.123 Age (years) 33.88 ± 3.44 31.18 ± 3.99 0.143 Body mass index (kg/m 2 ) 26.15 ± 3.46 25.59 ± 3.01 0.709 AMH (ng/ml) 1.89 ± 0.37 2.54 ± 0.56 0.389 FSH (mIU/mL) 5.8 ± 0.87 7.29 ± 1.16 0.371 LH (mIU/mL) 7.37 ± 0.95 5.56 ± 0.89 0.201 TSH (mIU/mL) 1.74 ± 0.27 2.09 ± 0.31 0.445 Endometrial size before treatment 5.64 ± 3.04 12.39 ± 8.74 0.069 Demographic and clinical characteristics of the patients. Three participants in the NAC group were excluded after randomization between 2021 and 2023; these individuals were excluded from the per-protocol analyses. However, the analysis was conducted using an intention-to-treat approach. Patients who experienced gastrointestinal complications required no special care other than discontinuation of the treatment. Finally, eleven participants in the NAC-treated group and fourteen participants in the placebo group were included in the analyses. A significantly higher level of TAC in serum was observed after NAC treatment ( P  = 0.031). Furthermore, the level of SOD was higher in infertile women with endometriosis after NAC treatment compared to before treatment; however, this difference was not statistically significant ( P  = 0.467) (Fig.  1 . Fig.1 Comparison of biochemical factor before and after intervention (the statistical significance of differences were assessed by Two-tailed t-test. The data are presented as mean ± SD). Comparison of biochemical factor before and after intervention (the statistical significance of differences were assessed by Two-tailed t-test. The data are presented as mean ± SD). Based on the results obtained from this study, the expression level of Bcl-2 increased in NAC-treated patients (0.0017 ± 0.0012) compared to the placebo group (0.0014 ± 0.0011) ( P  = 0.643). The expression of BAX decreased in the GCs of NAC-treated patients (0.0039 ± 0.00237) compared to the placebo group (0.0056 ± 0.00462). Similarly, the expression level of Caspase-3 decreased in NAC-treated patients (0.0066 ± 0.00316) compared to the placebo group (0.0072 ± 0.00735) ( P  = 0.387 and P  = 0.839, respectively) (Fig.  2 ). Fig. 2 Comparison of mRNA Expression expression in two groups (the statistical significance of differences were assessed by Two-tailed t-test. The data are presented as mean ± SD). Comparison of mRNA Expression expression in two groups (the statistical significance of differences were assessed by Two-tailed t-test. The data are presented as mean ± SD). The objective of this study was to investigate the effects of N -acetylcysteine (NAC) on the quantity and quality of oocytes retrieved during ovulation. Comparative analysis between the two groups showed no significant difference in the total number of oocytes obtained. However, a lower percentage of oocytes classified as defunct was observed in the NAC group (4.9%) compared to the placebo group (6.1%). Although the placebo group had a higher incidence of defunct oocytes, this difference was not statistically significant. Additionally, the researchers assessed the quality of embryos generated through ART for the study participants. Embryo evaluation was conducted using the Gardner scoring system. Statistical analysis revealed that the NAC group exhibited superior embryo quality compared to the placebo group (Table 2 ) but these differences were not statistically significant. In the ongoing randomized controlled trial (RCT), three patients in the NAC-treated group have achieved pregnancy, with one live birth reported to date. Table 2 The Oocyte and embryo quality and quantity in patients of both groups during ART cycle.  Drug (Mean ± standard deviation) Placebo (Mean ± standard deviation) P value Oocyte quality (number and types of oocytes retrieved during oocyte collection)  Oocyte No 7/62 ± 4/37 8/90 ± 3/83 0/51  G. V 0/37 ± 1/06 0/54 ± 1/29 0/76  MI 0/37 ± 0/51 0/72 ± 1/48 0/53  MII 6/50 ± 3/42 3/70 ± 7/09 0/72 Embryo quality  Embryo No 4/75 ± 3/10 4/81 ± 3/21 0/96  A 3 ± 2/64 2/42 ± 1/13 0/63  B 3/12 ± 2/53 2/70 ± 1/94 0/69  C 1 ± 1/53 2/50 ± 2/25 0/35 The Oocyte and embryo quality and quantity in patients of both groups during ART cycle. The functional interactions among genes were investigated through network analysis to elucidate molecular and cellular relationships. Results obtained from GeneMANIA indicated that Bax , Bcl-2 , and Caspase-3 interacted not only with each other but also with a group of additional genes, including BID, BCL2L11, BBC3, BCL2L1, DIABLO, NLRP1, BMF, BAD, ACIN1, FKBP8, APPL1, GZMB, BOK, DFFA, BAK1, BIK, PMAIP1, MCL1, AIFM1 , and STK3 , as illustrated based on STRING in Fig. 3 . The nature of these interactions was characterized as follows: 77.64% were physical interactions, 8.01% co-expression, 5.37% predicted interactions, 3.83% co-localization, 2.87% genetic interactions, and 1.88% pathway interactions. Fig. 3 Gene–Gene interactions: BAX, Bcl-2, and Caspase-3 exhibited interactions not only with each other but also with a cohort of additional genes. Gene–Gene interactions: BAX, Bcl-2, and Caspase-3 exhibited interactions not only with each other but also with a cohort of additional genes.

The findings of this study suggest that administration of N -acetylcysteine (NAC) may reduce apoptosis in granulosa cells of infertile patients with endometriosis. This effect appears to be associated with increased expression of Bcl-2 and decreased expression of BAX and Caspase-3, although these changes were not statistically significant. Additionally, NAC may increase serum levels of SOD and TAC. It should be noted that among all the markers studied, only TAC showed a significant increase. Although these results are preliminary, they are promising and should be considered in future studies. The reduction in apoptosis following NAC administration is associated with an increased number of oocytes, improved fetal quality, and consequently higher pregnancy and live birth rates in this patient group; however, these results are not statistically significant. A limitation of this study, which likely contributed to the lack of statistical significance, was the impact of the COVID-19 pandemic, which resulted in reduced patient recruitment. Conducting this study with a larger sample size would provide a clearer understanding of the true effect size of the NAC intervention.

Endometriosis is a multifaceted condition influenced by both genetic and environmental factors. A comprehensive understanding of these contributing factors, along with the effects of pharmacological interventions, may facilitate the development of effective therapeutic strategies 16 . One significant environmental factor associated with endometriosis is oxidative stress, which can trigger apoptosis in granulosa cells. The severity of endometriosis has often been shown to correlate with levels of oxidative stress markers. A reduction in antioxidant enzyme activity may be associated with severe cases of endometriosis. Some treatments for endometriosis also influence oxidative stress pathways. Therefore, assessing oxidative stress markers can help evaluate the effectiveness of these treatments 3 . SOD, an enzyme involved in oxidative stress, plays a crucial role in antioxidant defense. The decreased activity of SOD in the serum of women with endometriosis suggests a reduced antioxidant capacity in these patients. Similarly, lower levels of TAC have been observed in endometriosis patients compared to the control group 17 . The decline in both SOD and TAC may be related to the elevated levels of oxidative stress markers observed in these patients. Consistent with the literature, our study demonstrated a significant increase in serum TAC concentration after treatment with NAC. Additionally, we observed an increase in serum SOD levels following NAC treatment, which is likely an effect of NAC. On the other hand, antioxidant supplements such as NAC have been identified as potential agents for reducing apoptosis in cells due to their antioxidant properties 8 . In the intrinsic apoptotic pathway, the interaction between Bax , a pro-apoptotic gene, and Bcl-2 , an anti-apoptotic gene, is crucial in determining the initiation of apoptosis. The expression of Bax leads to the release of cytochrome c, which subsequently activates Caspase-9 and Caspase-3 , while Bcl-2 expression inhibits this cascade, thereby reducing apoptosis 18 . Our results showed that the level of Bcl-2 in granulosa cells was increased in NAC-supplemented patients compared to the placebo group. Additionally, our study demonstrated lower expression levels of BAX and Caspase-3 in the GCs of the NAC-treated group compared to the placebo; however, this reduction did not reach statistical significance. Yang et al. conducted research on COV434 human granulosa tumor cells treated with H2O2 to elucidate the molecular link between ROS and granulosa cell death. They observed that granulosa cells are sensitive to oxidative stress and the ROS generated from it. As excess ROS levels increased, cell death correspondingly increased due to elevated expression of Caspase-3 and Bax genes, along with decreased expression of the Bcl-2 gene. The addition of NAC to the medium prevented ROS production and inhibited Caspase-3 activity 12 . According to another 2017 study investigating granulosa cells in mice, NAC was administered intraperitoneally at a dose of 300 mg/kg for 7 days. The study found that NAC, in addition to normalizing abnormal hormone levels by reducing oxidative stress and promoting embryo implantation, reduced Caspase-3 gene expression and altered apoptosis levels in granulosa cells 19 . Yu and colleagues’ study on a rat model of kidney damage concluded that NAC increased Bcl-2 expression and decreased the expression of BAX and Caspase-3. These changes in gene expression in kidney cells were associated with reduced apoptosis 20 . The results of our study are consistent with the findings reported in these three studies. Our findings demonstrated that the number of apoptotic oocytes was higher in the placebo group compared to the NAC group. Supporting this observation, Yang et al. reported that apoptosis in granulosa cells disrupts intercellular communication and deprives oocytes of essential nutrients, ultimately leading to oocyte apoptosis and follicular atresia 12 . The results of our study regarding ovulated oocyte apoptosis are consistent with these findings, suggesting that NAC may decrease ovulated oocyte apoptosis; however, this result did not reach statistical significance, potentially due to the limited sample size. Additionally, a study by Fan et al., which included 58 mouse samples, indicated that NAC could enhance fetal quality by reducing oxidative stress and improving fertility rates 21 . In our investigation, the quality of fetuses in the NAC group was marginally superior to that in the placebo group; however, this difference was not statistically significant. Research focused on infertility-related conditions, such as endometriosis, aims to improve fertility outcomes for affected individuals. N -acetylcysteine, as an antioxidant supplement, is utilized for a range of health concerns, including female fertility. There is controversy regarding the effects of NAC on female fertility; some studies report mixed outcomes concerning its impact on endometriosis and fertility. In our study, we evaluated the impact of NAC on the fertility rates of infertile patients with endometriosis. Analysis of pregnancy rates among patients undergoing embryo transfer cycles suggested that NAC may enhance pregnancy rates, aligning with the findings of Fan et al. 21 and another study that reported a high pregnancy rate among 120 women who received NAC supplementation. Conversely, another study found no notable difference in pregnancy rates when 47 NAC-administered patients were compared to 45 non-supplemented patients in the control group 22 . Furthermore, a study involving 130 women with polycystic ovary syndrome (PCOS) demonstrated that NAC increased both the number of oocytes and the pregnancy rate in this population 23 . Nonetheless, our study was constrained by a limited patient sample, and further research with a larger cohort is necessary to comprehensively assess the effects of NAC on patients with endometriosis. Overall, the findings in this area suggest beneficial effects of NAC, particularly regarding the development of endometriosis.

This study was designed as a randomized clinical trial and was registered in the database of privately and publicly funded clinical studies conducted around the world ( https://clinicaltrials.gov/ ) under registration number NCT05460858 , at 15/07/2022. The project was conducted at the Royan Institute in Tehran, Iran, from 2021 to 2023 and was registered with the National Ethical Committee under number IR.ACECR.ROYAN.REC.1398.111, at 16.07.2019. In the 2023 study by Wicaksana et al. 24 , the average expression of the Bcl-2 gene in the control group (n = 17) was compared to that in the intervention group (n = 17), revealing a difference of 3 units. Based on this report, and assuming a statistical power of 80% and an alpha level of 0.05, the estimated sample size was 14 participants per group, accounting for a 10% dropout rate. Infertile women aged 22 to 38 years with endometriosis were assigned to receive either N -acetylcysteine (NAC) supplementation (n = 14) or a placebo (n = 14). However, there were three dropouts in the NAC group: two participants were discontinued due to gastrointestinal complications likely related to the supplement, and one participant withdrew for personal reasons. Therefore, the actual number of patients in the intervention group was 11 (Fig.   4 ). Written informed consent was obtained from all participants. The effervescent tablets, containing either the NAC supplement (600 mg) or a placebo, were prepared with identical packaging. Over a six-week period, coinciding with the initiation of ovulation induction, patients were administered two effervescent tablets daily (2 × 600 mg), resulting in a total daily dose of 1200 mg of either NAC or placebo for the entire study duration. Before and after supplementation, SOD activity and TAC concentration were measured in blood samples as the primary outcomes. Secondary outcomes included the expression levels of Bcl-2, Bax, and Caspase-3 in the granulosa cells of all participants. This study is a randomized, double-blind, placebo-controlled clinical trial with two parallel arms. Randomization codes were generated using the permuted block randomization method available on the website http://www.randomization.com . Block sizes varied, including blocks of 4 and 6. The drug and placebo were obtained from a pharmaceutical company (NAC supplements are produced by multiple pharmaceutical companies with Iranian FDA approval; we selected one of them) and were packaged identically. Labeling and coding were conducted in a double-blind manner at the start of the study by the correspondent. These codes were stored as randomization and grouping codes A and B in an Excel file. The drug or placebo was administered to patients according to the predetermined randomization sequence, and each patient was assigned a unique identification code that remained unchanged throughout the study. None of the project team members, except the correspondent, had access to the random assignment sequence, and they were completely blinded. The inclusion criteria for this study were as follows: participants were required to be between 20 and 42 years of age and to have moderate to severe clinical stages of endometriosis, specifically classified as stage III or IV according to the American Society for Reproductive Medicine (ASRM, 1997). Additionally, serum anti-Müllerian hormone (AMH) levels needed to fall within the range of 0.7 to 4.5 ng/ml, and participants were required to exhibit a normal hormonal profile, including follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH). Furthermore, participants had to undergo a standard long gonadotropin-releasing hormone (GnRH) agonist or antagonist ovulation stimulation cycle and possess a body mass index (BMI) of less than 30 kg/m 2 . Women were excluded from participation if they had congenital uterine malformations or a medical history of asthma. Additionally, if their spouse had severe male infertility requiring testicular sperm extraction (TESE) or percutaneous epididymal sperm aspiration (PESA), they were also excluded from the study. Blood samples in EDTA tube were obtained from all patients before and after the intervention to evaluate SOD (as an antioxidant enzyme) activities and TAC (as an OS marker) concentration. The blood samples were centrifuged at 1500 rpm for 10 min and serum was separated and then stored at − 80°C until evaluation. To investigate the levels of SOD and TAC, an enzyme-linked immunosorbent assay (ELISA) was performed. The levels of TAC were measured using commercial kits (Zell Bio GmbH, Wurttemberg and Germany) while the SOD levels were assessed using an ELISA kit (Abnova Corporation, Taiwan) in serum samples. Follicular fluid (FF) was collected during ovarian puncture approximately 34–36 h post-hCG (human chronic gonadotropin) injection and subsequently transferred to the research laboratory. Granulosa cells within the follicular fluid were isolated using a concentration gradient method. At the first step, the FF was centrifuged at 2000 rpm for 10 min at room temperature. The supernatant was discarded, and the pellet was resuspended in Tyrode’s solution. This suspension was then gradually layered onto a Sil-Select gradient (a 1:1 mixture of Sil-Select™ (Fertipro, Beernem, Belgium) and Tyrode’s solution) and centrifuged for 13 min at 3000 rpm. The layer formed between the Sill select gradient and the supernatant, which contained the granulosa cells, was collected and washed with 3 ml of DMEM/HamF12 medium (a 1:1 mixture of Dulbecco’s Modified Essential Medium (DMEM) and Ham’s F-12 Medium) supplemented with 10% fetal bovine serum (FBS). The resulting suspensions were centrifuged for an additional 13 min at 3000 rpm, after which the supernatant was removed from the pellet. Hyaluronidase enzyme was then added to the pellet and incubated for 5 min. Following this incubation, DMEM/HamF12 medium with 10% FBS was introduced to neutralize the enzyme, and the mixture was centrifuged at 1500 rpm for 5 min. After the supernatant was removed, the pellet containing the granulosa cells was prepared for RNA extraction. To extract total RNA, we utilized the RNeasy Plus Mini Kit (Qiagen, cat. no. 74134) in accordance with the manufacturer’s protocol (Tansey, M., 2024). In brief cell pellet in 350uL of BME + RLT lysis buffer and vortex. Transfer to qiashredder tubes and spin at 21300xg for 2min. Freeze flow-through if needed. Add 350uL of 70% ethanol and mix. Transfer 700μl of the sample to a RNeasy spin column, centrifuge, and discard flow-through. Add Buffer RW1, centrifuge, and discard flow-through. Add Buffer RPE, centrifuge, and discard flow-through. Repeat this step and then spin for 2 min. Place the column in a new tube, add 20μl RNase-free water, and centrifuge to elute RNA. Re-elute for greater yield. Store samples at − 80°. This kit is designed to purify total RNA from up to 10 7 cells and includes gDNA Eliminator columns to ensure that the extracted RNA is free from gDNA contamination. Subsequently, we employed the NanoDrop spectrophotometer (NanoDrop 2000) to assess the quantity and quality of the RNA, specifically evaluating the A260/A280 ratio. cDNA was synthesized from 500 ng of total RNA by using the Easy cDNA Synthesis Kit (Parstous, cat. no. A101161) and was stored at − 80°C being further processing. Following the synthesis of complementary DNA (cDNA), gene expression quantification was performed using a Real-Time PCR system, specifically the ABI Step-One RT-PCR system for quantitative reverse transcription PCR (qRT-PCR) (Applied Biosystems, USA). The reaction was conducted in a total volume of 10 μl, which comprised 2 μl of cDNA (25 ng/μl), 2.5 μl of deionized water (H2O), 3.5 μl of SYBR Premix Ex Taq II Kits (Takara, Japan), and 2 μl of specific primers (Table 1 ) at a concentration of 5 pmol/ml targeting Bcl-2 , BAX , Caspase-3 , and glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ). The primer for the control gene, GAPDH , was sourced from the primer bank of the Royan Institute. An evaluation of mRNA gene expression levels of Bcl-2 , BAX , and Caspase-3 was conducted between two groups: the NAC-treated group and the placebo group. This evaluation was performed by calculating ΔCt (threshold cycle) and 2 -ΔCt . Quantitative polymerase chain reaction (qPCR) was carried out using human-specific primers. The product sizes and primer sets are detailed in Table 4 . The GAPDH gene served as the internal control for comparison. The Gene Multiple Association Network Integration Algorithm (GeneMANIA) ( http://www.genemania.org/ ) was employed to generate gene interaction data, while STRING ( http://string-db.org/ ) was utilized to establish functional associations among the genes identified through GeneMANIA. GeneMANIA is an accessible online tool that integrates extensive genetic and protein information to investigate the functions of specific genes and present the findings (Xueting et al., 2024). The Kolmogorov–Smirnov test was employed to assess the normality of quantitative data across two groups. Following the confirmation of normal distribution, the quantitative data were expressed as mean ± standard deviation or standard error. An independent t-test was utilized to compare the means of the quantitative variables between the two groups, while the chi-square test was applied to compare the frequency (percentage) of qualitative variables. Additionally, Pearson’s correlation coefficient was used to evaluate the relationship between genes. A significance level of 0.05 was established for all statistical analyses.

Endometriosis is an estrogen-dependent disorder characterized by the ectopic growth of endometrial tissue outside the uterine cavity. This condition affects approximately 10% of women and girls of reproductive age 1 . Common symptoms associated with endometriosis include pelvic pain, dyspareunia, fatigue, dysmenorrhea, and infertility 2 , 3 . The disease is classified into four stages (I–IV), which provide a more comprehensive understanding of its progression. In stages I and II, adhesions are minimal; however, in stage III, adhesions may develop in the fallopian tubes or ovaries, with a more extensive presence of adhesions observed in stage IV. Infertility is notably more prevalent in stages III and IV 4 . Diagnosis of endometriosis involves a thorough examination of the patient’s clinical history and may incorporate imaging techniques such as ultrasound and magnetic resonance imaging (MRI) to enhance diagnostic accuracy. Treatment options aimed at alleviating symptoms include pharmacological interventions and surgical procedures 5 . Assisted reproductive technologies (ART), such as intrauterine insemination (IUI) and in vitro fertilization (IVF), may be used to address infertility associated with endometriosis 6 . The pathophysiology of endometriosis is influenced by multiple factors, including environmental elements such as oxidative stress and inflammation, as well as genetic and epigenetic factors 5 . Oxygen toxicity at elevated pressures can adversely affect the respiratory, cardiovascular, nervous, and digestive systems 7 . Reactive oxygen-containing substances, such as nitric oxide (NO) and reactive oxygen species (ROS), can have detrimental effects on the body, leading to cellular damage. ROS, which includes free radicals and oxygen intermediates, is produced in limited quantities within the body as a byproduct of the electron transport chain. However, excessive accumulation of ROS can induce oxidative stress within the organism 8 , 9 . Several studies investigating oxidative stress and antioxidants have revealed that an excess of oxidants, combined with an insufficient supply of endogenous antioxidants, may play a critical role in the development of endometriosis. The body contains several antioxidant enzymes, including superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase, and glutathione transferase, which neutralize reactive oxygen species (ROS). Among these enzymes, SOD is particularly important in protecting cells from oxidative damage by converting the superoxide anion radical into hydrogen peroxide, thereby safeguarding cellular integrity. However, SOD is not widely used in human medicine 3 , 10 . Antioxidant defense levels are commonly measured as total antioxidant capacity (TAC) 11 . Oxidative stress (OS) may contribute to the etiology of endometriosis by influencing various biological pathways and represents a potential therapeutic target for preventing and managing endometriosis symptoms 7 . Granulosa cells surrounding oocytes are particularly sensitive to ROS 12 . An increase in ROS, resulting from disruptions in multiple pathways, ultimately leads to decreased fertilization rates and an elevated risk of infertility. One such pathway is apoptosis; disruptions in this pathway and its associated gene expression can result in a diminished number of granulosa cells, a condition referred to as atherosclerosis 12 . Within the intrinsic apoptotic pathway, pro-apoptotic molecules such as cytochrome c are released into the cytosol from the mitochondrial intermembrane space in response to oxidative stress 13 . The anti-apoptotic proteins Bcl-2 and Bcl-XL , members of the BCL-2 family, inhibit the release of cytochrome c. In contrast, pro-apoptotic BCL-2 family proteins, such as BAX , promote cytochrome c release. Cytochrome c, together with Apaf-1 and procaspase-9, assembles into a multiprotein complex called the apoptosome, which activates the Caspase-3 signaling cascade through Caspase-9 activation, ultimately leading to apoptosis 14 . N -acetylcysteine (NAC) is an affordable and widely accessible antioxidant medication 3 . Furthermore, it is utilized as an antioxidant to mitigate oxidative stress in various diseases due to its potent free radical-scavenging properties 3 . NAC supplementation has been shown to directly reduce levels of reactive oxygen species (ROS) 15 . Additionally, NAC can indirectly decrease excess ROS by enhancing intracellular glutathione (GSH) levels 12 . Moreover, this compound has the potential to inhibit abnormal cell proliferation and modify cellular behavior from proliferation toward differentiation, thereby reducing endometrial masses 3 . Although previous studies have explored the effects of NAC in specific conditions, there is a paucity of research examining its impact on the expression of apoptotic genes such as Bcl-2 , Bax , and Caspase-3 in the context of endometriosis-related infertility. Therefore, this study aims to investigate the activity of SOD and TAC concentrations in serum, as well as to quantify the gene expression levels of Bcl-2 , Bax , and Caspase-3 in granulosa cells (GCs) using Real-Time Polymerase Chain Reaction ( ). Fig.  4 The consort flow diagram. Table   4 Primers used for qPCR analysis. Gene Primer sequence Size (bp) BCL-2 F ATT CCT GCG GAT TGA CAT TTC 116 BCL-2 R GCT GAT TTG AAA CTT CCC AAT G BAX F TGC TTC AGG GTT TCA TCC AG 146 BAX R CAT GTT ACT GTC CAG TTC GTC C CASPASE3 F AAG CAC TGG AAT GAC ATC TC 141 CASPASE3 R GAA ACA TCA CGC ATC AAT TCC The consort flow diagram. Primers used for qPCR analysis.