Altered transcriptome in cumulus cells of infertile women with advanced endometriosis with and without endometrioma.

RESEARCH QUESTION: Is the transcriptome of cumulus cells of infertile women with advanced endometriosis (EIII/IV), with and without endometrioma, altered? DESIGN: In this prospective case-control study, next-generation RNA sequencing was used to compare the transcript profile of cumulus cells among infertile patients undergoing ovarian stimulation for intracytoplasmic sperm injection with EIII/IV, with (n = 9) and without endometrioma (n = 9), and controls (n = 9). An in-silico enrichment analysis was conducted to establish the possibly altered pathways in cumulus cells of patients with endometriosis. RESULTS: Most of the differentially expressed genes (DEG) were found when cumulus cells from women with EIII/IV with endometrioma were compared with controls (DEG, n = 461). In women with EIII/IV without endometrioma, only 66 DEG were verified compared with controls. The enrichment analysis showed that some DEG in cumulus cells of endometriosis are involved in important pathways for the oocyte competence acquisition, such as oxidative phosphorylation, metabolism, mitochondrial function, acetylation and steroid biosynthesis. No DEG were found when cumulus cells from women with EIII/IV with and without endometrioma were compared. CONCLUSION: RNA sequencing results suggest that cumulus cells of infertile women with EIII/IV have an altered transcriptome, regardless of endometrioma. The present findings offer a better understanding of the genes and molecular mechanisms that may be involved in endometriosis-related infertility, mostly in the oocyte competence acquisition process.

Oocyte oxidative DNA damage may be involved in minimal/mild endometriosis-related infertility.

Early endometriosis is associated with infertility, and oxidative stress may play a role in the pathogenesis of disease-related infertility. This prospective case-control study aimed to compare the presence of oxidative stress markers in the follicular microenvironment and systemic circulation of infertile women with minimal/mild endometriosis (EI/II) versus individuals undergoing controlled ovarian stimulation for intracytoplasmic sperm injection (ICSI). Seventy-one blood samples (27 from infertile women with EI/II and 44 controls with tubal and/or male infertility factor) and 51 follicular fluid samples (19 EI/II and 32 controls) were obtained on the day of oocyte retrieval. Total hydroperoxides (FOX1 ), reduced glutathione, vitamin E, Superoxide dismutase, total antioxidant capacity, malondialdehyde, advanced oxidation protein products, and 8-hydroxy-2'-deoxyguanosine (8OHdG) concentrations were measured in both fluids. Women with EI/II showed higher FOX1 (8.48 ± 1.72 vs. 7.69 ± 1.71 µmol/g protein) and lower total antioxidant capacity (0.38 ± 0.18 vs. 0.46 ± 0.15 mEq Trolox/L) concentrations in serum, and higher 8OHdG concentrations (24.21 ± 8.56 vs. 17.22 ± 5.6 ng/ml) in follicular fluid compared with controls. These data implicate both systemic and follicular oxidative stress may in infertile women with EI/II undergoing controlled ovarian stimulation for ICSI. Furthermore, the elevated 8OHdG concentrations in follicular fluid of women with EI/II may be related to compromised oocyte quality.

PTGS2 down-regulation in cumulus cells of infertile women with endometriosis.

A deleterious effect of endometriosis on oocyte quality has been proposed. Evidence suggests that cumulus cells could be used as indirect biomarkers of oocyte quality. The PTGS2 gene, which encodes cyclooxygenase 2 (COX-2), is deregulated in endometriotic lesions and plays a crucial role in the acquisition of oocyte competence. To date, research evaluating PTGS2 expression in cumulus cells of infertile patients with endometriosis has not been conducted. The aim this study was to compare the expression levels of PTGS2 in cumulus cells of infertile women, with and without endometriosis, undergoing ovarian stimulation for intracytoplasmic sperm injection (ICSI). Therefore, a case-control study compared PTGS2 gene expression in the cumulus cells of 38 infertile patients with endometriosis and 40 without, using real-time polymerase chain reaction. For the first time, decreased expression of PTGS2 was found in cumulus cells of infertile women with endometriosis compared with controls (7.2 ± 10.5 versus 12.4 ± 15.7), which might be related to reduced levels of COX-2 in the cumulus cells of women with the disease. Consequently, we hypothesize that lower transcript levels of PTGS2 in cumulus cells may be involved in the impairment of oocyte quality, suggesting a possible mechanism involved in disease-related infertility.

Ultrastructural Evaluation of Eutopic Endometrium of Infertile Women With and Without Endometriosis During the Window of Implantation: A Pilot Study.

Endometriosis is frequently associated with infertility and it is believed that the impairment of endometrial receptivity may be one of the mechanisms involved in this condition. Pinopodes are endometrial cycle-dependent structures that seem to participate in embryo implantation, and alterations in their presence and/or morphology during the window of implantation could affect the endometrial receptivity and be involved in the disease-related infertility. However, the data on pinopodes in these women are scarce and inconclusive. This pilot study aimed to evaluate the cell pattern, including the presence and developmental stage of pinopodes, in eutopic endometrium of infertile patients with and without endometriosis during the window of implantation, using scanning electron microscopy (SEM). Endometrial biopsies were performed using a Pipelle catheter, and 12 samples classified in the window of implantation (6 infertile women with endometriosis and 6 infertile controls) were analyzed by SEM. The frequencies of cell types (microvilli, ciliated, and pinopodes) and the developmental stage of pinopodes were compared between groups using Mann-Whitney U test. Although the study was limited by its sample size, no large differences were detected between the groups regarding the presence and developmental stage of pinopodes, suggesting the absence of large structural changes in the endometrium of infertile women with endometriosis during the window of implantation.

Increased concentration of 8-hydroxy-2'-deoxyguanosine in follicular fluid of infertile women with endometriosis.

Impaired oocyte quality and oxidative stress might be involved in the pathogenesis of endometriosis-related infertility. To improve our understanding of the role of oxidative stress in this condition, we compare eight oxidative stress markers from each stage, including the simultaneous analysis of lipids, proteins and DNA damage, in the serum and follicular fluid of infertile women with endometriosis and infertile controls undergoing controlled ovarian stimulation for intracytoplasmic sperm injection. In total, 87 serum samples (43 with endometriosis, 44 controls) and 61 follicular fluid samples (29 with endometriosis, 32 controls) free of blood contamination upon visual inspection and presenting granulosa cells alone or granulosa cells plus a retrieved mature oocyte were collected on the day of oocyte retrieval. Total hydroperoxides, malondialdehyde, advanced oxidation protein products, glutathione, superoxide dismutase (SOD) and total antioxidant capacity (TAC) were determined by spectrophotometry, vitamin E by high-performance liquid chromatography and 8-hydroxy-2'-deoxyguanosine (8OHdG) by enzyme-linked immunosorbent assay. The endometriosis group showed higher serum concentrations of glutathione and SOD, lower serum concentrations of TAC and higher follicular concentrations of 8OHdG and vitamin E compared with infertile controls. These data indicate both systemic and follicular oxidative stress in infertile patients with endometriosis. For the first time, we demonstrate the presence of oxidative DNA damage, represented by higher 8OHdG concentrations in the follicular microenvironment of these patients, possibly related to compromised oocyte quality and associated with the pathogenesis of endometriosis-related infertility.

N-Acetyl-Cysteine and l-Carnitine Prevent Meiotic Oocyte Damage Induced by Follicular Fluid From Infertile Women With Mild Endometriosis.

This study evaluated the potential protective effect of the antioxidants, l-carnitine (LC) and N-acetyl-cysteine (NAC), in preventing meiotic oocyte damage induced by follicular fluid (FF) from infertile women with mild endometriosis (ME). We performed an experimental study. The FF samples were obtained from 22 infertile women undergoing stimulated cycles for intracytoplasmic sperm injection (11 with ME and 11 without endometriosis). Immature bovine oocytes were submitted to in vitro maturation (IVM) divided into 9 groups: no-FF (No-FF); with FF from control (CFF) or ME (EFF) groups; and with LC (C + LC and E + LC), NAC (C + NAC and E + NAC), or both antioxidants (C + 2Ao and E + 2Ao). After IVM, oocytes were immunostained for visualization of microtubules and chromatin by confocal microscopy. The percentage of meiotically normal metaphase II (MII) oocytes was significantly lower in the EFF group (51.35%) compared to No-FF (86.36%) and CFF (83.52%) groups. The E + NAC (62.22%), E + LC (80.61%), and E + 2Ao (61.40%) groups showed higher percentage of normal MII than EFF group. The E + LC group showed higher percentage of normal MII than E + NAC and E + 2Ao groups and a similar percentage to No-FF and CFF groups. Therefore, FF from infertile women with ME causes meiotic abnormalities in bovine oocytes, and, for the first time, we demonstrated that the use of NAC and LC prevents these damages. Our findings elucidate part of the pathogenic mechanisms involved in infertility associated with ME and open perspectives for further studies investigating whether the use of LC could improve the natural fertility and/or the results of in vitro fertilization of women with ME.