Endometriosis and Oxidative Stress. Do Reactive Oxygen Species Always Lead to Damages of Gametes and Embryos?

Research Article

Austin J Reprod Med Infertil. 2021; 7(1): 1054.

Endometriosis and Oxidative Stress. Do Reactive Oxygen Species Always Lead to Damages of Gametes and Embryos?

Török A and Máté G*

Pannon Reproduction Institute, Tapolca, Hungary

*Corresponding author: Gábor Máté, Pannon Reproduction Institute, H-8300 Tapolca, Bartók Béla str. 1-3., Hungary

Received: May 20, 2021; Accepted: June 30, 2021; Published: July 07, 2021


Reactive Oxygen Species (ROS) play a crucial role in the pathogenesis of many reproductive disorders, such as endometriosis on the one hand, but on the other hand they participate in different cellular proliferation processes, too. Endometriosis is an apoptotic endometrial, menstrual cells and lysed erythrocytes-induced inflammatory disease outside the uterine cavity, which activates macrophages leading to ROS production and oxidative stress. However, based on the available literature, the reproductive outcomes are still contradictory. In this study, the demographic, embryological and clinical results of 252 patients suffering from tubal infertility (control), ASRM I-II and III-IV endometriosis were analyzed. Endometriosis was associated with decreased anti-Müllerian hormone level and increased gonadotropin doses during stimulation (p<0.0001). In ASRM III-IV, reduced embryological parameters were observed, which resulted in 13.73% and 15.21% decrements in the implantation rates, 19.96% and 23.89% in the clinical pregnancy rates of patients suffering from ASRM III-IV endometriosis in comparison with control or ASRM I-II, respectively. In addition, miscarriage rates were 19.04%, 29.03% and 38.46% in control, ASRM I-II and ASRM III-IV, respectively. In our study, the supposed altered oxido-reduction environment of gametes and embryos obviously exerted negative effects on the embryological and clinical parameters, but these effects could not be observed in case of mild endometriosis with low level of stress.

Keywords: Antioxidants; Embryo development; Endometriosis; Oxidative stress; Infertility


8-OHdG: 8-Hydroxy-2’-Deoxyguanosine; AFC: Antral Follicle Count; AMH: Anti-Müllerian Hormone; ASRM: American Society of Reproductive Medicine; BMI: Body Mass Index; FISH: Fluorescence In Situ Hybridization; FSH: Follicle-Stimulating Hormone; H2O2: Hydrogen Peroxide; hCG: human Chorionic Gonadotropin; ICSI: Intracytoplasmic Sperm Injection; IU: International Unit; IVF: In Vitro Fertilization; LH: Luteinizing Hormone; O2●-: Superoxide Anion Radical; ●OH: Hydroxyl Radical; PCOS: Polycystic Ovary Syndrome; ROS: Reactive Oxygen Species


Every aerobic living organism suffers from the harmful effects of oxygen due to their mitochondrial respiration process. Namely, 1-5% of molecular oxygen is converted into the highly reactive and toxic superoxide anion (and further reactive oxygen species, ROS) but normally, it is neutralized by intracellular enzymatic or non-enzymatic antioxidant molecules. Their potential toxicity is determined by the balance between the ROS accumulation and antioxidants. So, oxidative stress can occur when the generation of ROS exceeds the amount of antioxidants or the synthesis of antioxidants is blocked [1]. Nevertheless, not only harmful effects of ROS are known, as they also have important intracellular signal transduction and regulatory functions in folliculogenesis, maturation of oocytes, dissolution of corpora lutea, implantation and embryo development [2,3].

Many human diseases are characterized by oxidative stress (e.g. inflammations, cardiovascular diseases, chronic pulmonary diseases, chronic kidney diseases, neurodegenerative diseases, cancers and infertility) [4]. Almost every type of infertility can be connected to oxidative stress. Among others, polycystic ovary syndrome, male infertility, advanced maternal age or endometriosis are associated with oxidative stress [5-7]. Endometriosis is an inflammatory disease, in which endometrial cells and tissues can be found outside the uterine cavity. Obviously, endometrial implants are hormonedependent, namely estrogen-dependent tissues [8]. Endometriosis is a very frequent finding among infertility patients. Since the diagnosis of endometriosis is often established only after several years, accurate statistics are not available on the frequency of the disease. However, it is commonly accepted that 10 to 15% of reproductive-age women suffers from endometriosis, but in infertility patients papers mention a frequency between 25-50% [9]. In infertile patients, the exact staging of the disease is very important. The classification recommended by the American Society of Reproductive Medicine (ASRM) [10] is used world-wide. In patients with ASRM I and II endometriosis, there is a good chance for a successful infertility treatment either using surgicalor medical treatment, their combination, or assisted reproductive techniques. Nowadays, there is no unified view on what kind of therapy could be recommended for patients at these stages. On the other hand, it is now unanimous that in the cases of stages III and IV, only the assisted reproduction techniques are justified. However, the success rate of assisted reproduction methods in these patients is significantly lower than that in stage I and stage II patients [11]. In a recent paper, Juneau et al. [12] demonstrated that aneuploidy rates of patients with endometriosis are equivalent to their age-matched peers in IVF population without endometriosis. On the other hand, Mansour et al. [13] found that endometriosis-related genetic errors leading to aneuploidy were significantly higher, in comparison to control. In addition, a plenty of indirect data suggest that via oxidative stress processes, endometriosis goes hand in hand with aneuploidy.

What make the topic really interesting are the contradictions of published results. In one half of the papers, clear evidence of endometriosis-induced oxidative stress and reduced embryological and clinical outcomes can be read, but in the other half, there is no difference between control and endometriosis patients. The aim of this study was to share our results on this field and show the importance of the exact classification of the disease because, depending on the stage of the disease, it might happen that we detect no differences and significant alterations, too, at the same time.

Materials and Methods

Study design

A retrospective analysis was carried out on the data of 73 women with stage I-II endometriosis, 70 women with stage III-IV endometriosis and 109 women with tubal infertility as control, who referred to Pannon Reproduction Institute, Tapolca, Hungary in 2017. Patients with endometriosis underwent laparoscopy or surgery before the IVF cycle and were scored based on the criteria of ASRM [10]. In control, only the patency of fallopian tube was observed (presence of blockages), laparoscopy of surgery did not happened and pain or pelvic adhesion did not implied to the presence of endometrioma. The objective of this analysis was to compare their clinical, embryological and pregnancy parameters.


For all patients, controlled ovarian hyperstimulation was performed using long protocol desensitization and gonadotropin stimulation. If the ovaries are affected by endometriotic tissues, the incidence of menstrual bleeding in the ovaries is reduced by agonist long protocols. For desensitization, 0.5 ml (0.525 mg) buserelin (Sanofi-Aventis, France) was administered per day subcutaneously (s.c.), starting in the midluteal phase of the previous cycle. When desensitization was achieved, the daily dose of buserelin was reduced to 0.25 ml (0.2625 mg) and stimulations were performed either using follitropin alpha (Gonal-F, Merck, Germany), or follitropin alpha - lutropin alpha (Pergoveris, Merck, Germany) s.c. in individual doses depending on the age, AMH level and antral follicle count (AFC) of the patient varying from 150 IU to 450 IU. Follicular growth was controlled by regular vaginal ultrasound folliculometry usually 2-4 times within a cycle. When the dominant follicle reached 18 mm in diameter, chorionic gonadotropin alpha (Ovitrelle, Merck, Germany) was administered s.c. in 500 μg dose to trigger the ovulation. 36 hours later, follicle aspirations were performed in general anesthesia with ultrasound guided needle using a vaginal probe. Luteal phase support was started on the day of follicle aspiration using progesterone gel intravaginally (Crinone, Merck, Germany) in 90 mg daily dose. Pregnancy tests were performed 12-16 days after the oocyte retrieval.

Embryo culture and transfer

pH-stabilized Origio® sequential media were used for oocyte denudation, fertilization, culture and transfer. Namely, Origio® FertTM was used for oocyte preparation and fertilization in the first 18-22 hours; Origio® CleavTM for embryo cleavage up to 72 hours; Origio® BlastTM for blastocyst formation up to 120 hours; and Origio® UTMTM was used for embryo transfer. Cells were incubated at 37°C, supplemented with carbogen gas containing 5% oxygen, 6% carbon dioxide and 89% nitrogen. ICSI was performed both in control and endometriosis groups, as we will discuss below, the residue of follicular fluid affected by endometriomas may exert a negative effect on the sperm cells during IVF. During the ICSI, 10% polyvinylpyrrolidone was used to facilitate the handling and immobilization of sperms. Embryo scoring was performed according to the methods of Baczkowski et al. [14]. Embryos from both test groups underwent evaluation of morphologic quality by grading from 1 to 3, where 1 is good quality, 2 is fair quality, and 3 is poor quality. Embryo transfers were performed with 1-3 embryos (if available) on day 3 or day 5.

Statistical analysis

Data were given as average and deviation. Shapiro-Wilks test was used to evaluate the distribution of the data. Non-normally distributed variables were examined using the non-parametric test of Kruskal-Wallis, while for multiple comparisons, Dunn’s test was applied. Differences between proportions or rates were evaluated with Fischer exact test. Graphpad InStat 7.0 software was used for statistical analysis.


The demographic parameters of the three investigated groups were almost the same (Table 1). The average value of age, BMI, AFC and the levels of main basal hormones did not differ significantly, but as a predictor of ovarian reserve, lower serum AMH levels were observed in both endometriosis groups, in comparison with control, 0.23-fold and 0.40-fold decreases were observed in ASRM I-II and III-IV groups, respectively. These alterations induced significant increment in the necessary amount of gonadotropin used for the stimulations, which was increased by 36.81% and 42.18%. In addition, some non-significant trends were observed: the levels of AFC and LH were non-significantly lower; and the level of FSH increased.