Introduction
In assisted reproductive technologies (ART), intracytoplasmic sperm injection (ICSI) has significantly increased conception rates, offering hope to couples facing fertility challenges. However, the interplay of various factors within the microenvironment of the female reproductive system remains a subject of ongoing research. Follicular fluid (FF) plays a vital role in the physiological development of oocytes within the ovarian follicle. This fluid surrounds the cumulus cells and oocyte, containing a mix of steroids, metabolites, polysaccharides, proteins, peptides, reactive oxygen species (ROS), and antioxidant enzymes. The interactions among various FF components are thought to promote follicular development and oocyte maturation [1,2].
Mitochondria are the primary sources of ROS; however, excessive ROS can impair their functions in oocytes and embryos, potentially leading to cell division arrest due to oxidative stress (OS) [3]. In ART cycles, the impact of OS is intensified by diminished cellular protective mechanisms. Multiple factors contribute to elevated ROS levels, including endogenous sources such as gametes, environmental factors, or interventions on embryos/gametes, as well as exogenous factors like laboratory conditions, culture conditions, and oxygen pressure. These factors collectively influence the outcomes of ART therapies [4]. Cellular malfunction and, in severe cases, cell death can result from the cumulative effects of oxidative processes. The reproductive system demonstrates the extensive physiological and regulatory roles of ROS, influencing processes such as folliculogenesis, oocyte maturation, luteal regression, and fertilization. Elevated oxidation levels in FF are associated with reduced embryo quality [4,5].
Additionally, elevated hydrogen peroxide (H2O2) levels in FF are associated with the production of low-quality embryos [4,5]. Additionally, increased levels of oxidized methionine and ubiquitinated proteins in FF have been observed in cases of unsuccessful pregnancies. Conversely, reduced levels of ROS and an increase in total antioxidant capacity (TAC) in FF are indicative of successful pregnancies achieved through ICSI [4].
The FF oxidation-reduction potential (FF-ORP), which has garnered attention in recent years, reflects the balance between oxidizing and reducing agents, indicating the oxidative state of the microenvironment [6]. The impact of FF-ORP on ICSI outcomes remains a topic of active discussion and investigation within the field of ART. Key considerations include its potential predictive value for assessing the quality of oocytes and embryos as a parameter reflecting the ovarian microenvironment [5,7]. Debates have arisen from the lack of consensus on how to interpret ORP values and the variability in study designs. This study aimed to explore the impact of FF-ORP on ICSI results. A deeper understanding of these relationships could lead to improvements in clinical practices, optimization of treatment approaches, and ultimately, higher success rates in assisted reproduction.
Methods
This cross-sectional study analyzed data from 341 ART cycles conducted between January 2021 and December 2023 at Hue University of Medicine and Pharmacy Hospital. The Ethics Committee of Hue University of Medicine and Pharmacy approved the study (approval number H2023/016). Informed consent was obtained from couples undergoing ART treatments using the ICSI technique, allowing the use of their FF data for research purposes. The exclusion criteria were as follows: women over the age of 38, individuals with ovarian cysts, adenomyosis, endometriosis, thyroid disorders, oocyte/sperm donors, and cases of male infertility characterized by severe oligozoospermia or azoospermia. However, cases of polycystic ovary syndrome were not excluded. This study collected comprehensive information on the general characteristics of the couples, various endocrine indices during the ovarian stimulation cycle, ORP, and pH levels in FF. Treatment outcomes, including oocyte retrieval results, fertilization rates, and blastocyst formation, were meticulously recorded. To explore the relationship between FF-ORP and ICSI outcomes, we categorized the groups based on whether their results were above or below the benchmark values of 80% for fertilization rate and 60% for blastocyst formation rate [8].
1. Controlled ovarian hyperstimulation and oocyte retrieval
Participants underwent controlled ovarian stimulation using a gonadotropin-releasing hormone antagonist protocol along with recombinant follicle-stimulating hormone (rFSH; follitropin alfa). An ultrasound performed on days 2–3 of the cycle assessed the antral follicle count (AFC) and checked for functional cysts. On this day, a starting dose of 150–225 IU of rFSH (Gonal F; Merck KGaA) was administered. Starting from the fifth day of stimulation up to the trigger day, participants received 0.25 mg/day of cetrorelix (Cetrotide; Merck KGaA). Oocytes were retrieved 35 to 36 hours after administering the human chorionic gonadotropin (hCG) trigger (Pregnyl 10,000 IU; Merck Sharp & Dohme Limited) under transvaginal ultrasound guidance.
2. Oxidative-reduction potential analysis
During oocyte retrieval in patients undergoing ART treatment, a minimum of 1.0 mL of FF from the first collected follicle was aspirated directly into a polystyrene round-bottomed tube (BD Falcon). This procedure was performed without using a flushing medium or introducing contaminated blood. The FF was then transferred to a 100 mm petri dish (BD Falcon) for oocyte collection. Afterward, the FF sample was returned to the tube, which was then covered with aluminum foil to protect it from light exposure. Subsequently, only FF samples without blood contamination were subjected to ORP analysis.
FF-ORP was measured using the MiOXSYS system (Caerus Biotechnologies), which utilizes electrochemical technology with an Ag/AgCl reference cell. The fluid sample was applied to the sensor and then inserted into the analyzer. The resulting static ORP measurement, expressed in millivolts, reflected the balance between total oxidants (ROS) and antioxidants in FF.
A 30-µL volume of thoroughly mixed fluid sample was transferred to the Sample Application Port of the inserted MiOXSYS sensor. The FF sample was absorbed through the filter at the sample port, passing over the working electrode and filling the reference cell, thereby completing the electrochemical circuit. Upon detection, the MiOXSYS analyzer began processing the sample, completing the analysis in approximately 2 minutes. The ORP analysis was performed quickly using the machine, which minimized the impact of external factors such as light exposure on the FF-ORP results. Furthermore, the pH of the FF was measured using the LAQUA pH1100 pH meter (Horiba) equipped with a pH electrode. The reported result was the average of three measurements.
3. ICSI, embryo culture, and grading
The oocyte-corona-cumulus complex was rinsed and then cultured for two hours in G-IVF PLUS (Vitrolife) at 37 °C, with an atmosphere containing 5.0% O2 and 6.0% CO2. Following this, the oocytes were denuded using 80 IU of Hyase (Vitrolife) and subsequently incubated for an additional hour in G-IVF PLUS prior to ICSI. Standard ICSI procedures were used for fertilization. Sperm preparation involved gradient concentration centrifugation using the Sil-select Plus density gradient system (45% to 90% layers; Fertipro). The sperm were then rinsed with SpermRinse (Vitrolife).
Following ICSI, embryos were individually cultured in G-TL media (Vitrolife), coated with Ovoil (Vitrolife), at 37 °C with 5% O2 and 6.0% CO2. The embryos were incubated in a benchtop incubator (IVFtech) until they reached the blastocyst stage on day 5.
The evaluation process adhered to the Gardner score system and the Istanbul consensus [9]. The fertilization assessment was typically conducted at 16 to 18 hours post-ICSI. Fertilization assessments were typically conducted 16 to 18 hours after ICSI. Blastocyst evaluations took place either 154 to 156 hours post-hCG injection or 112 to 124 hours after fertilization. This assessment included observation and imaging using an inverted microscope at ×200 magnification, conducted by two experienced embryologists. A blastocyst of good-quality was identified by the presence of a fluid-filled blastocoel cavity, a tightly compacted inner cell mass, and a cohesive trophoblast cell layer.
The general characteristics of patients were recorded during the ART treatment cycles. The outcomes of embryo culture were assessed based on several rates: the maturation rate, defined as the number of mature oocytes (metaphase II) divided by the number of oocytes retrieved; the fertilization rate, defined as the number of normal zygotes divided by the number of mature oocytes; the blastocyst formation rate, defined as the number of blastocysts formed divided by the number of embryos formed; and the good blastocyst rate, defined as the number of good-quality blastocysts divided by the number of embryos formed.
4. Statistical analysis
All statistical analyses were performed using SPSS ver. 23.0 (IBM Corp.). The Kolmogorov-Smirnov test was performed to determine whether the variables were normally or non-normally distributed. Continuous variables were presented as the mean and standard deviation for normally distributed data and the median and interquartile range for continuous indicators not following a normal distribution. Spearman correlation coefficients (r) were employed to evaluate the relationship between the parameters. Significant correlations were further analyzed using multivariable regression to indicate the strength of the predictors. Continuous variables were compared using the Student t-test when data were normally distributed. The Mann-Whitney U test was used for continuous variables that did not follow a normal distribution. All tests were two-tailed, and p-values less than 0.05 were considered to indicate statistical significance.
Results
This study involved 341 couples undergoing ART treatment. The baseline characteristics of the patients are detailed in Table 1. Both partners were relatively young, with an average infertility duration of approximately 5 years. We collected most of the endocrine test results during the ART cycle, including basal AFC, anti-Müllerian hormone (AMH), baseline follicle-stimulating hormone (FSH), estradiol (E2), luteinizing hormone (LH), prolactin, total FSH administered, and E2 levels on the day of triggering with hCG. The ICSI outcomes are also presented in Table 1. Across the 341 cycles, the average rates for different stages were as follows: maturation, 78.90%±17.42%; fertilization, 72.89%±20.38%; blastocyst formation, 61.86%±28.20%; and good-quality blastocysts, 38.93%±26.29%. Additionally, the mean values of pH and ORP in FF were crucial. The average FF-ORP was 85.24±25.97 mV, and the pH was 7.64±0.29.
The data in Table 2 indicated that the FF-ORP exhibited a statistically significant but weak negative correlation with the concentrations of E2 and prolactin, with correlation coefficients of –0.131 (p=0.015) and –0.108 (p=0.047), respectively. There was no significant correlation between the FF-ORP and other endocrine parameters or general patient characteristics. Additionally, the ORP index was negatively correlated with the pH in the FF (–0.183, p=0.001). Regarding the ICSI outcomes, the influence of FF-ORP was modestly significant, particularly in relation to the fertilization rate, where it showed a correlation coefficient of –0.126 (p=0.019). However, the multivariable regression test indicated that only pH could independently predict ORP in FF, as shown in Table 2.
Following the recommended practices of in vitro fertilization (IVF) laboratories, grouping samples based on lower and higher benchmarks revealed variations in FF-ORP across these subgroups (Tables 3, 4, Figure 1). There were no significant differences in general and endocrine characteristics between the groups categorized by fertilization rate benchmarks and those classified by blastocyst formation rate benchmarks. The group with a high fertilization rate exhibited a significantly higher number of normal zygotes and high-quality blastocysts than the low fertilization rate group. As shown in Table 3, the group with a lower fertilization rate (<80%) had a substantially higher FF-ORP (89.90 mV vs. 78.98 mV, p=0.030) than the group with a higher fertilization rate (≥80%). Figure 1 effectively illustrates these variations in FF-ORP. There were no statistically significant differences in ORP in FF between the groups with good blastocyst rates of less than 25% and those with rates of more than 25%, nor between the groups with blastocyst formation rates of less than 60% and exactly 60%, respectively (Figure 1).
We noted that the total FSH dose administered to the group with a blastocyst formation rate of less than 60% was higher compared to the group with higher rates (2,175.00 IU/L vs. 2,025.00 IU/L, p=0.010). Additionally, the group with a high blastocyst formation rate exhibited a greater number of normal zygotes and a significantly higher number of good-quality blastocysts compared to the other group (Table 4). Interestingly, the group that exceeded the benchmark demonstrated better ICSI outcomes than those that did not.
Discussion
In normal physiological conditions, there is a balance between the production of ROS and the complex antioxidant defense system. OS represents a form of ‘chemical stress’ in living organisms, primarily resulting from elevated ROS levels [10]. Hormonal, paracrine, and autocrine signaling pathways can directly affect the components of FF. It is believed that these changes may alter OS levels, potentially impacting oocyte maturation and fertilization, early embryo development, and subsequent pregnancy outcomes [11].
This study found no statistically significant correlation between FF-ORP and factors such as female age, body mass index, AFC, baseline FSH, AMH, LH concentration, total FSH dosage, or E2 on the day of trigger. However, a negative correlation was found between baseline E2, prolactin concentration, and pH in FF. Several studies have shown that female age is associated with OS levels in FF, with systematic antioxidant capacity tending to decrease as age increases [12,13]. However, there is a counterargument that age, despite promoting ROS production, does not affect the internal redox balance of FF [13]. Our research supports this view, indicating no correlation between age and FF-ORP. The endogenous antioxidants in FF function as a complex system, interacting with the main components of FF and external antioxidants to maintain the oxidative redox balance. For example, E2 acts as an antioxidant in cells by reducing peroxide production in mitochondria [10,14,15]. E2 is also thought to influence the oxidative redox status of FF, which reflects the number of oocytes retrieved in ICSI [16]. Potential issues in research on FF include the possibility that the concentration of substances in FF or clinical characteristics of the patient, such as age, lifestyle habits, or the type of ovarian stimulation, can influence oocyte quality and may be related to OS [17].
We observed that FF-ORP correlated with the fertilization rate but did not affect other ICSI outcomes. Several studies have reported findings similar to ours, although they used different instruments to measure OS levels [6,18-21]. We categorized groups based on the expected benchmark fertilization rate of an ART laboratory, following the 2017 European Society of Human Reproduction and Embryology (ESHRE) consensus [8]. According to this, the group with high fertilization rates (≥80%) had lower ORP values compared to the group with lower fertilization rates. FF-ORP was a predictive factor for 80% of fertilization. In the study by Sallam et al. [6], the authors also found a correlation between FF-ORP and fertilization rate, using a benchmark of 50%. The researchers suggested a cut-off point of 110 mV for fertilization and 90 mV for pregnancy. They observed no significant difference in FF-ORP between the pregnant and non-pregnant groups [6]. Our findings indicated that the average FF-ORP for the group with a fertilization rate below the expected threshold (80%) was 89 mV. Elevated ORP values may potentially affect the fertilization rate. Another study also showed that an elevated FF-ROS level was associated with better ICSI outcomes [22].
The processes of oocyte maturation and ovulation are associated with OS levels, indicative of elevated metabolic activity. Individuals with robust physiological systems are often able to manage this OS effectively, resulting in the production of high-quality oocytes and successful pregnancies. In contrast, oocytes that are less robust may struggle with OS management, leading to difficulties in embryo development and conception, and potentially resulting in fertilization failure [6]. Thus, there is often an inverse correlation between OS levels and IVF outcomes. Additionally, FF-OS is considered a predictive factor for the likelihood of fertilization and successful pregnancy in ART. Nevertheless, some studies have identified a correlation between ORP, gametes, and fertilization rates, without finding a link to embryo quality [13,23]. This discrepancy is attributed to the influence of OS and other factors on ART outcomes.
The combined effects of OS and immunological competence can lead to numerous clinical disorders that compromise women's reproductive potential [6]. In a study by Pekel et al. [24], malondialdehyde, superoxide dismutase, and TAC in the FF were measured, revealing diminished antioxidant activity in infertile individuals. The pathophysiology of conditions such as endometriosis, polycystic ovary syndrome, luteal phase deficiencies, tubal factor deficiencies, and poor placentation can be linked to various processes involving OS, which affect the redox balance in FF [13,25].
Direct techniques for assessing OS encompass a variety of methods, including chemiluminescence, the nitroblue tetrazolium test, cytochrome C reduction assay, fluorescein probe assay, electron spin resonance spectroscopy, and ORP measurement. Indirect methods for evaluating OS involve the Endtz test, lipid peroxidation assay, chemokine measurement, assessment of antioxidant compounds, microelements, vitamins, ascorbate levels, TAC, and DNA breakage analysis [2,26,27]. Each method has its distinct advantages and disadvantages. However, practical challenges such as feasibility, complexity, high costs, and the absence of standardized analytical techniques hinder their widespread implementation in clinical settings.
There are several methods for determining the oxidative redox balance in FF, all of which are entirely indirect. Commonly used techniques include thiobarbituric acid reactive substances (TBARS), ferric reducing antioxidant power (FRAP), and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) [13]. A relatively recent method for assessing OS in FF is measuring the overall balance between oxidative and antioxidative components [25]. This method is considered rapid, easily implementable, reliable, and more cost-effective than other approaches [28].
We recognize that the primary challenge of this study was selecting an appropriate ORP marker for evaluation and utilizing the MiOXSYS system for measuring FF-ORP. Due to the scarcity of prior research in this field, most studies have traditionally relied on conventional parameters. Since measuring ORP in semen is a common method for diagnosing OS in males, and because FF is similar to seminal plasma, it is reasonable to use the MiOXSYS system for ORP measurement in FF. A recent study also used the MiOXSYS system to measure ORP in blood plasma, documenting an average ORP of 136±14.9 mV. The authors noted that an ORP level within the range of 100 to 150 mV was associated with and predictive of embryo development [25].
In conclusion, our findings revealed a correlation between FF-ORP and fertilization rates, indicating its potential as a predictor for fertilization outcomes in ICSI. However, further investigation is needed to explore OS indices, their molecular biology and enzyme systems, and their interactions with oocyte maturation and embryo culture. Additional research is also essential to determine the impact of ORP on clinical outcomes, such as pregnancy and live birth rates.
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