Mitochondrial dysfunction and embryo quality: prospects for antioxidant correction containing myo-inositol and D-chiroinositol in a 5:1 ratio, manganese and folic acid

The intrafollicular environment, which encompasses the follicular fluid and cumulus cells, constitutes the oocyte microenvironment. The understanding of the functional role of this niche is continually being enhanced by new data, including insights into plastic and energy metabolism as well as various factors that directly influence oocyte maturation. On one hand, functional disorders of cumulus cells can contribute to the suboptimal response of ovaries to stimulation in assisted reproductive technology (ART) programs, while on the other hand, they can serve as markers of oocyte quality [1, 2]. In addition to examining the metabolome and gene expression profile of cumulus cells, a particularly promising area of research is mitochondrial dysfunction, which affects both the variability of biological aging and the decline in reproductive function in women. A strong correlation has been demonstrated between existing somatic diseases and mitochondrial dysfunction in cumulus cells [3–5].

The available research data offer a degree of optimism regarding the possibility of correcting mitochondrial dysfunction through the treatment of somatic diseases, lifestyle changes, and drug therapy. For instance, certain treatment and prevention methods not only have clinical effects in restoring reproductive function but also demonstrate metabolic effects related to the correction of mitochondrial dysfunction. Previous studies have shown that oral administration of D-chiroinositol (DCI) in women with anovulatory infertility and/or obesity correlates with restoration of ovulatory function and normalization of metabolic markers [6–10].

This study aimed to evaluate the functional activity of cumulus cell mitochondria and embryo quality in ART protocols among reproductive-aged patients with anovulatory infertility and obesity who were receiving supplementation with a complex of myoinositol (MI) and DCI in a ratio of 5:1, combined with manganese and folic acid.

Materials and methods

General study design

A single-center, open-label, prospective, comparative, randomized study was conducted from May 2023 to September 2024 to evaluate the effect of the dietary supplement Dikirogen (Pizeta Pharma S.p.A., Italy) on the functional activity of cumulus cells and embryo quality in patients diagnosed with anovulatory infertility and obesity and treated with assisted reproductive technology (ART) programs. The supplement contained a combination of 1000 mg myo-inositol (MI), 200 mg D-chiro-inositol (DCI), 200 μg folic acid, and 5 mg manganese in the form of manganese pidolate, an organic salt that is an inositol synergist. All patients included in the study met the inclusion criteria and did not meet the exclusion criteria. The control group for evaluating embryo quality and cumulus cell functional activity comprised oocyte donors.

Inclusion criteria were age between 24 and 36 years, primary anovulatory infertility, and body mass index (BMI) of 30–32 kg/m².

The exclusion criteria were general contraindications to ART, diabetes mellitus, and intake of medications affecting CYP3A4 function.

Administration of Dikirogen complex

Patients in the main group (n=11) received Dikyrogen according to the following regimen: one sachet taken twice daily after meals for 180±7 days.

Patient selection and randomization

Most patients were selected from our database of a previously conducted study assessing the effectiveness of the MI and DCI complex in a ratio of 5:1, in combination with manganese and folic acid, in reproductive-aged patients with irregular menstrual cycles. The sample size was expanded by including six primary patients [11]. Randomization was performed after signing informed consent using a sealed envelope method while adhering to the inclusion and exclusion criteria (Fig. 1).

Implementation of ART protocol

Stimulation of superovulation began on the 2nd or 3rd day of the menstrual cycle using recombinant follitropin alpha (Gonal-F, Merck Serono S.p.A., Italy). Ultrasound monitoring of folliculogenesis and endometrial growth was performed during ovarian stimulation. When the leading follicle reached a diameter of 14 mm, gonadotropin-releasing hormone antagonists (Cetrotide, Merck Serono S.p.A., Italy) were prescribed. Recombinant choriogonadotropin alpha (Ovitrelle, Merck Serono S.p.A., Italy) was used as a trigger for final oocyte maturation when at least two follicles reached a diameter of 18 mm. Transvaginal puncture of the follicles was performed under intravenous anesthesia 36 h after the trigger was administered.

Study of the functional activity of cumulus cells

This study was conducted according to previously described methods [12, 13], focusing exclusively on the follicular fluid surrounding mature oocytes. Using a surgical scalpel, a portion of the cumulus cells was cut from the oocyte-cumulus complexes for further analysis. The cumulus cell (CC) sample was placed in a test tube containing culture medium and HEPES buffer. The contents of the tube were resuspended by pipetting with a microdispenser for 20 s to 20 min, until the cumulus cell conglomerate was transformed into a homogeneous suspension.

Next, 2.0 ml of DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% streptomycin was added to each well of a six-well plate (Corning, USA). A sterile 24×24 mm, 0.17 mm thick cover glass was then placed in the wells of the plate. Then, the cumulus cell suspension (0.5 mL) was added, and the mixture was incubated at 37°C in 5% CO₂ for 20 h. During this incubation period, the cells settled on coverslips in each well.

After incubation, the contents of each well were replaced with serum-free DMEM/F12 medium supplemented with the mitochondrial counterstains MitoTracker CMTMRos and MitoTracker GreenFM for 30 min. All imaging steps were performed using a Leica TCS SP5 II SMD FLCS laser scanning confocal microscope with a HC PL APO 63x/1.30 GLYC CORR CS2 objective. In the volume scanning mode, 20 to 30 fields of view were captured for each preparation, with each cell being imaged closely using the digital zoom option. The fluorescence intensity of the dye accumulated in the mitochondria was measured using ImageJ with the Measure command.

Segmentation and morphometric analysis of the images were performed in Bitplane Imaris using the Filament Tracer module. The following parameters were estimated: (1) the average length of mitochondria, including all processes (Filament Length), excluding structures shorter than 0.7 μm to avoid segmentation errors, (2) the average number of mitochondria, and (3) the fluorescence decay time of the mitochondrial dye, measured using FLIM microscopy. Four to five fields of view were recorded for each preparation, with scanning conducted until 1000 photons were counted by the detector for the brightest point of the image. The data were imported into ImageJ using the PTU reader extension and analyzed using the FLIMj extension; (4) fluorescence intensity. Fluorescence intensity was estimated using the numerical value of brightness with 8-bit image encoding (ranging from 0 to 255 conventional units).

The ejaculate was prepared for fertilization in a sequential manner, first by centrifugation in a density gradient of silicone particles (80:40, SpermGrad, Vitrolife), followed by flotation. Embryos were obtained by adding the prepared sperm suspension at a concentration of 150 million/ml to a 500 μl drop of medium containing up to five oocyte-cumulus complexes. For oocyte fertilization, the in vitro fertilization (IVF) method was employed; the oocytes were denuded from the cumulus 17–18 h after fertilization. The resulting bipronuclear zygotes were placed in individual drops in the wells of EmbryoSlide dishes (Vitrolife, Sweden) for further cultivation. Embryos were cultured for 140 h in an EmbryoVisor multigas incubator with the TIME-LAPSE function (Vesttrade Ltd, Russia) in G-TL medium (Vitrolife, Sweden) coated with Ovoil oil (Vitrolife, Sweden) at 37°C in the presence of a gas mixture consisting of 5% O₂ and 6% CO₂. After culturing, the morphokinetic parameters of embryos from both groups were assessed using photos and videos throughout the entire development cycle from the zygote to the blastocyst stage. EmbryoVisor automatically recorded images in 11 focal planes every 10 min. In these cycles, t=0 was defined as the time of injection of the last oocyte. Annotation was performed by two trained specialists in accordance with published consensus definitions and recommendations [14]. The following main events after fertilization were identified and assessed: disappearance of both pronuclei (tPNf), formation of 2-, 3-, 4-, 5-, 6-, 7-, 8-, and 9-cell embryos (t2, t3, t4, t5, t6, t7, t8, and t9), time of onset of compaction (tSc), formation of a compact morula (tM), onset of blastulation (tSB), formation of a complete blastocyst (tB), and expanded blastocyst (tEB).

Statistical analysis

Data were tested for normality of distribution using the Jarque–Bera test. The statistical significance of differences between groups for continuous variables was assessed using the Student’s t-test or the Mann–Whitney U test, while categorical variables were evaluated using the Pearson χ² test. Differences between groups were considered statistically significant at p<0.05.

Ethical rules and standards

This study was approved by the Ethical Review Board of D.O. Ott Research Institute of Obstetrics, Gynecology, and Reproduction, St. Petersburg, Russia, dated 12.05.2023 and was conducted in accordance with the principles of the Declaration of Helsinki (1964).

Results

The patients in the main and comparison groups did not differ in terms of age or BMI (Table 1).

In the statistical evaluation of the effectiveness parameters of the embryological stage of the ART program, no differences were found in the number of obtained oocyte-cumulus complexes or fertilization efficiency. However, the blastulation rate in the main group was significantly higher than that in the comparison group (U=30, z=1.99, p=0.04) (Table 2).

A comparison of the timing of key events in embryo development showed that the disappearance of pronuclei; the formation of 2-, 3-, 4-, 5-, 6-, 7-, 8-, and 9-cell embryos; the time of onset of compaction; the formation of a compact morula; the onset of blastulation; the formation of a complete blastocyst; and the formation of an expanded blastocyst did not differ between patients in the main and control groups (Fig. 2).

A comparison of fluorescence intensity, which indirectly reflects the functional activity of mitochondria, showed that the emission intensity was highest in the control group, while the average values in the main group were significantly different from those in the comparison group (t=4.98, p=0.002). Notably, pronounced mitochondrial networks were identified in only 14.9% of the samples from the control group, whereas no mitochondrial networks were observed in the main and comparison groups (Fig. 3).

Note. 3a – Staining with the mitochondrial counterstaining dye Mitotracker GreenFM (×800); live functional mitochondria are stained green. 3b – Staining with the mitochondrial counterstaining dye Mitotracker CMTMRos (×800); active mitochondria, where ROS synthesis occurs, are colored red.

Discussion

Cultivation of patient embryos after supplementation with Dikirogen complex in incubators equipped with a continuous image capture system and analysis of the obtained video images allowed for tracing the key morphokinetic events of preimplantation development. This process enabled the recording and establishment of time data on the development of preimplantation embryos from the zygote stage to the formation of an expanded blastocyst. When comparing the timing of key events in the development of embryos from patients in the main group to the development of donor embryos, it was observed that the time intervals for the primary events of early embryogenesis, such as the disappearance of pronuclei and the formation of 2-, 3-, 4-, and 8-cell embryos as well as the onset of blastulation and the formation of an expanded blastocyst, did not differ between the embryos of patients in the main group and those in the control group.

Our previous data indicated that mitochondrial dysfunction of cumulus cells may be responsible for decreased oocyte quality, resulting in a lower number of optimal quality embryos [12].

The mitochondrial network is a source of reactive oxygen species (ROS). The development of oxidative stress depends on the production levels of each ROS, the rates of their relative neutralization, and the efficiency of their damaging effects on the cellular repair systems. An imbalance between these factors can result in severe mitochondrial dysfunction. Many studies have attempted to link metabolic markers with oocyte quality, including metabolites in follicular fluid or culture medium, gene expression of cumulus cells, and measurement of oxygen consumption in the environment surrounding oocyte-cumulus complexes. However, the informational value of these methods remains extremely low in the present context.

Confocal microscopy can be utilized for this purpose, as it allows the acquisition of high-resolution images of subcellular structures and provides detailed localization of the cell compartments of interest. Researchers can monitor intracellular and biochemical processes within cells using exogenous labels or molecular probes. However, the use of laser radiation complicates its clinical implementation because of the high probability of photodamage [15]. In our study, the assessment of cumulus cell emission intensity served as an indirect indicator of mitochondrial network function. This assessment is based on the intensity of laser-stimulated mitochondrial network emission and incorporates an integral index that considers both the total number of mitochondrial network cells and the intensity of their metabolic activity.

It is well known that oxidative stress can lead to alterations and damage to various cellular macromolecules, resulting in cell death. In this context, cell damage appears to be directly correlated with cellular ROS concentration. Typically, cells can manage ROS levels using various defence mechanisms that either prevent the formation of reactive species or remove them before they can cause fatal damage. In humans, studies on ROS and oxidative stress face methodological challenges when assessing the levels of oxidative stress markers in biological samples. Direct measurement of oxidative stress levels in cell-free biological fluids is particularly difficult because these fluids lack ROS-producing cells. Nevertheless, the effects of ROS can be demonstrated indirectly by assessing oxidative damage to the mitochondria.

Considering the above, it is clear that anovulatory infertility associated with obesity requires preconception preparation, specifically the administration of drugs aimed at correcting mitochondrial dysfunction. Both MI and DCI are phosphoinositides that influence intracellular metabolic processes by activating key enzymes involved in oxidative and non-oxidative glucose metabolism. During oocyte maturation, the primary role of MI derivatives is to generate calcium-mediated signals from gonadotropin-releasing hormone receptors [8, 9] and to prepare for successful oocyte activation at the time of fertilization [10]. Inositols also act synergistically with folates to regulate cellular DNA methylation. Manganese ions stimulate the secretion of gonadotropins and help maintain the normophysiological cycle by supporting the body's antioxidant resources [12], thereby maintaining the activity of Mn-superoxide dismutase, which fluctuates in a wave-like manner and reaches a maximum in the middle of the menstrual cycle. Clinical and epidemiological studies have demonstrated a correlation between nutritional deficiency of manganese and menstrual cycle disorders. In women aged 18–44 years (n=259), low manganese intake (<1.8 mg/day, OR 2.00; 95% CI 1.02–3.94) was associated with an increased risk of anovulation compared to the group with higher intake [13]. In Russia, the vitamin and mineral complex Dikirogen (Pizeta Pharma S.p.A., Italy) is registered, containing 1000 mg of MI and 200 mg of DCI (in a ratio of 5:1), along with 200 µg of folic acid and 5 mg of manganese. Dikyrogen is recommended for menstrual cycle disorders, premenstrual syndrome, hyperandrogenism (including polycystic ovary syndrome), and for pregravid preparation, including preparation for IVF and the use of ART. Manganese in Dikirogen is presented in the form of an organic salt (manganese pyroglutamate), characterized by good organoleptic properties, high bioavailability, and low toxicity. No side effects were associated with Dikirogen use.

Conclusion

The data obtained indicate that combination therapy with antioxidants, vitamins, and microelements, including 2000 mg MI, 400 mg DCI, 400 µg folic acid, and 10 mg manganese for 6 months before ART, reduces mitochondrial dysfunction of cumulus cells in patients with obesity and anovulatory infertility, leading to a decrease in oxidative stress and likely resulting in improved oocyte quality.