The influence of Myoinositol to improve biochemical manifestations of the serum and follicular fluid and ICSI outcomes in patients with PCOS: A prospective randomized research

doi:10.21203/rs.3.rs-4172354/v1

Purpose

The research investigated the capacity of Myo-inositol (MI) in order that it improves biochemical markers in serum and follicular fluid, and ultimately, intracytoplasmic sperm injection (ICSI) outcomes of women with PCOS.

Methods

Sixty infertile patients with PCOS who were undergoing ovulation induction for ICSI, were randomly divided to two groups. The MI group received 2000 mg Myo-inositol + 1 mg folic acid twice a day for 6 weeks with starting the ICSI cycle. For the same period, the control group received a placebo containing only folic acid (1 mg). Levels of hormonal profiles in serum and follicular fluid, as well as oxidative stress markers (MDA, TAC, GPx, and SOD) were estimated using an ELISA assay. Primary end points were ICSI cycle outcomes.

Results

Compared to the placebo group, the MI group demonstrated significant reduction in serum and follicular fluid levels of LH, LH/FSH ratio, total testosterone, AMH, and Androstenedione. Furthermore, the MI group exhibited meaningful increases in TAC, GPx, and SOD, but MDA significantly decreased. While the number of recovered and mature oocytes is not similar statistically among the groups, the MI group showed significant improvements in the percentage of immature oocytes, cleavage rate, and good embryo quality. A meaningful correlation was checked between follicular fluid AMH level and LH, FSH, total testosterone, Androstenedione, insulin, MDA, the number of recovered oocytes, and immature oocytes.

Conclusion

Our outcomes indicate that Myo-inositol administration in women with PCOS undergoing ART helps to improve their hormonal profiles, and the quality of oocytes and embryos. (Trial registration: IRCT202220921056008N1)

Myoinositol

Polycystic ovarian syndrome

Intracytoplasmic sperm injection

Reproductive outcomes

Myo-inositol administration in women with PCOS undergoing ART could improve their hormonal profile, and the quality of oocytes and embryos.

A complex and heterogeneous endocrine disorder is polycystic ovary syndrome (PCOS). It affects 7–14% of women in their reproductive years. This manifests as infertility, ovarian dysfunction, menstrual irregularity, and androgen excess, often accompanied by typical ovarian ultrasound features [1]. The etiology and diagnosis of PCOS remain disputed, with many contributing factors suspected, including the hypersecretion of luteinizing hormone (LH), hyperinsulinemia due to insulin resistance, ovarian hyperandrogenism, polycystic ovaries, and decreased fertility [2]. In recent years, extensive research efforts have been directed towards understanding the underlying mechanisms and developing effective treatments for PCOS [3].

Two key factors could be responsible for the low success rate of assisted reproductive techniques (ART) in patients with PCOS: (1) the limited quantity and poor quality of oocytes recovered following ovarian stimulation, and (2) the exact poor fertilization rate following intracytoplasmic sperm injection (ICSI) [4]. Various interventions explored reproductive consequences in inadequate responders undergoing ART, but unfortunately, convincing evidence for their effectiveness remains elusive. This includes insulin-sensitizing agents like pioglitazone, rosiglitazone, troglitazone, D-chiro-inositol, and most notably, metformin [5]. Therefore, any treatment capable of enhancing oocyte quality holds immense promise for achieving a "crowning achievement" in ART for PCOS patients [6].

Myo-inositol (MI), a cyclic sugar with six hydroxyl groups, acts as a precursor to inositol triphosphate, also known as an intracellular second messenger, that regulates hormones, such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and insulin [7, 8]. Beyond its role as a second messenger, inositol regulates exocrine gland secretion (pancreas, ovaries) and shows promise for assisted reproduction [9]. Notably, it may improve insulin sensitivity in PCOS, leading to raised glucose uptake [10]. MI supplements have shown promise in improving both the metabolic characterization and hyperandrogenism in PCOS patients, while also developing clinical pregnancy rates in infertile patients experiencing ICSI [11]. Prior investigations have provided evidence for its efficacy in restoring spontaneous ovarian activity and fertility in numerous patients diagnosed with PCOS [12]. MI has a vital role in different cellular functions, such as growth, survival, and reproduction [13, 14].

Evidence suggests that MI administration can enhance ovarian function, potentially reducing the FSH needed for ovarian stimulation and enhancing the quantity and quality of retrieved oocytes [15]. Notably, research has shown that MI can reinstate spontaneous ovulation and fertility in most PCOS patients [16, 17]. Furthermore, its potential benefits for insulin sensitivity, oocyte maturation, and embryo quality, have established MI as a clinical treatment for PCOS and a promising intervention for spontaneous ovulation restoration [18, 19, 11, 20]. While ART remains crucial for many PCOS women, poor oocyte quality is a major obstacle to successful ICSI cycles, with over two-thirds failing to achieve pregnancy [21]. Therefore, any treatment capable of enhancing oocyte quality would be a significant advancement in ART procedures. The research is aimed at estimating the impacts of MI on hormonal profiles, oxidative stress markers in serum and follicular fluid, and ICSI consequences in women of PCOS.

Study population

Researchers at Qom Infertility Treatment Center conducted a rigorous clinical test (randomized, double-blinded, and placebo-controlled) on a new infertility treatment from May 2022 to May 2023. Sixty infertile PCOS women with 25–35 years, experiencing Intracytoplasmic sperm injection (ICSI) were enrolled. Patients met the Rotterdam consensus criteria for PCOS (2003) [22]: two out of three clinical/biochemical hyperandrogenism, chronic oligo/anovulation, or polycystic ovaries on ultrasound. Criteria of prohibition were hypersensitivity to Myo-inositol, male infertility, infertility beyond anovulation, congenital adrenal hyperplasia, pelvic pathologies, thyroid dysfunction, androgen-secreting neoplasia, diabetes mellitus, Cushing's syndrome, hyperprolactinemia, medications influencing carbohydrate metabolism, severe hepatic/renal diseases, and hormonal analogues (except progesterone) within 2 months. Before the enrollment of patients, informed consent was obtained after approval through the Research Ethics Committee of Islamic Azad University, Varamin Pishva Branch (IR.IAU.VARAMIN.REC.1400.033).

Treatment design

Gynecologists conducted blinded examinations of all participants. Neither patients nor physicians knew which treatment regimen was administered. Throughout the study, participants were instructed to maintain their current levels of physical activity and nutriment and avoid taking new medication. Myo-inositol dosage and duration followed established protocols from Nazari et al. (2020) and Papaleo et al. (2009) [23, 5]. Eighty patients were randomly separated into two groups (n = 40): 1) Myo-inositol group (MI group): 40 patients received 2000mg Myo-inositol (Fairhaven Health-LLC, UK) and 1mg folic acid (Raha Co., IRAN) twice daily for 6 weeks. 2) Placebo group: 40 patients received 1mg folic acid only. Patients reported any side effects throughout the treatment period, and these were estimated at the end for frequency and severity. Twenty patients (10 per group) dropped out (Fig. 1), leaving 60 for the final analysis. A sample range of 60 was determined based on a 95% confidence interval and 80% power [24].

Controlled ovarian induction

Ovarian stimulation involved daily injections of recombinant gonadotropins (Cinal-F 75IU-CinnaGen, Iran) starting at 150–75 IU on the 2nd or 3rd day of menstruation, either spontaneous or induced. The dose was modified based on ultrasound monitoring of ovarian response. Once a controlling follicle got to 14 mm in diameter, a daily dose of GnRH antagonist (Cetroide, Merck Serono, Germany) was started and continued until ovulation was triggered. At least three follicles exceeding 18 mm in diameter were required for ovulation triggering with HCG injection (PDpreg5000IU-pooyesh darou, Iran). Oocytes were recovered 34–36 hours following hCG injection.

Before treatment, fasting blood samples were gathered twice: on day 3 of the menstrual cycle before the previous ovulation pick-up (OPU) and again during follicular aspiration. Each peripheral blood sample was directly centrifuged at 3000 rpm (10 min) (EBA20, Hettich, UK), and the serum was stored (at -70°C) for later examination. Coming after follicular aspiration and OPU, the follicular fluid (FF) was saved from the first aimed follicle without observable blood contamination. FF samples were centrifuged at 3000 rpm (10 min) using an EBA20 centrifuge (Hettich, UK). For further analysis, the supernatants were then saved at -70°C.

Oocyte retrieval, ICSI, and embryo culture

Semen analysis followed World Health Organization (WHO) guidelines [25]. Oocyte retrieval performed under ultrasound guidance using a thin, single-lumen needle (Reproline medical GmbH, Germany) inserted through the vagina. To prepare the oocytes for subsequent analysis, cumulus cells were enzymatically dislodged via a 30-second incubation in 20 IU/mL hyaluronidase (ART-4007A, SAGE BioPharma) followed by gentle mechanical pipetting. Mature oocytes, characterized by the presence of a first polar body, were then identified and isolated under a stereo microscope (Olympus Co, Japan). Only fertilized oocytes with two pronuclei (2PN), identified after overnight culture, were selected for embryo transfer. These embryos were then transferred to a pre-equilibrated cleavage medium (ART-1526, SAGE BioPharma) and cultured for three days. Finally, using an embryo transfer catheter (Cook Medical LLC, USA), the embryos were transferred back into the patient's uterus. Each patient received a maximum of three embryos during transfer. Fertilization was evaluated 12–16 hours following ICSI by confirming two different pronuclei. Cleavage rate was estimated 24–36 hours following fertilization. Quality of embryo was graded during third day of insemination using a three-point system [26]: Grade I: Symmetrical blastomeres without fragmentation; Grade II: irregular blastomeres with less than 25% fragmentation; Grade III: irregular blastomeres with over 25% fragmentation. Pregnancy was confirmed by serum β-HCG levels measured on days 14–15 after transfer. Clinical pregnancies were defined by the presence of both a gestational sac and heartbeat on ultrasound scans performed at 6 weeks post-transfer.

Assessment of clinical features and hormonal assays

Weight and height of patients were assessed for defining their body mass index (BMI). Obesity was defined as having a BMI greater than 30. The waist circumference was divided by the hip circumference to estimate the waist-to-hip ratio (WHR). The study measured blood pressure of patients. At the treatment conclusion, during the early follicular phase, participants' weight, waist and hip peripheries, and blood pressure were all reassessed. Serum and follicular fluid levels of the following hormones were evaluated by ELISA based on the manufacturer's guidance: total testosterone (TT, ng/dl; Abcam ab108666), thyroid stimulating hormone (TSH, mIU/ml; Abcam ab100660), follicle provoking hormone (FSH, mIU/ml; Abnova KA0213), luteinizing hormone (LH, mIU/ml; Abcam ab178658), estradiol (E2, pg/ml; Abcam ab108667), androstenedione (AE, ng/ml; Abcam ab178609), anti-Mullerian hormone (AMH, ng/ml; Abcam ab267629), prolactin (PRL, ng/ml; Abcam ab226901), and insulin (INS, mIU/ml; Abcam ab278123). Measurements were performed in duplicate on samples gathered during third day of the menstrual cycle and once during follicular aspiration.

Estimating the oxidative stress biomarkers

This study measured malondialdehyde (MDA), a biomarker of lipid peroxidation (LPO) [26], in both serum and follicular fluid using the thiobarbituric acid (TBA) colorimetric assay (TBARS Assay Kit, Abcam ab233471). MDA levels were expressed in micromoles per liter (µM). Additionally, total antioxidant capacity (TAC, µM; Abcam ab288592) and levels of activity in superoxide dismutase (SOD, IU/ml; Abcam ab277415) and glutathione peroxidase (GPx, ng/ml; Abcam ab123158) were measured in both samples by ELISA methods based on the manufacturers' guidance. Samples collected on the third day of the menstrual cycle and once during follicular aspiration were measured in duplicate.

Statistical analysis

Kolmogorov-Smirnov was used to test the distribution of changes in the two groups for normality. Demographic and experimental data were then presented as mean ± SEM if normally distributed, or reported using other descriptive statistics if not. For normally distributed data, Statistical comparisons were conducted with Student's t-tests for repeated measures, with a two-tailed p-value < 0.05 considered significant. Chi-square tests analyzed categorical data when appropriate, while Pearson's correlation coefficients assessed relationships between variables. SPSS software (version 24.0) was used for all analyses.

Clinical and demographic features

Before treatment, the two groups demonstrated no significant differences in any baseline characteristics, including age, marital duration, infertility duration, waist circumference, BMI, hip circumference, waist-to-hip ratio, or prevalence of oligomenorrhea and hirsutism (Table 1).

Hormonal, biochemical characteristics, and oxidative stress markers in the serum

After a comparison of the MI group with the placebo group, serum levels of LH (P = 0.001), LH/FSH ratio (P = 0.001), total testosterone (P = 0.001), androstenedione (P = 0.01), and AMH (P = 0.001) showed significant changes, while levels of FSH (P = 0.085), estradiol (P = 0.574), prolactin (P = 0.124), insulin (P = 0.230), and TSH (P = 0.459) remained stable (Table 2). Similarly, Table 3 indicates meaningful differences in the adjusted change of serum MDA (P = 0.001), TAC (P = 0.001), SOD (P = 0.01), and GPx (P = 0.01) between the MI and placebo groups.

Table 1. Baseline and clinical characteristics of the two groups of PCOS patients (n=80).

Data are shown as mean ± SEM. Analysis was performed by Student’s t- test. NS. No differences were observed between the mean of variables in the two groups (P > 0.05). *Analysis was performed by Pearson Chi-Squared test for comparisons.

Placebo group, folic acid; MI group, Myo-inositol plus folic acid; BMI, body mass index.

Parameters	Treatment groups
Parameters	MI	Placebo	*p value*
Age (25-35 years)	27.77±3.3	28.43±3.07	0.423^NS
Duration of marriage (year)	6.8±2.46	7.18±3.24	0.608^NS
Duration of infertility (year)	5.93±2.36	5.22±2.37	0.246^NS
Waist size (cm)	90.9±13.2	91.7±15.4	0.931^NS
Hip size (cm)	106.4±13.1	108.4±13.4	0.970^NS
Waist/Hip ratio (WHR)	0.84±0.04	0.83±0.05	0.366^NS
BMI (kg/m²)	27.32±3.48	28.13±2.99	0.853^NS
No. of Oligomenorrhea patients (%) ^*	8 (26.7)	7 (23.3)	0.677^NS
No. of Hirsute patients (%) ^*	7 (23.3)	6 (20)	0.654^NS

Table 2. Oxidative stress markers and hormonal profile of baseline and after treatment in the serum of PCOS patients and their adjusted changes.

Data are shown as mean ± SEM. Analysis was performed by Student’s t- test. Significant differences for the comparison between treatments are in bold type.

Placebo, folic acid group; MI, Myo-inositol plus folic acid group; FSH, Follicle stimulating hormone; LH, luteinizing hormone; PRL, Prolactin; AMH, Anti-Mullerian hormone; TSH, Thyroid stimulating hormone; E2, estradiol. MDA, malonaldehyde; TAC, Total Antioxidant Capacity; SOD, Superoxide Dismutase; GPx, Glutathione Peroxidase.

Parameters	Treatment groups
	MI (n=30)			Placebo (n=30)			*p value for change comparison*
	Baseline	After treatment	change	Baseline	After treatment	change	*p value for change comparison*
LH (mIU/ml)	9.3±0.25	5.7±0.24	- 3.6±0.31	10.9±0.24	10.4±0.18	1.07±0.18	0.001
FSH (mIU/ml)	4.9±0.15	4.8±0.9	- 0.13±0.19	5.01±0.17	5.3±0.18	0.33±0.17	0.085
LH/FSH ratio	1.9±0.06	1.2±0.05	- 0.7±0.07	2±0.05	2.1±0.07	0.11±0.08	0.001
Total Testosterone (ng/dl)	65.28±1.8	49.58±2.3	- 15.7±1.3	64.32±1.7	82.53±1.5	18.2±1.2	0.001
E2 (pg/ml)	56.5±1.9	61.15±2.1	4.7±1.5	54.8±2.1	57.9±2.5	3.12±2.1	0.574
PRL (ng/ml)	17.8±0.7	16.7±0.7	- 1.1±0.35	18.23±0.7	18.07±0.7	- 0.16±0.5	0.124
Androstenedione (ng/ml)	2.9±0.2	2.09±0.16	- 0.86±0.14	2.85±0.19	3.1±0.16	0.24±0.13	0.01
Insulin (mIU/ml)	19.67±0.7	19.07±0.6	- 0.6±0.2	19.44±0.6	19.28±0.6	- 0.16±0.2	0.230
AMH (ng/ml)	8.5±0.27	6.9±0.2	- 1.6±0.25	9.03±0.27	11.78±0.3	2.7±0.3	0.01
TSH (mIU/ml)	2.2±0.2	2.01±0.2	- 0.19±0.07	2.3±0.25	2.2±0.27	- 0.08±0.1	0.459
MDA (μM)	1.9±0.1	1.2±0.09	- 0.7±0.04	2.06±0.09	4.02±0.09	1.9±0.13	0.001
TAC (μM)	0.79±0.09	1.64±0.1	0.85±0.05	0.77±0.06	0.3±0.05	- 0.47±0.07	0.001
SOD (IU/ml)	44.4±0.7	48.4±0.8	3.97±0.2	44.1±0.6	29.1±0.9	- 14.9±1.1	0.01
GPx (ng/ml)	35.39±0.6	39.31±0.6	3.92±0.3	35.91±0.5	27.74±0.5	- 8.17±0.5	0.01

Table 3. Comparison of the hormonal profile and oxidative stress markers in the follicular fluid of patients with polycystic ovary syndrome.

Data are shown as mean ± SEM. Analysis was performed by Student’s t- test. Significant differences for the comparison between treatments are in bold type.

Placebo, folic acid group; MI, Myo-inositol plus folic acid group; FSH, Follicle stimulating hormone; LH, luteinizing hormone; PRL, Prolactin; AMH, Anti-Mullerian hormone; TSH, Thyroid stimulating hormone; E2, estradiol; MDA, malonaldehyde; TAC, Total Antioxidant Capacity; SOD, Superoxide Dismutase; GPx, Glutathione Peroxidase.

Parameters	Treatment groups
Parameters	MI (n=30)	Placebo (n=30)	*p value*
LH (mIU/ml)	0.89±0.05	1.18±0.07	0.03
FSH (mIU/ml)	6.04±0.28	5.6±0.32	0.350
LH/FSH ratio	0.16±0.01	0.24±0.02	0.01
Total Testosterone (ng/dl)	6.8±0.37	8.2±0.6	0.047
E2 (pg/ml)	483.43±32.7	451.33±26.3	0.448
PRL (ng/ml)	18.5±0.8	18.9±0.8	0.753
Androstenedione (ng/ml)	404.73±25.6	487.23±27.9	0.034
Insulin (mIU/ml)	6.6±0.4	7.73±0.5	0.082
AMH (ng/ml)	438.9±23.6	526.07±29.4	0.024
TSH (mIU/ml)	1.63±0.1	1.6±0.1	0.794
MDA (μM)	1.96±0.09	4.7±0.36	0.001
TAC (μM)	2.6±0.1	1.9±0.07	0.001
SOD (IU/ml)	42.91±0.9	39.64±0.9	0.02
GPx (ng/ml)	39.77±0.7	36.83±0.5	0.01

Table 4. Distribution of retrieved oocytes, the quality of oocytes and embryos, and pregnancy outcome in PCOS patients.

Data are shown as mean ± SEM. Analysis was performed by Student’s t- test. Significant differences for the comparison between treatments are in bold type. * Analysis was performed by chi-square test for multiple comparisons (P < 0.05). NS. No differences were observed between the mean of variables in the MI group compared with the placebo group (P > 0.05).

Parameters	Treatment groups
Parameters	MI (n=30)	Placebo (n=30)	*p value*
No. of oocytes retrieved	9.5±0.69	11.03±0.95	NS
No. of immature oocytes (GV+MI)	2.2±0.2	3.1±0.37	0.038
No. of mature oocytes (MII)	7.33±0.68	7.93±0.67	NS
MII/total oocytes retrieved	0.74±0.03	0.73±0.02	NS
No. of fertilized oocytes (2PN)	7.01±0.6	5.7±0.4	NS
No. of cleaved embryos	6.8±0.5	5.2±0.4	0.027
No. of embryos Grade I	3.27±0.42	1.53±0.15	0.001
No. of embryos Grade II	2.17±0.2	1.8±0.2	NS
No. of embryos Grade III	1.33±0.17	1.83±0.18	NS (0.06)
No. of Good quality of Embryos (I+II)	5.43±0.5	3.33±0.3	0.01
No. of clinical pregnancy (%) *	11 (36.6)	6 (20)	NS

Hormonal, biochemical characteristics, and oxidative stress biomarkers in the follicular fluid

Similar to serum levels, follicular fluid (FF) concentrations of LH (P = 0.001), LH/FSH ratio (P = 0.01), total testosterone (P = 0.047), Androstenedione (P = 0.034), and AMH (P = 0.024) significantly increased in the MI group in comparison with the placebo group (Table 3). Conversely, levels of FSH (P = 0.350), estradiol (P = 0.448), prolactin (P = 0.753), insulin (P = 0.082), and TSH (P = 0.794) remained stable (Table 3). Furthermore, adjusted changes in FF levels of MDA (P = 0.001), TAC (P = 0.001), SOD (P = 0.02), and GPx (P = 0.01) were significantly different between the MI and placebo groups (Table 3).

Estimation of oocytes morphology and embryos

While the whole of oocytes recovered and mature oocytes did not indicate significant alteration between the MI and placebo groups (P > 0.05), the whole of immature oocytes (MI + GV) significantly reduced in the MI group (P < 0.01). Cleavage rates were significantly higher in the MI group in comparison with the placebo group (P < 0.01), while the number of fertilized oocytes (2PN) remained similar (P > 0.05). Notably, the MI group raised the formation of good quality embryos (Grade I) on day 3 (P < 0.01), with no significant difference detected in Grades II and III. Overall, the number of good quality embryos (Grade I + II) was significantly higher in the MI group (P < 0.01). However, there was no significant difference in the clinical pregnancy rate between the groups (P > 0.05) (Table 4).

Correlations between follicular fluid variables

In the placebo group, follicular fluid (FF) levels of AMH indicated significant positive correlations with LH (r = 0.766; p = 0.0001), insulin (r = 0.512; p = 0.004), total testosterone (r = 0.642; p = 0.0001), androstenedione (r = 0.773; p = 0.0001), MDA (r = 0.438; p = 0.016), the whole of retrieved oocytes (r = 0.569; p = 0.0001), and immature oocytes (MI + GV) (r = 0.739; p = 0.0001). Notably, a significant negative correlation was observed between FF AMH and FSH (r= -0.563; p = 0.001). However, in the MI group, these associations were attenuated or absent. FF AMH levels no longer exhibited significant correlations with LH (r = 0.283; p = 0.029), FSH (r= -0.037; p = 0.845), insulin (r = 0.098; p = 0.607), total testosterone (r= -0.015; p = 0.937), androstenedione (r = 0.096; p = 0.612), or MDA (r = 0.175; p = 0.345). Similarly, there were no significant correlations between AMH and the whole of retrieved oocytes (r= -0.126; p = 0.507), or immature oocytes (MI + GV) (r = 0.182; p = 0.335). These consequences indicated that MI treatment modifies the relationships between AMH and other follicular fluid factors, potentially contributing to improved ICSI outcomes in PCOS patients (Figs. 2, 3).

This study showed that Myo-inositol significantly reduced serum and follicular fluid (FF) levels of LH, LH/FSH ratio, full testosterone, AMH, and androstenedione in comparison with the placebo group. Conversely, significant increases were observed in the levels of TAC, GPx, and SOD in both serum and FF of the myo-inositol group compared to the placebo group. Additionally, Myo-inositol significantly lowered MDA levels in both serum and FF. Notably, there was no significant difference between two groups in serum or FF levels for FSH, TSH, prolactin, insulin, and estradiol.

Because of the established link between insulin resistance and PCOS symptoms, insulin sensitizing agents are often used to address hormonal imbalances [3]. Artini et al. considered the impacts of myo-inositol (MI) in 50 obese PCOS women. Twelve weeks of treatment led to statistically significant decreases in blood levels of LH, prolactin, testosterone, and insulin, along with a reduction in the LH/FSH ratio. Moreover, insulin sensitivity significantly raised, and menstrual cyclicity was saved in the whole of amenorrheic and oligomenorrheic participants [27]. Similarly, Genazzani et al. presented similar findings in a study with 20 obese PCOS women. Following 12 weeks of MI administration, they observed significant decreases in plasma levels of LH, prolactin, testosterone, and insulin, as well as improved insulin sensitivity [28].

Our study investigated the efficacy of 6 weeks of myo-inositol (MI) treatment at 4 g/day on hormonal parameters in serum of PCOS patients. Compared to the placebo group, MI treatment significantly reduced total testosterone, androstenedione, LH, LH/FSH ratio, and AMH levels, while serum FSH, prolactin, TSH, estradiol, and insulin remained unchanged. Supporting the findings of previous studies [28, 29, 27], these results demonstrate that MI administration significantly improves hormonal and metabolic aspects in PCOS subjects. This confirms MI's potential as a safe and effective alternative for PCOS patients undergoing ICSI, with no observed side effects at the standard dosage. Notably, discrepancies with other studies may be attributed to differences in treatment duration of MI and genetic variations within the studied populations.

Analyzing the molecular profile of follicular fluid (FF) offers valuable insights into PCOS and its impact on oocyte quality. Intricately intertwined with the oocyte, this biological complex harbors a diverse array of bioactive molecules, critically driving follicle development and maturation [30]. In PCOS, elevated FF insulin levels may trigger local androgen production, potentially compromising oocyte quality. Dysregulated FF composition, characterized by high levels of LH, androgens, AMH, TSH, and leptin, and an imbalance between pro-oxidative (ROS) and antioxidant (TAC) molecules, hinders reproductive success by decreasing fertilization and implantation rates, increasing embryonic fragmentation, and raising miscarriage rates [31]. Our study demonstrates the significant effectiveness of 6-week MI treatment in reducing FF levels of LH, LH/FSH ratio, total testosterone, AMH, and androstenedione compared to the placebo group. While some contradictory reports exist [5, 32], these discrepancies could likely stem from variations in patient selection criteria, drug dosage, as well as ovulation induction protocols. However, oxidative stress, an imbalance in free radicals and antioxidants of cells, plays a vital role in PCOS development. Numerous studies have demonstrated elevated levels of oxidative stress markers in both serum and follicular fluid (FF) of PCOS patients, potentially linking it to disruptions in cellular organelles and molecular and biochemical procedures [33, 34]. Consistent with previous findings [35, 26, 33, 36], our study confirms elevated oxidative stress in PCOS patients. We observed significant alterations in serum levels of MDA, TAC, SOD, and GPx activity in the MI group in compared to the placebo group.

Several late researches have presented that myo-inositol, alone or combined with folic acid, can improve ovulation rates and regulate menstrual cycles [37, 38, 17]. This suggests that, beyond its known impact on fertilization rates, myo-inositol supplementation might also enhance overall oocyte quality and pregnancy outcomes in supported reproductive technologies (ART) [35]. In our research, we observed a significant development in the whole of immature oocytes in patients treated with myo-inositol. Additionally, both the percentage of grade I embryos (high quality) and the overall good quality embryo rate (grade I + II) significantly improved in the cured group, along with a higher cleavage rate. However, the difference was not statistically significant, although the pregnancy rate was higher in the treated group. This finding aligns with existing literature on the effects of MI [23, 37, 35, 11, 26]. One potential mechanism by which Myo-inositol enhances fertilization and embryo quality could be its ability to boost oocyte capacity for the crucial oscillatory Ca2 + response during fertilization and early embryonic improvement [5].

Calcium (Ca2+) oscillations is vital for normal fertilization and embryonic development [17]. Myo-inositol, an insulin-sensitizing molecule, has shown promise in PCOS women by improving insulin resistance, steroidogenesis, ovarian stimulation parameters, high-quality embryo formation, and even spontaneous ovulation [21, 39, 26]. However, some review studies suggest that patients may respond differently to myo-inositol therapy based on their specific PCOS phenotypes [32]. To optimize results, further research could explore adjustments in dose, treatment duration, sample size, patient inclusion criteria, and ovulation induction protocols.

Anti-Müllerian hormone (AMH) makes a vital contribution to ovarian follicle improvement and is often elevated in PCOS, correlating with various reproductive and metabolic/endocrine alterations [40]. A better perception of this link could help us develop better treatments for PCOS [41]. Several studies have shown elevated AMH in PCOS women compared to healthy individuals, potentially due to abnormally high levels of LH, androgens, and insulin [41]. Additionally, research has established significant correlations between follicular fluid (FF) AMH and key factors in PCOS pathophysiology, including LH, FSH, testosterone, DHEA-S, BMI, insulin, and oxidative stress [42, 43, 40]. In our study, we observed these same correlations between FF AMH and the biochemical features (LH, FSH, insulin, total testosterone, androstenedione, MDA), immature oocytes, and total retrieved oocytes in the placebo group. These findings support previous studies [44, 43, 40].

Several lines of evidence extra provide the idea that AMH is in a causal way involved in PCOS pathophysiology, demonstrating a near link between alterations in AMH concentrations and improvements in PCOS symptoms in the answer to therapy [45, 41]. This study strengthens that connection by showing that myo-inositol not only induced positive alterations in both serum and follicular fluid hormonal parameters but also led to reductions in LH, AMH, elevated androgenic values, and oxidative stress markers. Consequently, we observed improved oocyte and embryo quality. These findings solidify myo-inositol's position as a safe and effective alternative for managing ovulation, hyperandrogenism, and hormonal parameters in PCOS patients, potentially leading to better outcomes. Overall, myo-inositol offers a promising, side-effect-free treatment option. However, this study also has constraints. The sample size is underpowered, and we were unable to track individual ICSI outcomes for oocytes retrieved from the dominant follicles.

Based on current evidence, myo-inositol stands as an effective therapeutic option for PCOS patients. Its benefits extend beyond improving hormonal profiles in both serum and follicular fluid. It also promotes oocyte maturation, embryo quality, and potentially, ICSI outcomes. Notably, myo-inositol supplemental materials may raise the whole of embryos having more quality, potentially leading to increased pregnancy rates among PCOS women. This aspect warrants further investigation in future studies.

Acknowledgments: The authors are grateful to the members of the IVF unit of the infertility treatment center of the ACECR, Qom.

Author contributions: All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Zeynab Yazdanpanah, Ebrahim Cheraghi, Mitra Heydari Nasrabadi, and Masoud Salehipour. The first draft of the manuscript was written by Zeynab Yazdanpanah, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding: No funding was received for conducting this study.

Availability of data: Data underlying this article will be shared on reasonable request to the corresponding author.

Conflict of Interests: The authors have no relevant financial or non-financial interests to disclose.

Consent to participate: Written, informed consent was obtained from the patients.

Consent for Publication: No identifying details of the participants are published in this manuscript; hence, consent for publication is not obtained.

Ethical approval: This study was approved by the research ethics committee (Islamic Azad University, Varamin Pishva Branch [IR.IAU.VARAMIN.REC.1400.033]).

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The influence of Myoinositol to improve biochemical manifestations of the serum and follicular fluid and ICSI outcomes in patients with PCOS: A prospective randomized research

Status:

Version 1

Abstract

Purpose

Methods

Results

Conclusion

Figures

What does this study add to the clinical work?

Introduction

Methods

Study population

Treatment design

Controlled ovarian induction

Oocyte retrieval, ICSI, and embryo culture

Assessment of clinical features and hormonal assays

Estimating the oxidative stress biomarkers

Statistical analysis

Results

Clinical and demographic features

Hormonal, biochemical characteristics, and oxidative stress markers in the serum

Estimation of oocytes morphology and embryos

Correlations between follicular fluid variables

Discussion

Conclusions

Declarations

References

Status:

Version 1