Is There An Association between Dyslipidemia and The Risk of Ovarian Hyperstimulation Syndrome in A Population of Non-Obese Polycystic Ovary Syndrome Patients? A Cross-Sectional Study.

It seems that polycystic ovary syndrome (PCOS), one of the most complicated endocrine disorders, plays an important role in the anovulatory infertility. The prevalence of PCOS in women of reproductive age ranges from 6 to 20% ( 1 ). PCOS is a metabolic syndrome characterized by obesity, insulin resistance, increased oxidative stress and chronic inflammation, and dyslipidemia ( 2 ). Dyslipidemia is the most common PCOS-related metabolic dysfunction, which includes being abnormal in one or more types of plasma lipids, such as elevated total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and triglyceride (TG) concentrations, as well as reduced highdensity lipoprotein cholesterol (HDL-C) levels ( 3 ). Obesity is common in women with dyslipidemia. However, some individuals have metabolic abnormalities and dyslipidemia without being obese, which is known as normal weight dyslipidemia ( 4 ). Women’s dyslipidemia is frequently linked with increased ovarian inflammation and oxidative stress, which can disrupt the normal procedure of cytoplasmic and nuclear maturation of an oocyte, follicular steroidogenesis, and ovulation ( 5 ). Increased levels of lipid metabolic parameters have recently been linked to diminished oocyte quality and embryo developmental potential, as well as poor clinical outcomes ( 5 ). The harmful effect of dyslipidemia on female fertility is widely acknowledged, but the precise mechanisms by which it influences ovarian functions and an oocyte quality remain unknown. Women with dyslipidemia are thought to have a high level of inflammatory cytokines ( 6 ). The inflammatory state caused by dyslipidemia is idiosyncratic and distinct from classical inflammation ( 7 ). Dyslipidemia enhances the release of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-18 (IL-18), both of which are prominent mediators of inflammation in women with PCOS-metabolic dysfunctions ( 8 ). It has been proposed that TNF-α and IL-18 have affected the processes of ovulation, fertilization, and embryo implantation ( 8 , 9 ). In addition, emerging evidence suggests that ovarian hyperstimulation syndrome (OHSS) is associated with dyslipidemia and inflammatory cytokines ( 10 , 11 ). OHSS is a severe complication of controlled ovarian hyperstimulation (COH), with a significantly increased OHSS risk in PCOS patients ( 11 ). The presence of inflammatory cytokines in follicular fluid (FF) has been associated with oocyte quality, embryo development, and clinical outcomes following ART cycles ( 7 ). Because follicular somatic cells directly interact with FF, the inflammatory cytokines found in FF may contribute to increase androgen production and influence oocyte growth and maturation processes ( 12 ). For the first time, Niu et al. ( 13 ) demonstrated an association between FF cytokines and embryo quality in PCOS patients with metabolic syndrome. Therefore, the relationship between lipid metabolites and inflammatory cytokines in serum and FF required to be further explored ( 5 ). In this regard, the current study aims to investigate the relationship between dyslipidemia and pro-inflammatory cytokines in blood and FF with ART outcomes and the risk of OHSS in non-obese PCOS patients undergoing intracytoplasmic sperm injection (ICSI).

A total of 210 couples were assessed for their eligibility to participate in the current study, of which 80 couples met the exclusion criteria and 130 couples, 51 in the normal lipid and 79 in the dyslipidemia group, participated in the present study. In term of serum evaluation, there was no significant difference between the two groups in the levels of anti-Mullerian hormone (AMH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and FSH/LH. However, mean age and BMI were significantly higher in the dyslipidemia group in comparison with the normal lipid group (P=0.021, P<0.001, respectively). Also, serum levels of estradiol were significantly higher in the normal lipid group compared to the dyslipidemia group (P=0.025). In addition, serum lipid metabolic parameters, including TC, TG, LDL-C, and HDL-C, were significantly higher in the dyslipidemia group in comparison with the normal lipid group (P<0.001, Table 1 ). Demographic characteristics Data are presented as mean ± SD and median (inter quartile range) for estradiol at day of trigger. BMI; Body mass index, AMH; Anti-Mullerian hormone, FSH; Follicle-stimulating hormone, LH; Luteinizing hormone, TC; Total cholesterol, TG; Triglyceride, LDL-C; Lowdensity lipoprotein cholesterol, and HDL-C; High- density lipoprotein cholesterol. There was no significant difference between our groups in the FF levels of TC, TG, and HDL-C ( Table 2 ). However, the FF level of LDL-C in the dyslipidemia group was significantly higher than the normal lipid group (P=0.007). Follicular fluid lipid metabolic parameters Data are presented as mean ± SD. For statistical test use t test. TC; Total cholesterol, TG; Triglyceride, LDL-C; Low-density lipoprotein cholesterol, and HDL-C; High-density lipoprotein cholesterol. There was no significant difference between the two groups in the serum level of IL-18. However, the serum level of TNF-α was significantly higher in the dyslipidemia group in comparison with the normal lipid group (P<0.001). Furthermore, the FF levels of IL-18 and TNFα were significantly higher in the dyslipidemia group than in the normal lipid group (P<0.001, P=0.005, respectively, Table 3 ). Serum and follicular fluid cytokines concentrations Data are presented as mean ± SD. For statistical test use t test. IL; Interleukin, and TNF- α; Tumour necrosis factor-alpha. The results demonstrated that the incidence of OHSS was higher in the normal lipid group than in the dyslipidemia group ( Table 4 ). Risk of OHSS For statistical test use chi-square test. OHSS; Ovarian hyperstimulation syndrome. Table 5 represents the ART results and clinical outcomes of the groups studied. There was no significant difference between our groups in different parameters. In the current study, 48 FET cycles were conducted in the normal lipid group. In addition, 73 FET cycles were performed in the dyslipidemia group. Embryo transfer procedure did not take place for nine couples, because of their ultimate decision on canceling ET. Cleavage-stage embryos were transferred in 19 cases of the normal lipid group and 33 cases of the dyslipidemia group. Furthermore, for 29 cases in the normal lipid group and 40 cases in the dyslipidemia group, blastocyst-stage embryos were transferred. There was no significant difference in the chemical and clinical pregnancy rates between the groups studied. ART and clinical outcomes Data are presented as mean ± SD or median (interquartile range) or frequency (%). Statistical tests: for sperm characteristics, number of normal morphology injected oocytes, number of dysmorphic injected oocytes, number of highly dysmorphic injected oocytes, use Mann Whitney U test, for number of retrieved oocytes, number of MII oocytes, fertilization rate and cleavage rate, blastocyst development rate, number of embryos transferred, use t test and for chemical and clinical pregnancy rate use Chi-square test. *; P<0.05, IVF; In vitro fertilization, MII; Metaphase II. Our findings revealed a correlation between FF cytokines and ART outcomes in PCOS patients, regardless of lipid status ( Table 6 ). Pearson's correlation analysis showed that the number of retrieved oocytes in PCOS women was negatively correlated with FF levels of TNF α (P=0.001) and IL18 (P=0.049). In addition, the number of MII oocytes in PCOS women was negatively correlated with the FF level of TNFα (P=0.011). Pearson's correlation analysis of fertilization and cleavage rates revealed a negative correlation with FF levels of TNFα (P=0.002, P=0.003 respectively) and IL18 (P=0.042, P=0.049 respectively). Furthermore, there is a negative correlation between blastocyst development rate and FF level of TNFα (P=0.042, Table 6 ). Inflammatory cytokines markers and ART outcomes٭ ٭; Pearson’ correlation analysis between follicular fluid cytokines with assisted reproductive technologies (ART) outcomes among PCOS patients regardless of lipid status, IL; Interleukin, and TNF- α; Tumor necrosis factor-alpha. In the present study, the levels of FF lipid metabolic parameters, including TC, TG, HDL-C, and LDL-C, in the dyslipidemia group was higher than in the normal lipid group, and also the level of LDL-C was statistically significant. In addition, serum TNFα levels and FF levels of TNFα and IL-18 in POCS patients were significantly higher in the dyslipidemia group compared to the normal lipid group. According to the findings, the dyslipidemia profile did not affect ART outcomes. The current study found a strong correlation between FF inflammatory cytokines and ART outcomes, including the total number of oocytes and metaphase MII oocytes, fertilization rate, cleavage rate, and blastocyst development rate. The risk of OHSS was higher in the normal lipid group than in the dyslipidemia group.

Dyslipidemia is a vital metabolic phenotype that influences ART outcomes in PCOS patients ( 1 ). An elevated LDL, cholesterol and glycerol in serum, are present in the follicular fluids of poly cystic ovaries. This is due to compromised lipase expression and altered lipolysis triggered by insulin resistance ( 11 ). The lipotoxic effect of the dyslipidemia complex causes a low-grade inflammatory situation ( 20 ). Obesity causes macrophage infiltration into adipose tissue and, consequently, alteration of macrophage phenotype from anti-inflammatory to pro-inflammatory status ( 7 , 21 ). In addition, non-obese women with PCOS display a rise in lipid-induced cytokine secretion from mononuclear cells ( 4 ). González et al. ( 20 ) demonstrated that in there is an increased level of, TNFα, IL-6, and IL-1β cytokines in the absence of obesity in patients with PCOS. IL-18 is a proinflammatory cytokine initiates the cascade of other inflammatory cytokines, including TNFα, IL-1β, and IL-6 ( 22 ). Most studies on chronic low inflammation in PCOS have focused on serum rather than FF ( 20 ). Some studies have demonstrated that IL-18 is present in the FF of PCOS patients ( 22 , 23 ), and the present study discovered that IL-18 and TNFα levels in the FF of PCOS patients are higher in the dyslipidemia group compared to the normal lipid group. TNFα and IL-18 are likely to be cytokines involved in the pathophysiology of PCOS, possibly in non-obese women with dyslipidemia. Because FF contains some factors accountable for ovulation and oocyte maturation, disturbances in its composition can influence folliculogenesis ( 24 ). It seems that inflammatory microenvironment changes in the FF of women with PCOS may be a crucial factor that may have a detrimental impact on the biology of oocytes ( 25 ). Although, there were no statistical differences in ART outcomes or clinical pregnancy between our groups, the present study found a correlation between FF inflammatory cytokines and ART outcomes regardless of lipid status. OHSS is a potential complication of IVF/ICSI cycles. In this case, the ovaries swell, and fluid leaks into the peritoneum, which could be fatal. This condition is more frequent in PCOS women undergoing IVF/ICSI cycles ( 26 ). The pathological mechanism of OHSS is currently not well understood. It seems, that capillary permeability enhancement may be a main pathophysiological mechanism, which results in fluid transfer from the intravascular space to extravascular parts ( 10 ). Dyslipidemia has been shown to cause vascular wall damage ( 10 ). In addition, because of their vasoactive possessions, inflammatory cytokines have been identified as potential mediators in developing OHSS ( 27 ). In a retrospective study, Liu et al. ( 10 ) showed that dyslipidemia increases the severe OHSS incidence in PCOS patients undergoing ICSI cycles compared to PCOS patients in the normal lipid group. In a study involving 1,470 PCOS patients, Jiang et al. ( 3 ) found no significant differences in the incidence of OHSS between dyslipidemia and normal lipid groups. Interestingly, the current study indicated that the risk of OHSS was higher in the normal lipid group than in the dyslipidemia group. Although we evaluated the risk of OHSS occurrence rather than its incidence, our findings contradict the mentioned studies. Previous research has identified low BMI, young age, and a high serum E2 level as risk factors for OHSS ( 28 ). Therefore, one explanation for the high risk of OHSS in the normal lipid group is young age and low BMI, which were significantly lower in our normal lipid group. Furthermore, the normal lipid group had higher serum E2 levels on the trigger day. There is reasonable evidence that a high E2 level is associated with an increased risk of OHSS ( 29 ). Low BMI, young age, and high serum E2 levels may increase the risk of OHSS in the normal lipid group. Therefore, more research is needed to assess the correlation between OHSS and dyslipidemia in PCOS. We are of the opinion that dyslipidemia in non- obese PCOS women may influence ART outcomes due to increasing pro-inflammatory cytokines. Our study was limited by the small study-population size. Also, the present study was not enough comprehensive to examine different risk factors, and also, was not used the total groups that were very similar for equating, risk factors such as age, BMI, and serum E2 levels.

The present study underscored the association between lipid metabolic disorders in non-obese PCOS and heightened inflammatory cytokine levels, correlating with ART outcomes but not with OHSS risk.

The present cross-sectional study began in January 2023 and ended in April 2024 at the Roya Infertility Clinic in Qom, Iran. This study protocol was approved by the Royan Institute’s Ethics Committee, Tehran, Iran (IR.ACECR.ROYAN.REC.1401.050). All participants provided written an informed consent. PCOS patients who met the Rotterdam Criteria ( 1 ) and also, experienced intra-cytoplasmic sperm injection (ICSI) cycle were assessed for eligibility to participate in the present study. According to the Rotterdam consensus, a patient is categorized as a PCOS affected that shows at least two of the following criteria: i. Clinical or biochemical hyperandrogenism or both, ii. Oligo-anovulation, and iii. Polycystic ovary morphology (PCOM) ( 1 ). Based on plasma level of fasting TC and TG, patients were divided into two groups, dyslipidemia and normal lipid groups. Dyslipidemia group was defined as patients with TG ≥150 mg/dL or/and TC ≥200 mg/dL ( 14 ). Patients with serum estradiol level of ≥3000 pg/mL on the day of trigger (36 hours before ovums pick-up) and more than 15 oocytes on the day of oocytes pick-up were considered to be at risk of OHSS ( 15 , 16 ). Women with a history of using anti-hyperlipidemia and anti-inflammatory medications, infectious diseases, sexually transmitted diseases, hypothyroidism, autoimmune disorders, tubal factor infertility, endometriosis, chronic inflammatory diseases, a history of ectopic pregnancy or miscarriage, myomas, polyps, adhesions, previous pelvic surgeries, cancer diagnosis, thrombophilic disorders, anemia, TG ≥300 mg/dL, TC ≥250 mg/dL and body mass index (BMI) ≥30 kg/m2 were all excluded. In addition, participants with an ovarian stimulation cancelation cycle, no oocyte on the day of oocyte pick-up, chromosomal abnormalities and severe male factor of their spouses were also excluded. The gonadotropin releasing hormone (GnRH) antagonist protocol was used to stimulate the ovaries. Transvaginal ultrasound-guided ovum pick-up (OPU) was performed 36 hours after 0.2 mg Triptorlin subcutaneous (Triptorlin, Ferring Gmbh, Germany) injection. Ultrasound-guided transvaginal oocyte retrieval (TVOR) was employed with a single-lumen needle (Reproline Medical, Rheinbach, Germany). Cumulus cell-oocyte complexes (COCs) were retrieved and washed in buffered medium (G-MOPS™ PLUS, Vitrolife Co., Sweden). Oocyte denudation was performed 2 hours after retrieval utilizing hyaluronidase for 30 seconds (HYASE-10X™, Vitrolife Co., Sweden) followed by mechanical dissection. Under a stereomicroscope (Olympus, Japan), mature (MII) oocytes were specified by the presence of the first polar body. Only MII stage oocytes were used for ICSI. Semen specimens were collected by masturbation into sterile polypropylene containers after 2-5 days of sexual abstinence. Semen processing was performed by density gradient ( 17 ). The processed sperm suspension was transferred to a 50-mL droplet of polyvinylpolypyrrolidone (PVP, ART-4006-A; SAGE BioPharma) immediately before ICSI. Two-three hours after oocyte retrieval, a single motile sperm with apparently normal morphology ( 17 ) was immobilized and used for ICSI. Inseminated oocytes were transferred to the fertilization medium (ART-1520; SAGE BioPharma), covered with mineral oil (Reproline Medical) followed by incubation under 37°C in a humidified atmosphere of 6% CO2 condition. Fertilization was assessed the following day, and fertilized oocytes were transferred to the equilibrated cleavage medium if two pronuclei (2PN) were present (ART-1526; SAGE BioPharma). The embryo was incubated at 37°C in a humidified atmosphere, 6% CO2. Some laboratory key performance indicators (KPIs) were measured according to the Vienna consensus ( 18 ): Blood and FF samples were collected at day OPU. Blood-free FF from the leading and first-punctured follicles were collected. Blood and FF samples were centrifuged for 10 minutes at 2000 rpm at 4°C to separate plasma from whole blood and to eliminate cell debris from the FF samples. All samples were stored at -80°C within one hour of oocyte retrieval procedures until laboratory analysis. All embryos were frozen to prevent late OHSS in PCOS patients and then underwent frozen embryo transfer (FET) cycles. Three-day-old embryos vitrified in the commercial media (Kitazato BioPharma Co., Japan) for FET cycles were thawed in commercial media (Kitazato BioPharma Co., Japan) based on the manufacturer’s protocol. The embryos on day three were placed in an embryo culture medium (SAGE 1-Step™, CooperSurgical Co., USA) after the thawing process and incubated at 37°C in 6% CO2 until embryo transfer. According to the opinion of the treatment team of the patients, embryos that had the ability to become blastocysts were cultured (SAGE 1-Step™, CooperSurgical Co., USA) and incubated at 37°C in 6% CO2 until day 5. Top-quality day three embryos or good-quality blastocyst ( 19 ) was selected for FET cycle. Based on the American Society for Reproductive Medicine (ASRM) guidelines, embryo transfer was performed by an expert gynecologist using an embryo transfer catheter (Cook, USA) under ultrasound guidance. Chemical pregnancy was assessed with serum beta human chorionic gonadotropin (hCG) using an ELISA kit (Idealdiagose, Iran) 14 days after embryo transfer. Women were considered chemically pregnant if they had a positive blood beta HCG test. The chemical pregnancy rate was calculated as number of positive ßHCG test at 2nd weeks per number of FET cycles ×100. The clinical pregnancy rate was calculated as the number of detected gestational sacs by ultrasonography at 7th weeks of ET per number of FET cycles ×100. Lipoprotein concentrations in serum and FF, including TC were measured using the Colorimetry (CHOP-pop method, BT1500). Using Colorimetry (GPO pop method, BT1500), TG was evaluated and HDL level was measured by the turbidimetry method (Refurbished, Biotecnica, BT1500, Italy). The LDL level was calculated by the formula: LDL=TCTG/ 5- HDL. Inflammatory markers, including TNFα and IL-18, were measured using an ELISA kit (Karmania Pars Gene, Kerman, Iran). For statistical analyses, the SPSS statistical software package SPSS 22 (IBM Corp., Armonk, NY, USA) was used. Kolmogorov-Smirnov test verified the normality of the variables. If the variables were normal, the relationship between them was assessed using an independent sample t test and we reported mean ± standard deviation (SD); otherwise, the Man Withney test and median (interquartile range) was used if the variables were not normal. Categorical variables were assessed with the Chi-Square test and these variables were expressed as a number (%). The correlation between serum and FF cytokines and ART outcomes was investigated using Pearson’s correlation analysis. The P<0.05 was considered as statistically significant.