Altered expression of kisspeptin, dynorphin, and related neuropeptides in polycystic ovary syndrome: A cross-sectional study

Abstract Background Since kisspeptin (KISS1) in the hypothalamus is affected by the inhibitory effect of dynorphin, it raises questions about the controlled balance of these 2 neuropeptides in women with and without polycystic ovary syndrome (PCOS). Objective This study compares the expression levels of KISS1, dynorphin, neurokinin-B, leptin, and neuropeptide-Y in women with and without PCOS. Materials and Methods In this cross-sectional study, the peripheral blood samples of 20 women with PCOS and 20 women without PCOS who referred to Yamin Kencana Clinic, Cipto Mangunkusumo hospital, Jakarta, Indonesia were enrolled from August-December 2022. mRNA relative expression of genes related to the central factors associated with PCOS, such as leptin, neuropeptide-Y, KISS1, tachykinin-3, and prodynorphin (PDYN), in PCOS and non-PCOS populations were examined. Gene quantification was carried out by the quantitative polymerase chain reaction method. Results The KISS1/PDYN ratio was significantly higher in the PCOS group than in the control group (p = 0.02), and the PDYN was lower in the PCOS group than the control group (p < 0.001). Moreover, the positive correlation between KISS1 and the KISS1/PDYN ratio was significantly stronger in the PCOS group than in the control group (R = 0.93; p < 0.001 vs. R = 0.66, p < 0.001). Conclusion Our results suggest that an increased KISS1/PDYN ratio in PCOS women is related to diminished dynorphin expression. Low expression of the gene encoding dynorphin and a high KISS1/PDYN ratio is highly specific to PCOS.


Introduction
The identification of the kisspeptin-neurokinin-B-dynorphin (KNDy) neuron network within the hypothalamus has provided valuable insights into the intricate roles played by these neurons in the pathogenesis of polycystic ovary syndrome (PCOS).PCOS is the most common endocrinopathy in women of reproductive age.It is associated with abnormal uterine with an increased risk of PCOS development (5).A recent meta-analysis also reported higher serum KISS1 levels in subjects with PCOS than those without (6).Any disturbance in KISS1 regulation also can lead to a metabolic imbalance, such as obesity (7).Moreover, hormonal profile differences in women with PCOS are still unclear (8,9).
Differences in hormone profiles in PCOS women may be related to differences in leptin (LEP) and neuropeptide-Y (NPY) gene expression levels.
These recent studies enhance our understanding of the genetic basis of PCOS and shed light on the potential contributions of these genes to the development of the syndrome.
While some studies have measured the serum levels of neuropeptides in PCOS women (4-9), few studies have focused on the expression of the genes encoding these neuropeptides in peripheral blood (6,10).Previous studies have stated that the mRNA expressions of these genes were detected in peripheral blood (11)(12)(13).Since the secretion of KISS1 is influenced by many factors, such as dynorphin, NKB, LEP, and NPY, it would be interesting to study their role in the context of women with PCOS.

Knowledge of gene expression levels in women
with PCOS is required to identify gene variation and its effect on phenotypic hormone variations as a basis for further research.This study aimed to compare KISS1, PDYN, TAC3, LEP, and NPY expression levels in women with and without PCOS.and c) polycystic ovaries on ultrasound examination (14).

Subjects
Women diagnosed with PCOS were included in the PCOS group, while women without PCOS served as the control group (n = 20/each).
Participants were excluded if they were either pregnant, on medications that could alter metabolic parameters for the past 2 months, or had endocrine abnormalities.

Sample size
The sample size was calculated using the Lemeshow formula.The prevalence of PCOS in Indonesia is 8-10%, so the proportion value of 10% is used.The percentage or errors used in this study was 5% ( = 5%).The precision used in this was 20% (d = 0.2).Based on these calculations, 10 samples are sufficient for each group.However, we used 20 participants in each group for the power of our study.

Ethical considerations
All research participants gave their permission to be part of this study in the form of written informed consent.The Ethics Committee of the Indonesia and Cipto Mangunkusumo hospital, Jakarta, Indonesia has approved the study (Code: 0449/UN2.F1/ETIK/2018).to the KISS1/PDYN and KISS1/TAC3 ratio with a significant level of < 0.05.

Demographic and hormonal profiles
For this study, a total of 40 women were consecutively screened for eligibility based on the inclusion and exclusion criteria, and none of them were found to be ineligible or refused to participate.Finally, the analysis included 20 women in the PCOS and 20 women in the control group.
The mean age of the participants was 30.00 ± 5.00 yr in the PCOS and 28.83 ± 1.25 yr in the control group.The 2 groups had no significant differences in age, body mass index, and hormonal profiles, except for a longer menstrual cycle in the PCOS group.Moreover, no significant difference was observed in the total testosterone levels, SHBG levels, and free androgen index in the 2 groups (Table I).

Correlation between KISS1 and its ratio to dynorphin and NKB
We sought the correlation of the relative expression of KISS1 to the KISS1/PDYN and KISS1/TAC3 ratio to reveal the controlled balance of these neuropeptides since KISS1 in the hypothalamus is affected by the inhibitory effect of dynorphin and the stimulatory effect of NKB.By comparing the relative expression of KISS1 against dynorphin and NKB, we get a ratio that reflects the controlled balance of these 2 neuropeptides in the hypothalamus.
Correlation analysis was performed separately for the control and PCOS groups (Figure 1).Neuropeptides expression in PCOS

Discussion
In this study, we could not establish a higher KISS1 expression in the PCOS group than in the control group.The level of protein or peptide contained in the body significantly and positively correlated with the mRNA expression of its genes (17).Despite the absence of studies that have determined the KISS1 mRNA expression in PCOS women, the results of our study are in line with the studies on KISS1 serum levels.Several recent publications reported higher serum levels of KISS1 in subjects with PCOS than in controls (6,(18)(19)(20)(21)(22), while 2 reported the contrary (10,23).Ibrahim et al.
report that serum levels of KISS1 were significantly higher in PCOS women than in the control group (22).
Previous research suggests that KISS1 is secreted in a pulsatile manner from the hypothalamus and peripheral organs such as the pancreas, ovaries, adipose tissue, and adrenal glands (24).In contrast to KISS1's intrahypothalamic functions, little is known about KISS1's physiological importance in extrahypothalamic tissues.KISS1 appears to function in the peripheral systems that control reproduction in mammals, but more research is needed (25).The current knowledge does not rule out the potential that extrahypothalamic KISS1 acts as an autocrine/paracrine factor in the peripheral tissues to fulfill its physiological function.Furthermore, we also found a significant correlation between the KISS1 and KISS1/PDYN ratio, which was stronger in the PCOS groups than in the control groups.This is thought to be related to the reduction of dynorphin expression, which then affects the increase in GnRH pulsatility as the pathophysiology of PCOS.Meanwhile, the correlation between KISS1 and KISS1/TAC3 was weaker and non-significant in the PCOS groups.The main limitation of this study is that there is no evidence to clarify that alterations in mRNA levels of KNDy peptides in the blood accurately reflect the levels of gene expression in the hypothalamus.To obtain a more meaningful understanding of PCOS, the findings of this study must be supported by future studies that directly tie blood levels of KNDy peptides to those in the hypothalamus.Moreover, this study only observed a small number of subjects.Increasing the sample size in future studies would allow better intergroup analyses.Furthermore, peripheral blood testing is expected to be a potential non-invasive alternative diagnosis for PCOS.

Conclusion
The low expression level of the mRNA gene encoding dynorphin is highly specific for PCOS.These findings were followed by a high KISS1/PDYN ratio specific to the PCOS group.Furthermore, a significant positive correlation was established between KISS1 and the KISS1/PDYN ratio.This study indicates that the expression of these neuropeptide genes in peripheral blood is associated with PCOS.
it critically for important intellectual content.
Anom Bowolaksono contributed substantially in supervising laboratory workup, interpretation of data, design of laboratory workup, and revising the article.Each author agrees to be personally accountable for the entire work.
bleeding and infertility due to ovulation disorders and abnormal androgen production.Diagnostic criteria for PCOS based on the 2003 Rotterdam consensus include hyperandrogenism, oligomenorrhea/anovulation, and polycystic ovarian morphology (1).Increased pulse amplitude and frequency of gonadotropin-releasing hormone (GnRH) in the hypothalamus cause relatively persistent and non-pulsatile luteinizing hormone (LH) secretion, resulting in chronic anovulation and hyperandrogenism, which leads to PCOS (2).The amplitude and frequency of GnRH pulsatile secretion are affected by several neuropeptides, such as kisspeptin (KISS1), neurokinin-B (NKB), and dynorphin, which are expressed by the KISS1, tachykinin-3 (TAC3), and prodynorphin (PDYN) genes, respectively (3).The discovery of the KNDy neuron network in the hypothalamus helps us understand the role of these neurons in regulating GnRH secretion at puberty and during reproductive age.Numerous studies have investigated the genetic components of PCOS (4-9); for instance, Chaudhary et al. reveal that polymorphism in several genes play a role in the steroidogenesis pathway results in the phenotypic expression of PCOS (4).In another study, Farsimadan et al. found that polymorphisms within KISS1 were associated Blood samples were collected at the Yamin Kencana Clinic, Cipto Mangunkusumo hospital, Jakarta, Indonesia following the World Health Organization standard blood collection procedure(15).Sample processing was conducted at the Human Reproductive, Infertility, and Family Planning Laboratory of the Indonesian Medical Education and Research Institute, Faculty of Medicine, University of Indonesia, Jakarta.The authors confirm that all experiments were performed in accordance with relevant guidelines and regulations.Gene quantification was carried out by the quantitative polymerase chain reaction method using the SensiFAST SYBR Green PCR Master Mix [Bioline] kit and Prime Pro 48 quantitative polymerase chain reaction system from Cole-Parmer Ltd [TechneTM] polymerase chain reaction machine based on the given protocols.The housekeeping gene used in this study was β-actin.The primary base sequence of the LEP, NPY, KISS1, TAC3, PDYN, and β-actin genes was obtained from the National Center for Biotechnology Information database.Relative gene expressions of each gene were obtained by processing the cycle threshold (Ct) value with the Livak method (16).The Livak method calculates the relative gene expression by comparing the expression of target genes with reference genes.The reference gene Ct normalized target gene Ct and control gene Ct, resulting in ΔCt target and ΔCt control , respectively.Then ΔCt target was subtracted against the ΔCt control to obtain the ΔΔCt.The value obtained was a relative comparison to the control using a 2-based exponential (2 −ΔΔCt ).In addition to assessing relative gene expression, this study measured hormone levels as demographic variables.Specifically, testosterone, sex hormone-binding globulin (SHBG), and free androgen index were measured to evaluate hormonal profiles in women with and without PCOS.Peripheral venous samples were collected and subjected to centrifugation to obtain serum for the quantitative measurement of testosterone and SHBG concentrations.The enzyme-linked immunosorbent assay method was employed for this purpose.Commercial fluorescence enzyme immunoassay kits were used for the determination of serum testosterone (ST AIA-Pack Testosterone, TOSOH, Japan, Cat.No. 0025204) and SHBG (ST AIA-Pack SHBG, TOSOH Bioscience, Japan), following the manufacturer's instructions.The assay utilized a sandwich-type approach with a pre-coated 96-well plate and enzyme-labeled secondary antibodies.A sample volume of 300 µl was required for analysis.Before measurement, each sample was labeled and prepared in a sample cup and a test cup.The prepared sample and test cups were then inserted into the TOSOH instrument.The free androgen index was calculated as the ratio of total testosterone to SHBG and reported as a percentage.Baseline demographic characteristics and hormonal profiles were compared to see if there were any significant differences between groups that could interfere with the interpretation of the study results.Potential confounding variables, such as age, body mass index, and hormonal level, were identified.Potential confounding variables due to disease and metabolic abnormalities were controlled using the exclusion criteria.

Pearson's correlation
test found a significant positive correlation between KISS1 mRNA expression and the KISS1/PDYN ratio.The correlation of KISS1 and KISS1/PDYN ratio was revealed to be stronger in the PCOS group (Pearson R = 0.93, p < 0.001) than in the control group (Pearson R = 0.66, p < 0.001).On the other hand, a positive and significant correlation was observed between KISS1 and the KISS1/TAC3 ratio in the control group (Pearson R = 0.56, p = 0.01) but was not found in the PCOS group (Pearson R = 0.22, p = 0.35).

Figure 1 .
Figure 1.Correlation between kisspeptin-to-prodynorphin ratio in the A) Control and B) PCOS groups, kisspeptin-to-tachykinin-3 ratio in the C) Control and D) PCOS groups.Pearson correlation test.

Furthermore,
KISS1 neurons' development and function can be influenced by metabolic factors at various stages of development.The metabolic changes that KISS1 neurons undergo can be temporary or permanent (for example, epigenetic modifications) (26).This makes it evident that peripheral changes can affect the increase in hypothalamic KISS1 production in PCOS.This study's foremost, and novel finding was that dynorphin expression was lower in typical PCOS women than in controls.These results demonstrated a significantly higher KISS1/PDYN ratio in PCOS women than in controls.To our knowledge, a similar finding regarding lower dynorphin levels in PCOS women has not been published before.Research on these genes in the pathophysiology of PCOS has not been widely carried out.Although no studies have examined dynorphin levels or the KISS1/PDYN ratio in PCOS women, our findings align with the basic theory of regulatory systems in which KNDy neurons regulate GnRH pulsatility.It is known that KISS1, while the inhibitory function of dynorphin on KISS1, works to control KISS1 secretion with NKB as a balance regulator.Thus, reduced dynorphin production will undoubtedly have an impact on increasing KISS1 secretion, which in turn causes an increase in the frequency and amplitude of the pulsatility of GnRH secretion(27).