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Testosterone level correlates significantly with luteinizing hormone to follicle-stimulating hormone ratio among women with polycystic ovary syndrome: A cross sectional study [version 2; peer review: 1 approved with reservations]

Background: Polycystic ovary syndrome (PCOS), an endocrinological problem among women in the reproductive age, is characterized by chronic ovulatory dysfunction, hyperandrogenism, and elevated luteinizing hormone: follicle stimulating hormone (LH-FSH) ratio. The goal of this study was to examine if the blood LH-FSH ratio and total testosterone (TT) levels in Sudanese women with PCOS were linked. Methods: This cross-sectional study included 300 women with confirmed PCOS based on Rotterdam criteria. PCOS women mean (standard deviation): age 29.1(5.8) years; body mass index (BMI) 27.9±4.6 kg/m 2 . Each participant underwent a clinical history, physical examination, and ovaries ultrasonogram. ASYS Expert Plus Microplate was used to quantify serum LH, FSH, and TT levels in fasting blood specimen drawn during the follicular phase of the menstrual cycle of women with PCOS. Results: More than two-thirds of the participants (71.0%) had an aberrant LH-FSH ratio (cut-off>1.0), and 58.3% had hyperandrogenemia (TT>109.5 ng/dL). Hyperandrogenemic women had significantly increased LH-FSH ratio (P= 0.000). to distinguish between hyperandrogenic and non-hyperandrogenic PCOS women.


Introduction
Polycystic ovary syndrome (PCOS) is a prevalent endocrine condition affecting women of reproductive age, with a reported frequency of 6 to 15%. 1,2 PCOS is characterized by a female sex hormone imbalance and increased androgen production, which results in irregular or extended menstrual periods, obesity, and excessive hair growth. 3 Genetic and epigenetic factors, as well as environmental variables have been identified as risk factors for intra-ovarian hyperandrogenism. 3 PCOS is a challenging disorder to diagnose since it is a diverse condition with different characteristics. It is currently diagnosed using revised Rotterdam criteria, which has been recently approved by an international evidencebased PCOS guideline. 4,5 According to certain studies, the brain is both a regulator and an impacted organ in PCOS (the brain phenotype). The brain, which contains multiple receptors, and neurons with their neurotransmitters, produces a higher pulse frequency of gonadotropin. As a result, people with PCOS have more luteinizing hormone (LH) than follicle-stimulating hormone (FSH) secretion. 6 Increased ovarian androgen production is caused by a change in the LH-FSH ratio. A number of studies have proposed appropriate cut-off values for the LH-FSH ratio in PCOS patients. [7][8][9][10] However, the optimal cut-off threshold remains unclear because of the varying sensitivity and specificity. Hsu et al. suggested that an LH-FSH ratio of >1 offered the best combination of sensitivity and specificity for the diagnosis of PCOS. 7 while, in contrast, Papaleo et al. postulated that an LH-FSH ratio of ≥2 is the characteristic feature of abnormal gonadotropin secretion in PCOS patients. 10 Furthermore, hyperandrogenemia lowers estrogen's negative feedback to the hypothalamus, resulting in increased LH pulse frequency. As a result, a vicious cycle is set in motion, causing a variety of clinical symptoms of PCOS, including hyperandrogenism. 11 The goal of this study was to see possible link between the LH-FSH ratio and total testosterone levels in Sudanese women with PCOS.

Study design
This was an observational study with a cross-sectional design. The study was conducted between October 2020 and September 2021 in Khartoum-Sudan. Various infertility centers clinics were visited to select the study sample, using a convenience sampling method. The inclusion criteria were women with confirmed PCOS based on Rotterdam criteria, 5 where at least two of the following criteria were fulfilled: oligomenorrhoea/anovulation (delayed menses >35 days or <8 spontaneous hemorrhagic episodes/year), hyperandrogenism (clinical hirsutism using modified Ferriman-Gallwey score of ≥8 or biochemical) and morphology of polycystic ovaries on ultrasonography (12 or more follicles in each ovary measuring 2 to 9 mm in diameter, and/or increased ovarian volume>10 mL 3 ). Women with systemic disease (cardiovascular disease and diabetes mellitus), on medication prior to the study (oral contraceptives, glucocorticoids, ovulation induction agents, and estrogenic or anti-androgenic) were excluded from the study. 4,5 Sample size calculation At the time of the study, there was no published data about the prevalence of PCOS in Sudan. For this, the prevalence of PCOS in unspecified populations 3-10%. 12 The sample size was determined based on the following equation 13 :

REVISED Amendments from Version 1
In this new version we did some changes based on reviewers' suggestions. We have added Statements in an introduction about the different cut-off value of LH-FSH ratios from different studies. Moreover; in discussion; some findings discussed the impact of obesity on reproductive.
Any further responses from the reviewers can be found at the end of the article

Ethical considerations
The study protocol was approved by the Federal Ministry of Health at Khartoum-Sudan. This study was conducted in compliance with the ethical standards of the responsible institution on human subjects as well as with the Helsinki Declaration. The study objective was explained to potential participants, and those who agreed to participate provided a signed consent before the start of the study.

Procedure
After signing an informed consent form, the socio-demographic characteristics, medical and gynecological history were taken from each patient using a questionnaire. The detailed medical and gynecological history (menstrual pattern, fertility, hirsutism) were taken from all patients included in the study. Then full general and pelvic examinations were performed.
Following standard protocols, weight was measured twice. After calibration, OMRON BF508l Body Fat Scales (China) were utilized. Women were told to remove their bulky attire. The weights were calculated to within 0.1 kg. After calibration, ladies were requested to remove their shoes and a portable stadiometer (SECA-213 model, Germany) was used to measure their height twice. Quetelet's BMI was estimated using a conventional formula (weight in kilograms divided by height in meters 2 ). Underweight (<18.5 kg/m 2 ), normal (18.5-24.9 kg/m 2 ), overweight (25.0-29.9 kg/m 2 ), and obese (30 kg/m 2 ) were the BMI categories used by the WHO. 14 During the follicular phase, ultrasonography was performed by Mindray (model: DP-50, Germany) and vaginal transducer where presence or absence of ovarian cysts were conformed or excluded. If cysts were seen, then the volume as well as the number of small follicles were determined, in each ovary.

Methods of sampling and analysis
Women were asked to come back on days 2-5 after a spontaneous menstruation or on a convenient day if they had amenorrhea. In a fasting state, 5 mL venous blood was collected between the hours of 8 a.m. and 9 a.m. The blood was centrifuged using Hettich (D-78532 Tuttlingen: Germany), then plasma obtained, and kept at -20°C until the assay. Enzymes linked immunosorbent assay (ELISA); ASYS (model: Expert Plus; type G020150; serial nr: 28382; Austria) was used to quantify serum LH, FSH by indirect method, and TT competitive methods.

Statistical analysis
Data was coded, and statistical analysis was performed with social package of statistical science version 26.0 (SPSS Inc., IBM, Chicago, IL, USA). The Kolmogorov-Smirnov was used for testing the normality of continuous data. All data was skewed. Continuous variables were presented as mean with standard deviation, and median with interquartile ranges. Whereas qualitative data was expressed with frequency (%). Mann-Whitney U test, and Chi-square test for qualitative variables were used to assess the relationship between LH-FSH ratio groups and androgen status. The association between the LH-FSH ratio, serum TT, and BMI was tested using Spearman's correlation test. The link between the LH-FSH ratio>1 and other factors was investigated using binary logistic regression analysis. The serum LH-FSH ratio was studied using a receiver-operating characteristic (ROC) curve to see if it might distinguish androgen status.
Women with LH-FSH ratio >1 (based on their serum LH-FSH ratio) had significantly increased serum TT level (P=0.000), and 70.0% of them had TT> 109.5 ng/dL. 55.4% (n=118) of them had positive family history to PCOS, compared with counterpart. There were no significant differences in age, BMI, and menstrual cycle frequency irregularity between groups (Table 1).
In comparison to their counterpart, women with hyperandrogenemia (serum TT > 109.5 ng/dL) had significant increase in LH-FSH ratio (P=0.000). Furthermore; obesity was found in 30.9% (n=54) of them based on their BMI ( Table 2).
As shown in Figure 1, the Spearman's correlation revealed that serum LH-FSH ratio was significantly correlated with serum TT (ng/dL) (r= 0.329, P=0.000). However; both LH-FSH ratio and TT were insignificantly correlated with BMI among women with PCOS (results not shown on figure). Table 3, the serum LH-FSH ratio (odds ratio (OR) (95% confidence interval (CI)): 2.308 (1.698-3.139, P=0.000) was independently related to androgen status and can be used as a predictor in women with PCOS.

Discussion
In the present study, the link between serum LH-FSH ratio and total testosterone level were investigated in a crosssectional analysis of 300 Sudanese women with PCOS. The study revealed that 30.3% of women were obese based on their BMI. Obesity was found in 50-80% of women with PCOS, according to McCartney and Marshall. 16 Obesity augments the androgen production by stimulating LH, which in turn leads to hyperandrogenism. 17 Leptin, an appetitecontrolling adipokine has a direct impact on the neuroendocrine and reproductive function of obese PCOS women. 18 This may differ depending on race, ethnicity, location, and environmental factors (physical activity, diet, stress).  Values are numbers and percentages; * P-value were obtained using Mann-Whitney test; and P # were obtain using Chi-Square test.
The most common clinical symptom of women diagnosed with PCOS, according to Malini and George, 19 is a greater LH-FSH ratio. The LH-FSH ratio in healthy women is 1:1. In PCOS, the LH level is higher than the FSH level, resulting in increased ovarian androgen production and ovulatory failure. Various LH-FSH ratio cut-offs have been proposed from <1 range to 4.6-5.5 the hormonal imbalances in PCOS are a dependence of insulin, LH and testosterone. 7 However, the cut-off more than 1.0 was found to be the most successful in diagnosing PCOS in terms of sensitivity and specificity. 7,20 Moreover, Tola et al found that in Sudanese women with PCOS the cut-off more than 1 has a diagnostic power of PCOS with sensitivity and specificity 72%, 76% respectively. 2 According to our findings, 71.0% of women with PCOS exhibited an abnormal LH-FSH ratio with a cut-off of >1.
Morshed et al. reported that 71.0% PCOS patients (cut-off more than1.0) exhibited an aberrant LH-FSH ratio. 21 According to Nath et al., 22 70.58% of women with PCOS have an increased LH-FSH ratio. As a result, the authors proposed that the LH-FSH ratio is one of the defining characteristics of PCOS women. 22 Table 2. Characteristics of the study population (n= 300) based on androgenmia.
Variables Androgenemia (cut-off > 109.5 ng/dL) P-value   Study revealed 58.3% of women with PCOS had hyperandrogenemia (cut-off TT more than 109.5 ng/dL). Furthermore, the findings showed a statistically significant difference in the frequency of hyperandrogenemia between the changed and normal LH-FSH ratio groups demonstrated that an altered LH-FSH ratio with a cut-off of 1.0 was associated with androgen status in women with PCOS. Moreover, the LH-FSH ratio was linked to a higher level of serum TT. Alexiou et al., reported hyperandrogenemia was present in 78.2% of PCOS. 23 Livadas and his colleagues reported that prevalence of hyperandrogenemia was 58.8%. 24 The imbalance in LH: FSH causes proliferation of ovarian theca cells leading to increased steroidogenesis, and ultimately leading to hyperandrogenemia in PCOS women. 25 In the hyperandrogenic group LH-FSH ratio was higher and differed significantly from the value of the normoandrogenic group. This may suggest that LH-FSH ratio has an independent relation with TT, which is consistent with previous studies. 19,26 In regression analysis our study observed that LH-FSH ratio>1 was significantly positive independently associated to hyperandrgenemia. Moreover, ROC analysis indicated that serum LH-FSH ratio>1 was significantly discriminate androgen statuses among PCOS women with cut-off to serum TT 109.5 ng/dL. The studies 19,26 also demonstrate that an LH-FSH ratio greater than 1.0 is enough to generate significant testosterone production from the ovaries, resulting in hyperandrogenemia.
There are several solid points in the present research. A high sample size was used in this study. It looked at the relationship between the LH-FSH ratio>1 and TT in Sudanese women PCOS. It is the first study to use an LH-FSH ratio greater than 1 to distinguish hyperandrogenic status in PCOS Sudanese individuals. Our research can be used as a starting point for further research. To compute the free testosterone index, serum sex hormone binding globulin or free testosterone were not assessed. Furthermore, we lack Sudanese population-specific data on serum TT reference ranges.

Conclusions
Sudanese women with PCOS are prone to androgenemia. Furthermore, among women with PCOS, the LH-FSH ratio was found to be substantially linked with total testosterone. The finding of this study may aid in a better understanding of the pathophysiology and management of hyperandrogenemia in PCOS women of Sudanese descent.