Effects of empagliflozin on metabolic parameters in polycystic ovary syndrome: A randomized controlled study

Empagliflozin is a sodium‐glucose‐cotransporter‐2 inhibitor that improves cardiovascular risk and promotes weight loss in patients with type‐2 diabetes. Polycystic ovary syndrome (PCOS) is associated with obesity and increased cardiovascular risk; therefore, empagliflozin may be of benefit for these women. The aim of this study was to compare the effects of empagliflozin vs metformin on anthropometric and body composition, hormonal and metabolic parameters in women with PCOS.


| INTRODUC TI ON
Polycystic ovary syndrome (PCOS) is a heterogeneous endocrine disorder featured by hyperandrogenism, menstrual irregularities and polycystic ovaries that affects women of reproductive age. 1 PCOS is also associated with infertility, obesity, metabolic disturbances and increased cardiovascular risk. 1,2 Accordingly, the treatment of PCOS is commonly symptom-based, while the ideal treatment would address both the reproductive and metabolic abnormalities related to PCOS. 1,3 Hormonal contraceptives alone or combined with anti-androgens have been the cornerstone for managing menstrual disturbances and clinical or biochemical hyperandrogenaemia 4 ; however, some hormonal contraceptives may unfavourably affect the lipid profile in PCOS 5 and increase the risk of thrombosis and cardiovascular events in the general population. 6 Glucose lowering agents including metformin and thiazolidinedione have been shown to be effective in managing the metabolic abnormalities (ie, insulin resistance, hyperinsulinaemia, and diabetes mellitus) and chronic anovulation; however, their use has been inconsistently associated with improvements in weight loss and body composition, menstrual irregularity or clinical symptoms of excess androgens. 7,8 Empagliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, is a new treatment option for adults with type 2 diabetes 9 ; however, its effects in PCOS have not been previously explored. Its principal action involves inhibition of glucose reabsorption by the kidney, and therefore, glucose excretion via urine. Notably, this action mechanism is insulin-independent; as such it does not increase the risk of hypoglycaemia, making it attractive for use in normoglycaemic individuals. 10,11 Recent trials have demonstrated that treatment with empagliflozin promotes weight loss, 12 exerts positive effects on arterial stiffness, vascular resistance and blood pressure and decreases the relative risk for cardiovascular and all-cause mortality in patients with type 2 diabetes. 10,13 Evidence from preclinical studies suggests that these cardio-protective effects may be due to the reduction in oxidative stress and suppressed markers of inflammation and fibrosis. 14,15 In humans, the cardiovascular benefits may also be mediated by reductions in HbA1c, insulin resistance, plasma volume, weight/fat mass and inflammation. 16,17 Given that these pathological features are also common in PCOS 1,2 ; empagliflozin may be of potential benefit for this population.
Therefore, the aim of this study was to explore and compare the effects of empagliflozin vs metformin on anthropometric, body composition, hormonal and metabolic parameters in women with PCOS.

| MATERIAL S AND ME THODS
An open-label, randomized, comparative study in women with PCOS was performed in the Academic Diabetes, Endocrinology and Metabolism research centre at Hull Royal Infirmary. All participants were women, aged between 18 and 45 years, had a body mass index (BMI) ≥25 kg/m 2 , were diagnosed with PCOS based on the Rotterdam criteria [biochemical hyperandrogenism, as indicated by a free androgen index (FAI) >4, and self-reported oligomenorrhea (cycle length >35 days and 9 or fewer periods per year) or amenorrhoea (absence of menses for a period ≥3 months). 18

| Procedures
Height and weight were recorded with participants wearing light clothing and no shoes using a weighing scale with attached stadiometer Medical Ltd). Compliance with the treatments was calculated by counting the returned medications at the end of the 12-week period.

| Blood sampling and biochemical analyses
Following an overnight fast, blood samples were collected both at the baseline and final visit (end of the 12-week period). The fasting venous blood was collected into fluoride oxalate and serum gel tubes. Blood samples were separated by centrifugation at 3500 g for 15 minutes at 5°C, and the aliquots were stored at −80°C within 1 hour of collection.
Serum insulin was assayed using a competitive chemiluminescent immunoassay performed on the manufacturer's DPC Immulite

| Statistical analysis
There are no previous studies on the effect of empaglifozin or any other SGLT-2 inhibitors on women with PCOS. Sample size was based on an independent t test with an arbitrary level of 5% significance (2-tailed) and power of 80%. Assuming a common standard deviation of 16% for radial augmentation index and a 4% reduction as a significant change, 21 a sample size of 16 patients per group allowed us to detect a between-group mean difference of 4%. To allow for loss-to-follow-up, we aimed to recruit 20 patients per group.
All data were checked for normality according to the Shapiro-Wilk test. Mean differences for all parameters expressed as % change from baseline between women with PCOS in the empagliflozin group and the metformin group were analysed with independent t test or Mann-Whitney U-test for normally and non-normally distributed data, respectively. Mean differences between baseline and 12-week follow-up within each treatment group were analysed with a paired t test or a signed-rank test for normally and non-normally distributed data, respectively. Values are presented as mean SD, if the variables were normally distributed, or median and interquartile range, if the F I G U R E 1 Flow diagram of the study variables were skewed. All statistical analyses were performed using IBM-SPSS version 24.0 (Chicago, IL) with P-values ≤0.05 considered to be significant. Linear regression analysis using function ln R was used to confirm the findings of univariate analysis after adjustments for confounders. Since this was a randomized controlled trial, we only adjusted for age and age + BMI, where relevant.

| RE SULTS
Forty-two participants with PCOS were screened; two participants were excluded from participation because they did not meet inclusion criteria, as such 40 participants were randomized to the two treatment arms (empagliflozin, n = 20 and metformin, n = 20; TA B L E 1 Changes in anthropometric, body composition, hormonal and metabolic parameters following 12 wks of empagliflozin and metformin treatment   Table 1 and   Table S1. Compliance was over 90% in both groups. There were no adverse events or serious adverse events in the metformin group. In the empagliflozin group, two patients reported adverse events (headache and dizziness, n = 1; mild rash, n = 1), which were, however, unrelated to the study drug.
F I G U R E 2 Percentage changes from baseline in anthropometric and body composition parameters after 12 wks with empagliflozin and metformin treatment. *P < 0.05; BMI, body mass index; BMR, basic metabolic rate; FFM, fat free mass; HC, hip circumference; TBW, total body water; WC, waist circumference. Body composition data (BMR, body fat %, fat mass, FFM and TBW) are presented for 18 participants in the empagliflozin group with available data When data were expressed as percentage change from baseline in each group, significant differences in weight (  Figure 2). The proportion of women with PCOS who experienced (a) a decrease >5%, (|b) an increase >5%, or (c) a change ≤5% from baseline in anthropometric and body composition parameters are presented in Table S2.

| Hormonal and metabolic parameters
In the empagliflozin group, significant increases in SHBG (P = 0.049) and oestradiol levels (P = 0.032) were seen after 12 weeks of treatment (Table 1). There were no other hormonal changes for either group (Table 1). No differences were seen in percentage change from baseline for any of the hormone parameters between groups (Table 1, Figure 2).
There were no changes following 12 weeks of treatment in blood pressure, endothelial function (RHI, AI), insulin sensitivity (insulin, fasting glucose, HOMA-IR), fasting lipid profile or hs-CRP in either treatment arm (Table 1). Between groups, comparisons did not reveal any differences in percentage changes from baseline for any of these metabolic parameters (Table 1, Figure 2).

| D ISCUSS I ON
In this first study on the comparative effects of the SGLT2 inhibitor, empagliflozin and metformin in overweight and obese women with PCOS, we demonstrated that treatment with empagliflozin over a 12-week period, resulted in significant reductions in weight, BMI, waist and hip circumference, total body fat mass and BMR, compared to treatment with metformin, but did not differentially affect hormonal or metabolic parameters.
Obesity is prevalent among women with PCOS and has been associated directly and/or indirectly with negative metabolic, cardiovascular, endocrine, reproductive and mental health outcomes. 1,2 Weight reduction exerts positive effects on PCOS-related outcomes; therefore, it is a primary goal of the management of the condition. 3 In the present study, women with PCOS in the empagliflozin group had a mean weight loss of 1.5 kg, which is similar to the weight loss reported in previous short-term trials. 12 Mechanistically, initial weight loss in empagliflozin studies is attributed to the calorie loss (approximately 200-300 kcal/d) associated with glucose excretion, but also to the mild diuretic effects of the drug. 9,12 Conversely, the steady-state weight loss associated with SGLT2 inhibitors treatment may result from fat loss. 22,23 In animal models, SGTL2 inhibitors have been shown to cause reduction in body weight and fat mass by enhancing lipolysis, fatty acid oxidation and adipose tissue browning. 14,24 These findings coincide with the alterations seen in substrate utilization from carbohydrates to lipids and potentially, ketone bodies. 9,25 Reductions in other measures of adiposity including waist and hip circumference, visceral and subcutaneous fat depots or indices, which may better reflect risk for metabolic disturbances and cardiovascular disease, have also been demonstrated in patients with type 2 diabetes following treatment with SGLT2 inhibitors, 22,23 with these findings being in agreement with the improvements in waist and hip circumference seen in our women with PCOS assigned to receive empagliflozin.
In the present study, the metformin group experienced modest increases in body weight. Studies on the effect of metformin on body weight in women with PCOS have yielded mixed results. 8 women in the UK with a mean BMI of 38 kg/m 2 and showed that it is no different than placebo in terms of weight reduction. 26  were shown in a meta-analysis (10 studies, total n = 6203 participants) on the efficacy and safety of empagliflozin only. 29 In addition to glycaemic control, use of SGLT-2 inhibitors results in a reduction in TG levels and increases in HDL-cholesterol levels, but also LDLcholesterol levels, possibly due to the shifted metabolism favouring lipid utilization. 30,31 In contrast to these beneficial effects on glycaemic control and less pronounced lipids effects of SGLT-2 inhibitors reported in type 2 diabetes, we did not observe significant changes in fasting glucose, insulin fasting lipids or hs-CRP at 3 months after empagliflozin treatment compared to baseline or any differences between our treatment groups. These results may be related to the short duration of the study or to the baseline characteristics of our participants with PCOS, who were young and did not have diabetes.
Further evidence from studies in patients with type 2 diabetes suggest that empagliflozin, and other SGLT2 inhibitors such as canagliflozin and dapagliflozin cause reductions in blood pressure, as a result of their natriuretic effects or due to the intensification of anti-hypertensive therapy. 32,33 No such blood pressure changes were demonstrated in our women with PCOS, though these subjects were normotensive and changes may not have been expected. Similarly, measures of endothelial function (RHI) or arterial stiffness (AI) were not altered compared to baseline in either treatment groups. Empagliflozin has been shown to improve endothelial dysfunction in preclinical studies in diabetic rat models, 34,35 but human data are scarce. A recent 16-week study demonstrated that dapagliflozin add-on therapy to metformin improved endothelial function, as evaluated by flow-mediated dilation, in patients with inadequately controlled early-stage type 2 diabetes mellitus. 36 Although there are no comparative data from studies that have investigated the effects of SGLT-2 inhibitors in women with PCOS, the results of our 12-week intervention contrast those of a longer study which demonstrated that metformin treatment for 6 months improved or even normalized abnormal flow-mediated dilation on the brachial artery and improved plasma endothelin-1 levels in women with PCOS. 37 The discrepancies in these results may be at least partially explained by differences in study duration and the use of different endothelial function measures.
There were significant increases in the SHBG and oestradiol levels in the empagliflozin group, but no significant reductions were seen in FAI and serum total testosterone levels. The % changes from baseline in hormonal levels did not differ to metformin. Metformin use in women with PCOS has been associated with improvements in hormonal levels. A recent meta-analysis demonstrated that metformin treatment resulted in small improvements in serum testosterone, but no changes in free testosterone, FAI, SHBG, DHEAS LH, FSH, LH/FSH ratio, oestradiol or progesterone compared to placebo in women with PCOS. 38 Metformin may also have some beneficial effects on ovulation and menstrual frequency. 8 Given the short follow-up of the present study, we did not assess these parameters, which is a limitation of the present study.

| CON CLUS IONS
Empagliflozin treatment over a 12-week period had beneficial effects on weight, BMI, waist and hip circumference and total body fat in overweight and obese women with PCOS compared to metformin, but no differences were seen in hormonal and metabolic parameters including insulin resistance and androgen levels. Placebo-controlled and comparative treatment randomized

ACK N OWLED G EM ENTS
This study was supported internally by the Academic Diabetes Endocrinology and Metabolism Unit at University of Hull. We