Ovarian hyperstimulation syndrome (OHSS) is an iatrogenic, serious complication in women undergoing assisted reproductive technologies (ARTs) for fertility treatment.1–3 It occurs due to exaggeration of ovarian response, which is characterized by high estradiol (E2) levels, enlarged ovaries with increasing numbers of large follicles, and enhancement in vascular permeability and shifting of intravascular fluids into third space.4

The high basal E2 level has a predictable value in high-risk patients who are young, underweight, have a medical history of OHSS or polycystic ovarian syndrome, and have high follicles numbers and sizes.5–7 The incidence is estimated to be 0.6% to 5% in moderate to severe OHSS in vitro fertilization (IVF) cycles,8 and reaches up to 20% in high-risk patients.9 E2 plays an important role in the pathophysiology of OHSS by increasing vascular permeability (VP) and vascular endothelial growth factor (VEGF) and its receptors numbers which is, in turn, enhanced by gene stimulation over time.10

Signs and symptoms of OHSS are hypovolemia, and metabolic and thromboembolic complications, which are mainly observed in severe OHSS cases needing hospitalization.11–13

Fenugreek (Trigonella foenum-graecum) is an annual herb that belongs to the family Fabaceae. Approximately 90% of the world production comes from India, China, Iran, Pakistan, and Palestine.14 It is a daily food component for several nations and countries, without any notable, unpleasant effect.15,16 It has been used in folk medicine as lactation stimulant.17 Recently, the various medical benefits of fenugreek have been extensively studied18 with further emphasis on its effects on female gonadal hormones and their reproductive functions.19,20 It reduces the serum levels of E2 and the number of ovarian follicles.21,22 Moreover, recent studies found that fenugreek can reduce VP by decreasing VEGF expression in hepatocyte cytoplasm.23

Preventing OHSS requires interrupting the pathological process, such as controlling the levels of serum E2. Hence, this study aimed to evaluate the effect of fenugreek seed aqueous (FSA) extract on serum E2 levels in an OHSS rat model. We assumed that the FSA extract prevents OHSS development in treated animals by decreasing E2 levels.

Methods

This study was conducted over two weeks. The study protocol was approved by the Faculty of Medicine and the Integrated Center for Research Animal Care and Use.

A total of 34 immature Sprague Dawley female rats were used. The rats were obtained from an authorized supplier and kept for three days in the animal laboratory for acclimatization under maintained conditions on 12-hour cycles of light and darkness (lights on from 07:00–19:00) and at room temperature 24 oC. During the study period, the rats had free access to water and standard rat diet (rat food pellets). On the first day of the experiment, the rats were randomly divided into two control groups, negative (NC) and positive (PC), and a treated (T) group. The experiment started when the rats were 18 days postnatal (DPN), with a body weight 40.0±5.0 grams.

Fenugreek seeds were collected from a local market. The fenugreek seeds species were identified and deposited in Herbarium of Faculty of Pharmacy (voucher number PIIUM 0226-1). FSA extract was prepared following the method given by Khalki et al.24 Dry, clean fenugreek seeds were ground into a fine powder and dissolved in distilled water at a ratio of 1:20 (g/mL). The suspension was stirred on a magnetic stirrer hot plate for 24 hours at room temperature. The mixture was then centrifuged at 5000 rpm for 15 minutes at room temperature. Eventually, the extract was freeze-dried for five days.

All rats were weighed daily and received a standard rat diet. In addition to the normal diet, the T group was given a daily oral dose of 1500 mg/kg body weight of FSA extract25 at 09:00 a.m. from day one to day 13 of the experiment [Figure 1]. The following equation was used to calculate the doses:

FSA extract dose = body weight of rat (g) x 15000 / 1000

The extract was given orally, using a metallic oral gavage (20 gauge).

In the second week of the experiment (day eight), OHSS was induced in both PC and T groups using the animal model published by Kitajima et al.26 Rats in the PC and T groups were injected subcutaneously with pregnant mares’ serum gonadotropin 50 IU/day (PMSG; Merck, US) for four consecutive days (25–28 DPN), followed by human chorionic gonadotropin 25 IU (hCG; N.V. Organon, Nederland) on 29 DPN. After 48 hours (31 DPN), blood samples were collected, and the rats were decapitated under general anesthesia.

Under general anesthesia (ketamine, tiletamine and xylazine + zolazepam; UKM, Malaysia), approximately 3 mL of blood sample were obtained from each rat from retro-orbital sinus using capillary tubes. The sera were separated by centrifugation of blood samples at 3000 rpm for 15 minutes and kept at -20 °C for subsequent E2 immunoassays analysis. It should be noted that pre-treatment blood sample collection as a baseline in this model (immature rats, body weight 40.0±5.0 grams) is impossible before FSA treatment. According to rodents’ nonterminal blood collection guidelines,27 blood collection is limited up to 10% of total circulating blood volume in healthy, normal, adult animals on a single occasion and collection may be repeated after three to four weeks. To measure the E2 level, at least 50 µL of serum from each rat was needed which was not practically possible in our model. Therefore, the T group was compared with the two control groups (PC and NC groups).

Serum E2 was assayed using rat’s E2 enzyme-linked immunosorbent assay kit (ab108667-17 beta estradiol ELISA Kit, Abcam, USA). The detected concentration range for this kit is 20–2000 pg/mL. The 1X Washing Solution was prepared one day before the assay, while the other kit solutions were ready-to-use and stored at 4 oC. The serum samples, standards, and controls were equilibrated to room temperature one hour before use; 25 μL of standard and sample were added to their corresponding wells and 200 μL of 17 beta Estradiol-HRP conjugate. After incubation, the microplate wells were washed three times using 300 μL diluted washing solution, and 100 μL TMB substrate solution was added into each well and incubated for 30 minutes at room temperature in the dark. A blue color developed. Finally, 100 μL of Stop Solution was added into all wells and shook gently. The blue color turned yellow. All assays were done in duplicate.

The absorbance of the samples was detected at 450 nm wavelength within 30 minutes. E2 concentrations were calculated using an ELISA analysis online calculator28 using optical density values against standard curves calibrated with known concentration values. The assays were done in duplicate. The absorbance of the samples was measured at 450 nm.

The results are presented as median. The significant differences between and among the groups were determined using the Kruskal-Wallis test. A p-value < 0.050 was considered statistically significant.

results

The effect of FSA extract on serum levels of E2 in the T group (median 47.48 pg/mL) was nearly three times lower than the PC group (median 127.42 pg/mL). Also, the maximum value of the concentration recorded in the T group (491.36 pg/mL) was five times lower than the PC group (2701.94 pg/mL) [Table 1].

In addition, we observed an increase in E2 concentrations in the OHSS model; PC and T groups compared to the NC group. The median E2 concentrations in the T group (47.48 pg/mL) was nearly five times higher than the NC group (9.94 pg/mL). While the PC group (127.42 pg/mL) was 13-times higher compared to the NC group. The change in E2 concentrations is considered to be a diagnostic value of the model success.

The Kruskal-Wallis test showed that there was a statistically significant difference in serum E2 levels between all groups (p-value < 0.050) and among groups when compared with each other; namely NC and PC (p-value < 0.001), PC and T (p-value = 0.005), and NC and T (p-value = 0.001) groups [Figure 2].

Discussion

FSA extract significantly decreases serum E2 level, nearly three times in the OHSS model, which is consistent with findings of some experimental studies.20,21 In these studies, the authors found a reduction in serum E2 level due to FSA extract effect.

E2 plays an important role in the pathophysiology of OHSS by increasing VEGF and VP. Controlling serum E2 levels is effective in decreasing the incidence of OHSS cases in IVF cycles.29

E2 was found to regulate VEGF through its effect on gene expression depending upon the length of exposure.30 Also, E2 is an indicator of patients at high risk of developing OHSS.31

Normally, in premenopausal women, estrogens are synthesized in the ovaries, which are under the cyclic control of pituitary gonadotropins. Both cytochrome CYP17 (P-450 17α-hydroxylase) and cytochrome CYP19 (P-450 aromatase) are involved in estrogen biosynthesis. Estrogens are mainly catabolized by hydroxylation reactions.32 Estrogen synthesis takes place in the ovarian granulosa, which is stimulated by the gonadotropins, luteinizing hormone (LH), and follicle stimulating hormone (FSH). Their response to FSH signaling increases enzymes responsible for estrogens synthesis.33 Aromatase converts testosterone to E2.34

Another study demonstrated that herbs with estrogenic property could alter the action of pituitary gland through peripheral modulation of LH and FSH.35 As a result, these actions decrease the serum concentration of E2. Another mechanism suggested that estrogenic property can affect E2 substrate supplementation during its synthesis. The production process is subsequently affected.35

A review addressed the biological role of phytoestrogens in the modulation of estrogen synthesis and metabolism. It stated that phytoestrogens are structurally similar to E2.36 In vitro, in vivo, and human studies have proved that phytoestrogens may also control estrogen function by affecting estrogen synthesis and metabolism or hormonal signaling within the hypothalamus-pituitary-gonadal axis. Phytoestrogens can also affect estrogen levels in ovarian tissue by interfering with estrogen-synthesizing and metabolism enzymes such as aromatase and estrogen receptors signaling pathways.36

Similarly, such estrogenic properties have been found in fenugreek seeds extract.37 Furthermore, the findings of a study by Bakrim,20 found that fenugreek seed extract reduces FSH and LH, which may be the mechanism of reducing serum E2 level and could explain our results.

Testosterone is considered an E2 precursor in the aromatase pathway. Fenugreek extract is thought to act through aromatase inhibition, leading to decreased testosterone breakdown to estrogen,38 which may also explain our results.

Although there is a significant increase in follicles numbers and size in FSA extract treated OHSS models, E2 levels are reduced. This may be due to the effect of FSA extract on the function of granulosa cell, where the E2 level is considered a marker of granulosa cell activity.35,39

conclusion

We observed a significant decrease in serum E2 in treated OHSS model, which was almost three times compared to non-treated OHSS model. Thus, the FSA may have the ability to prevent OHSS. Findings in this study have important implications for the development of female infertility adjuvant drugs for safe ARTs cycles in terms of OHSS prevention.

Disclosure

The authors declared no conflicts of interest. This research was funded by Research Initiative Grant (RIG) from IIUM Research Management Center (RIGS– 15 -083).

Acknowledgements

Authors would like to thank IIUM Research Management Center. The authors gratefully acknowledge the services and facilities provided by Integrated Centre for Research Animal Care and Use IIUM. We are gratefully thankful to Professor Raul Gómez and Mr. Ahmed Ben Hameid for their assistance and guidance. Also, we are thankful for Mr. Suhaimi Bin Hashim the senior laboratory technician for his help and assistance.

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