There are many ovulation inducing drugs is prescribed nowadays, Clomiphene citrate (CC) is the first choice for women treatment with ovulatory disorder or those with Polycystic Ovaries (PCOS) and have been widely used since 1962 until today. Clomiphene citrate or Clomid^® tablets are orally administered, nonsteroidal land usually given on the third or the fifth day of cycle after spontaneous or progesterone induced withdrawal bleeding with 50 mg for five days. The effective dose of CC ranges from 50, 100, 150 mg/day, doses excess 250 g/day is not approved by the FDA [1-3]. During CC treatment which binds to oestrogen receptors(ER) in reproductive system the levels of both (FSH & LH) is indicated to rise and effect ovarian stimulation to produce one or more dominant follicles emerges and matures [2]. Recently some studies suggests that is GH with IGF-1 have an important role in human follicular regulation development and steroidogenesis, any abnormal defect in IGF-1 can leads to folliculogenesis disturbance in PCOS [4,5]. Oral administration of CC does cause a decreasing of IGF-1, GH and Insulin levels or not change it in PCOS [6,7]. Evaluation of GH and IGF-1 can be measured by serum concentration level which is not differ between males and females in healthy adults, whereas GH higher in females [8,9]. The aim of the present study is to evaluate the effect of CC on Insulin, IGF-1 and GH levels in offspring of treated mothers.

Animals

All experimental procedures with mice were approved by the ethical guidelines of the animal care and use committee of King Abdul-Aziz University. Twenty five virgin albino mice of SWR strain female, at age (8 week old) and weighing (23-25 g) were used in the present study.

Mice were obtained from animal house unit of king Fahad Medical Research Centre, King Abdul-Aziz University, Jeddah, Saudi Arabia. Animals were acclimatized to laboratory conditions for one week before to the initiation of experimental treatments and were housed in standard plastic cages and maintained in controlled laboratory conditions room, temperature (20 ± 1ºC), and light: Dark cycle (12:12 h) and humidity (65%). Mice were feed ad libitum with standard diet and had free access to tap water.

Experiment design

Animals were divided in to two groups: 1-Control group (five females). 2-Clomiphene citrate treated group (20 female). Mice were oral injected with 0.2 and 0.3 mg/day of CC daily for 2 week, after 2 week every female were housed with a male for mating after female get pregnant males were taken out. On the day 26th after weaning and 8 week blood samples were taken from orbital sinus of mice offspring (22 pre-pubertal, 22 post-pubertal male and 23 pre-pubertal, 23 postpubertal female) for Insulin, IGF-1 and GH levels were determined by using (Elba Science) ELIZA kits, according to the manufacturer's instructions.

Statistical analysis

Statistical Package for Social Sciences (SPSS version 20) was used. Data were presented as mean (standard deviation). The continuous variables between more than 2 groups as comparison between control, male and female were compared using Onaway ANOVA (LSD) test, and between 2 groups as pre-pubertal and post-pubertal were made using unpaired student "t" test. A probability (P) <0.05 was considered significant. Graphs were made using Prism software for statistics version 5.

Males group

Table 1 and Figure 1 as well for the analysed data showed that Premales vs. Pre-control significant difference increase in IGF-1(6.75 ± 3.01 vs. 2.90 ± 0.01, ¹P=0.021), Insulin (14.92 ± 5.59 vs. 5.51 ± 0.01, ¹P=0.0002) and GH (17.42 ± 5.99 vs. 8.31 ± 0.01, ¹P=0.001), but lower significant increase in IGF-1(6.03 ± 2.44 vs. 2.4 ± 0.01, ¹P=0.028) and Insulin (16.75 ± 3.88 vs. 8.89 ± 0.01, ¹P=0.007) in Post- males vs. Postcontrol group. While the Pre-males vs. Post- males showed high significant increase in GH only (17.42 ± 5.94 vs. 6.04 ± 2.21, ²P=0.0001).

Table 1: Comparison of the measured hormones in different studied groups of male mice.

Figure 1: Comparison illustration of measured IGF-1, GH and Insulin levels pre-pubertal and post-pubertal male versus female mice offspring with control.

Females group

In Table 2 and Figure 1 analysed data shown low significant increase in Insulin and GH only in Pre-females vs. Pre-control Insulin (10.39 ± 4.70 vs. 4.20 ± 0.01, ¹P=0.030), GH (13.47 ± 5.75 vs. 6.80 ± 0.01, ¹P=0.016). But there were no statistically significant difference in Post-females vs. Post-control group. While in Pre-females vs. Post-females the significant decrease was found in GH only in this group GH (13.47 ± 5.75 vs. 10.01 ± 2.91, ²P=0.010).

Table 2: Comparison of the measured hormones in different studied groups of female mice.

Male and Female Groups

All results in Table 3 and Figure 1 as well are shown high significant difference in IGF-1, Insulin and GH levels in all males and female groups in both stages per-pubertal & post-pubertal as the following:

Table 3: Comparison of the measured hormones in treated pre-pubertal and post-pubertal male versus female mice.

Pre-males vs. Pre-females Insulin (14.92 ± 5.59 vs. 10.39 ± 4.70, ¹P=0.005), GH (17.42 ± 5.94 vs. 13.47 ± 5.75, P¹=0.029). Post-males vs. Post-females Insulin (16.75 ± 3.88 vs. 11.43 ± 4.69, ²P=0.0001), GH (6.04 ± 2.21 vs. 10.01 ± 2.91, ²P=0.0001. Pre-males vs. Post-females Insulin (14.92 ± 5.59 vs. 11.43 ± 4.69, P³=0.029), IGF-1 (6.75 ± 3.01 vs. 4.87 ± 2.47, P³=0.028), and GH (17.42 ± 5.94 vs. 10.01 ± 2.91, P³=0.0001). Pre-females vs. Post-males Insulin (10.39 ± 4.70 vs. 16.75 ± 3.88, P4=0.0001), GH (13.47 ± 5.75 vs. 6.04 ± 2.21, P4=0.0001).

Although, Clomiphene Citrate (CC) considered one of the safest drug given to women with evolutionary disorder since the 60s, rises concern in the medical field on its effects on the newly offspring. Results of this study have shown considerable evidences of disparity in IGF-1, GH and Insulin levels in treated mother’s offspring. Where, CC causes significant increasing in GH and insulin levels in males and females comparing to control and to each other, while the significant increase and differ concentration level for IGF-1 were found in males comparing to control and to females as well, but not in females comparing to control. These findings are consistent with results of previous studies CC treated PCOS patients, were they had higher level of IGF-1 than healthy women [4]. On other hand there were many outcomes of research results showed disagree with present study as what reported in a successful treated cycles by CC in PCOS were resulted significant reduction in IGF-1 level, and no changes in insulin plasma concentrations [10]. Similar findings are also recorded where CC does not affect insulin level in PCOS, but IGF-1 level was decreased [6]. Oral administration of CC lead to 30% decrease in IGF-1 level in PCOS women [7]. Lower level of IGF-1 was found in females comparing to with Growth Hormones Deficiency (GHD) males [11]. In acromegalic patients male the IGF-1 levels was decreased by 40% comparing to normal males level [12]. Blood transmission form the treated mothers to their offspring may cause these highly significant increases and changes in hormones concentrations levels. In conclusion, blood biochemical analysis indicated that CC causes alterations on IGF-1, GH and insulin hormones in treated mother’s offspring in albino mice.

The authors are extremely grateful to King Abdul-Aziz University for encouragements and unlimited support.