Ping-Ping Sun,Na Sun,Yue-Min Zhang,Jin-Guang Wang,Hua-Gang Ma＊

1The Reproductive Medicine Centre of Weifang People's Hospital,Weifang 261041,Shandong,China.

Keywords:Clomiphene citrate,Ovulation promotion,Overweight,Obesity,Multiple follicle,Endometrium thickness

Background

Both overweight and obesity are increasing worldwide and have detrimental influences on several human body functions including the reproductive health.It has already been confirmed that overweight affected the female reproductive system by contributing to anovulation,irregular menses,adverse oocyte quality,endometrial alterations,and hormonal imbalances.In particular,obese women undergo perturbations of the‘hypothalamic pituitary ovarian axis’,and frequently suffer of menstrual dysfunction leading to anovulation.For infertile women with anovulation,ovulation induction is considered the treatment of choice.Clomiphene citrate (CC) has been widely used as the first-line drug for ovulation induction [1].Normally，CC is taken orally from fifth day of the a spontaneous or progestin-induced menses [2].The initial dose is a single 50 mg tablet daily for a five-day period [3].However,the regular dosage of clomiphene can cause side effects such as thinner endometrium,abnormal cervical mucus,polyfollicular development and persisting ovarian cyst formation,which makes its consecutive monthly treatments in the clinic impossible[4–6].

M von Wolff [7]performed that the therapy with 25 mg clomiphene per day allows very few side effects and significantly increases transfer rate per initiated cycle in natural-cycle in-vitro fertilization.Whether it could mitigate the side effects by reducing CC dosage without affecting the clinical effects is an interesting question that has not been answered.In this study,we used 25 mg of CC daily instead of 50 mg to compare the effects and side effects.

Materials and methods

Patients selection

A prospective cohort study was approved at the Medical 84 Ethics Committee of Weifang People’s Hospital,from January 2015 to January 2016(E20141106).The Medical 84 Ethics Board of Weifang People’s Hospital ratified the study protocol,and all patients provided written informed consent.

The patients whose body mass index ≥24 kg/m2were recruited for this study [8].The inclusion criteria were as follows:(1) female ≤35 years with irregular menstruation;(2) basal follicle stimulating hormone(FSH) ＜ 10 U/L;(3) did not take any hormone treatment or OI therapy in the past three months;(4)the female at least one patent fallopian tube at hysterosalpingography or laparoscopy.

The exclusion for this study were as follows:(1)patients detected with ovarian cysts (according to ultrasound tests on day 3 of menstruation);(2)patients with weight gain due to other pathological reasons;(3)the females were excluded if they had double sided tubal pathology or other endocrine disorders.

Treatment plans

A random figure was created by using SPSS 22.0 software,and the participants were numbered and randomly divided into group regular-dose CC and low-dose CC according to the criteria of inclusion.All participants started taking CC from the 5thday of the menstrual cycle for 5 days.For the regular-dose CC group,the oral administration of CC was 50 mg per day,but the daily dose reduced to 25 mg in the low-dose CC group.Transvaginal ultrasound was used to detect the growth of follicles and endometrium on the 10thday of menstrual cycle.According to the developmental state of the follicles,HMG (75 IU or 150 IU) was added as appropriate to induce follicular development.Human chorionic gonadotropin (HCG,10000 IU)was administered once up to the diameter of follicle ≥18 mm and ultrasound was used again to check if ovulation had occurred after two days of injections.Luteal-phase supplementation was started in the evening of the day of ovulation with 10 mg twice a day oral dydrogesterone.Pregnancy tests were performed after 14 days of ovulation.

Detection methods

The information about menstruation,fertility history,body weight and height of all participants were collected.Transvaginal ultrasound was used to measure the endometrium thickness on the 3rdday of menstrual cycle and HCG day seperately,count the number of dominant follicles (≥16 mm in diameter),and check if ovulation occurred after HCG injections.Levels of FSH,luteinizing hormone (LH),estradiol(E2),and testosterone were measured in the forearm venous blood samples (2–3 ml,taken between 8:00–10:30 am in fasting patients) on the 3rdday of menstrual cycle and 10thday of menstrual cycle.The primary endpoints were endometrial thickness,the number of dominant follicles on HCG day and cancellation cycle rate(cycle would be cancelled if the number of dominant follicles were more three).The secondary endpoints were ovulation rate and biochemical pregnancy rate per initiated cycle.

Statistical analysis

Statistical analysis was done using SPSS 22.0.The measurement data that fulfilled the normal distribution were presented as mean ± standard deviation,and analyzed by t-test and analysis of variance.The data that did not fulfill the normal distribution were presented as median (interquartile range) and analyzed using the rank sum test.The count data comparison was conducted by χ2test or Fisher exact test.P＜0.05 was considered as statistically significant.

Results

Study subjects and clinical information

During the study period,212 women were assessed for eligibility.Of these,6 women did not meet the inclusion criteria and 6 women refused to participate.The remaining 200 women were randomly allocated to group regular dose CC (n=83) or group low-dose CC(n=105) group.Four women in the two groups were subsequently lost to follow-up and two women excluded from the analysis.Moreover,six patients quite(Figure 1).

The baseline characteristics of the participants,age,body mass index,basal hormone levels (FSH,LH,E2,and testosterone) and endometrium were comparable between the two treatment groups.There were no statistically significant differences(P＞0.05)(Table 1).

The serum hormone levels before and after treatment in the two patient groups

The hormonal values before and after each treatment are shown in Table 2.The FSH,LH and E2 levels of regular-dose CC group were significantly increased after taking CC 5 days (P＜0.001 for all).In the low-dose CC group,the mean level FSH was not significantly increased after 5 days of treatment (P=0.69),while the LH and E2 levels were significantly increased after 5 days treatment (P＜0.001 for both).Moreover,the levels of FSH in the regular-dose CC group were significantly increased in compared with the low-dose CC after 5 days of medication.But there were no significant differences in the levels of LH and E2 between the two groups(Table 2).

The ovulation induction outcomes in the two groups.

Compared with the group regular-dose CC,the group low-dose CC demonstrated significantly thicker endometrium on the HCG day,fewer dosage and shorter days of HMG,less numbers of mature follicles and higher ovulation rate (P＜0.05).There were also no cancellation cycles (caused by increased quantities of follicles) observed in the group low-dose CC.In addition,the diameter of dominant follicle was larger in the group low-dose CC at 5 days after treatment,while there was no difference in follicular size between the two groups on the HCG day.After HCG injections,there was similar biochemical pregnancy rates in the two groups(Table 3).

Figure 1 Flow chart showing recruitment and analysis of the study population

Table 1 Clinical feature and endocrine and metabolism parameter among

Table 3 Endometrial thickness on the HCG day and follicular development outcomes after treatment of different doses of CC

Discussion

The classical view of ovarian follicle development is that it is regulated by the hypothalamic-pituitary-ovarian axis.As known,there is a gonadotropin-releasing hormone (GnRH) pulse generator in the mediobasal hypothalamus,where GnRH is released in a pulsatile manner [9]The variations in GnRH pulse frequencies and amplitudes have differential effects on FSH and LH synthesis and release by the anterior pituitary [10,11].FSH is preferentially stimulated at low GnRH pulse frequencies,whereas LH is preferentially stimulated at high GnRH pulse frequencies.

Many factors can affect the GnRH pulse frequencies.Studies have shown that overweight or obesity strongly influenced hypothalamic pituitary ovarian axis [12],which can decrease GnRH pulse frequencies and further cause the reduce of the pituitary release of the two gonadotropins:FSH and LH.Secondary less FSH and LH in turn acted on the ovaries,which eventually affected ovarian hormone synthesis,folliculogenesis and lead to irregular ovulation.It was suggested that CC constantly exerted its effect by directly stimulating Kiss-1 gene expression in the ARC region of the hypothalamus and thus stimulated the pulsatile release of GnRH [13],ultimately stimulating follicular growth by potentiating the release of gonadotropins.So CC was recommended as the first-line drug for overweight infertile women with anovulation.

CC is a selective estrogen receptors (ERs)modulator that binds to ERs and plays dual roles as an estrogen agonist and antagonist at the level of the hypothalamus[14].In women who have a certain level of E2,CC exerts an antagonistic effect on ERs.It inhibits the negative feedback of estrogen at the level of the hypothalamus and pituitary.The increased LH and FSH secretion induces follicular development [15,16].But its antiestrogen effects on the cervical mucus and endometrium result in be difficult to sperm penetration and unreceptive to the embryo.In addition,the increased pulse frequency after CC treatment stimulated multiple follicular development and cyst formations,especially in consecutive stimulation cycles and higher dosages[17,18].

We evaluated in this study,the efficacy of low dose clomiphene protocol in obese women with anovulation.The study showed that low dose clomiphene protocol were better than regular dose protocol in terms of endometrium,single follicular development,the amount of total dose of HMG used and ovulation rate.There are fewer ERs occupied by low-dose CC,and the estrogen which produced by follicular development binds to the unoccupied ERs to exert the estrogen effect.Meanwhile,it was found that the low-dose CC led to the lower serum FSH level after 5thday post-drug treatment.This is mainly because endogenous estrogen produced by developing follicle can combine with undepleted ERs to start up the negative feedback on FSH.As a result,with the same FSH threshold value,low-dose CC is more beneficial for monofollicular development,reducing the risks associated with the development of multiple follicles.Reports had shown that clomiphene inhibited positive feedback at the level of the hypothalamus and resulted in lower ovulation rate [19].Low dose CC attenuates the inhibition of hypothalamic positive feedback,and exogenous HCG can easily stimulate LH surge and induce ovulation.

In addition,although the CC dose was reduced to half,there was no difference on the diameter of follicles of HCG day and biochemical pregnancy rates in both two groups.In the low-dose CC group,less HMG dosage and days may be related to larger follicular diameter after 5 days of administration,and the specific mechanism needs further study.

The current study has some limitations.The trial did not include a control group without medication,and lack the comparison of clinical pregnancy rates.Larger studies incorporating these factors should be conducted in the future.

Overall,the present study show that low-dose CC can achieve the same clinical efficacy as regular-dose CC and cause less side effects.Therefore,our current study indicated that low dose CC treatment has obvious advantage in ovulation induction in females with overweight and has significant clinical application values.