Clomiphene citrate or Letrozole as a first line treatment of anovulatory sub fertile polycystic ovarian syndrome women

Background : PCOS is associated with anovalation and a common cause of infertility affecting 4-6 % women in reproductive age , Clomiphene citrate has traditionally used as a drug of choice in treating women with anovulatory PCOS , in the last decade letrozole , an aromatase inhibitor has emerged as an alternative ovulation induction agent . Objective : to compare letrozole and clomiphene citrate as an ovulation induction drug in infertile PCOS women . study design : prospective study . Patient and method :


Introduction
PCOS was first described by Dr. Stein & leventhal in 1935, it is the most common cause of female infertility and ovulatory dysfunction, it is a syndrome of ovarian dysfunction along with cardinal features of hyperandrogenism and polycystic ovary morphology.Its clinical manifestations may include menstrual irregularities, signs of androgen excess and obesity, insulin resistance and elevated serum LH levels are also common features in PCOS (1).prevalence:-(4-12%).The prevalence of polycystic ovaries seen on ultrasound is much higher, around 25% in normal and 87% in patients with oligomenorrhea.(1) Pathophysiology The etiology of PCOS remain unclear.Women with PCOS have abnormalities in the metabolism of androgens and estrogen and the control of androgen production .High serum concentrations of androgenic hormones , such us testosterone , androstenedion, and dehydroepiandrostorone sulfate (DHEA_S) may be encountered in these patients however , individual variation is considerable, and a particular patient might have normal androgen levels.(2) PCOS is also associated with peripheral insulin resistance and hyper-insulinemia , and obesity amplifies degree of both abnormalities .Insulin resistance in PCOS can be secondary to a post binding defect in insulin receptors signaling pathways , and elevated insulin levels may have gonadortopin-augmenting effect on ovarian function .Hyperinsulinemia may also result in suppression of hepatic generation of sex hormonebinding globulin (SHBG) , which in turn may increase androgenicity.(3)A proposed mechanism for anovulation and elevated androgen levels suggests that , under the increased stimulatory effect of luteinizing hormone (LH) secreted by the anterior pituitary ( a result both of disordered ovarian-pituitary feedback and exaggerated pulses of GnRH from the hypothalamus) stimulation of the ovarian theca cells is increased , these cells in turn increase the production of androgens (e.g.testosterone , androstenedione).
Because of the decrease level of (FSH) follicle stimulation hormone relative to LH, the ovarian granulosa cells cannot aromatize the androgens to estrogens, which leads to a decreased estrogen levels and consequent anovulation (2).(GH) growth hormone and insulin like growth factor -1(IGF-1) may also augment the effect on ovarian function.Some evidence suggests that patients have a functional abnormality of cytochrome P450c17 , the 17 hydroxylase , which is the rate-limiting enzyme in androgen biosynthesis.PCOS is genetically heterogeneous syndrome in which the genetic contribution remain incompletely described , PCOS is an inherently difficult condition to study genetically because of its heterogeneity.Studies of family members with PCOS revealed that about 50% of first-degree relatives have PCOS suggesting that an autosomal dominant mode of inheritance occurs for many families with this disease.(4) Clinical features (5) Oligomenorrhea / amenorrhea (65-75%) Hirsutism (30-70%) Sub-fertility(75%) Obesity (40%) and metabolic syndrome.

Diagnostic criteria
A l990 expert conference sponsored by the National Institute of Child Health and Human Development (NICHD) of the united states national institutes of health (NIH) proposed of the following criteria for the diagnosis of PCOS:-

Imaging tests
Ovarian ultrasound preferably using trans-vaginal approach:-US Criteria has been defined also by Rotterdam consensus as an ovary with 12 or more follicles 2-9 mm in diameter and increased ovarian stroma and volume >10 ml.It was postulated that the hyperandrogenic environment within PCO results in an increased recruitment of growing follicles of 2-5 mm, followed by the arrest at 6-9 mm.(8) Pelvic CT-scan or MRI to visualize the adrenals and ovaries .

Treatment of
Anovulation in polycystic ovaries syndrome .

1-Weight loss:
-obesity is observed in more than (50) percent of women with PCOS.The body fat is usually deposited centrally (android obesity ).Normalization of menstrual cycle and ovulation could occur with weight reduction include diet and life style modification (5) .

2-improvement in metabolic function :-
Polycystic ovarian syndrome is associated with metabolic derangements, and treatment of this leads to resumption of ovulation .Insulin resistance is thought to play an important role in the pathogenesis of PCOS in the subset of patient who have either incremental (BMI ) or hyperinsulinemia and /or significant hyperandrogenism .Medications that have been tested in patients with PCOS include metformin, thiazolidine and ions , vitamin D and statins (7) .

3-Ovulation induction Agents:-
The recommended first choice of treatment remains clomiphene citrate (CC) , if CC use fails to result in pregnancy ; the recommended second line intervention is the exogenous gonadotropins or laparoscopic ovarian drilling ( LOD).

Clomiphene citrate
It was first synthesized in 1956, and it was approved for infertility treatment by the United States Food and Drug Adminstration (FDA) in 1967.It is available as a racemic mixture of two stereo isomers, En(Trans)-clomiphene and Zu(Cis) clomiphene , its zuisomer exists in tissues for weeks and contributes to its metabolic life where are en-isomer is more potent one and is responsible for its biological action.It is non-steroidal triphenylethylene derivative that exhibits estrogen receptors antagonist properties, most commonly used as first line treatment for IO in women with anovulatory PCOS.CC is readily absorbed with half life of 5 days, and primarily excreted in the feces.(9)

Mechanism of action :-
Clomiphene exerts its major effects on the hypothalamus, pituitary, ovary and uterus .
Hypothalamus and pituitary:-Most evidence suggests that primary site of CC action is the hypothalamus where it appears to bind to hypothalamus estrogen receptors there by blocking the negative feedback effect of circulating estrogen as a consequence, GnRH pulsatility and gonadotrophin secretion are enhanced, which further results in the stimulation of follicular recruitment, selection, assertion of dominance, and rupture.In vitro data suggest that CC also has a pituitary site of action where it causes an increase in the gonadotrophin response to GnRH.(10) Ovary :-The ovarian action of CC are for the most part secondary to the effects of elevated FSH and LH on ovarian follicular development .Direct effects of CC on the ovary are not well understood but probably exist .Uterus and cervix:-Clomiphene citrate acts primarily as an anti-estrogen in the uterus , cervix and vagina this may at least partially explain the low pregnancy rates observed cycles .Some investigators have observed significantly decreased sonographic endometrial thickness and altered morphometric endometrial histology (decreased gland number and increased vacuolated cells) in clomiphene cycles.Data on the effect of CC on cervical mucosa are conflicting; while one study found a decrease in the quality and quantity of cervical mucosa at all CC doses, in a meta -analysis , detrimental effect was seen only with doses greater than or equal to 100 mg/day (2) .

Indications (2)
 Anovulation:-is the most important indication for CC treatment .In addition, treatment is indicated for women with oligomenorrhea, or amenorrhea, who responded to progesterone treatment with withdrawal bleeding.These women belong to the WHO group II, the majority of these women have the characteristics of PCOS.However, given its hypothalamic site of action, CC ineffective in women with hypogonadotropic hypogonadism (WHO group I). Luteal phase deficiency:-progesterone levels are typically higher after CC treatment than in spontaneous cycles, reflecting improved preovulatory follicle and corpus luteum development.
 Unexplained infertility:-In couples whose infertility remains unexplained, empiric treatment with CC may be justified, particularly in young couples with a short duration of infertility. IVF:-when multiple follicle development is required.

Doses
The starting dose of CC generally should be 50 mg/day for 5 days, but a dose of 25 mg can be considered for lower weight patients, while a dosage of 100 mg may be more appropriate for obese patients, starting from cycle day 2-5 of a spontaneous menstruation or after progestin induced withdrawal bleeding .CC commenced on day 2 of the menstrual cycle rather than day 5 , result in more rapid follicular growth , a longer CC-free period before ovulation .The dose of CC is increased by 50 mg in every subsequent cycle in the absence of ovulation.Maximum dose of CC which has been reported to be used is 250mg/ day , however the recommended maximum dose is 150 mg/day as there is no clear evidence of efficacy at higher doses and this is in accord with FDA recommendation of 750mg/ treatment cycle.( 1)

Monitoring
The following procedures may be used for monitoring:  Follicular monitoring with vaginal ultrasound, starting 4-6 days after last pill.Serial transvaginal ultrasound can reveal the size and number of developing follicles. Serum estradiol levels, starting 4-6 days after last pill. Urine LH surge tests started 3-4 days after last clomiphene pill and continue until ovulation is indicated ( positive test) or through cycle day 18.  Mid-luteal progesterone, with at least 10ng/ml 7-9 days after ovulation being regarded as adequate.
The practice in many centers is to monitor the first cycle by follicle monitoring or urinary LH surge to allow adjustment of the dose in subsequent cycle.In the absence of complete cycle monitoring an ultrasound is recommended to evaluate ovarian and endometrial morphology on the start of CC.There is no evidence that administrate of (HCG) in mid cycle improves the chances of conception.(11)

Results
Comiphene citrate is a very efficient ovulation inductor ; approximately 75-80% of patients with PCOS will ovulate after CC.However, there appears to be discrepancy between ovulation and pregnancy rates and life table analysis of largest and most reliable studies indicates a conception rat of only up to 22% per cycle in those ovulating on CC.This discrepancy occurs due to:- peripheral anti-estrogenic action on the endometrium. Antiestrogen effects on the cervical mucus.The mechanism responsible for initiation of malignancy is possibly repetitive trauma of ovarian surface and exposure of this surface to estrogen rich follicular fluid , which may stimulates mitotic cell proliferation.The risk of ovarian cancer after CC therapy increases with the number of treatment cycles, when only six CC cycles are applied the risk of ovarian cancer will not exceed that of other women, but more than 12 cycles of use in a life time has been associated with increase in risk of future ovarian cancer (relative risk RR=1.5-2.5) .Therefore after 6-9 cycles of CC use this drug should preferably be discontinued (14) .
10-The evidence of fetal and neonatal abnormalities for patients on CC is similar to that seen in the general population.There is no data to suggest a higher rate of congenital anomalies or spontaneous abortions after using this drugs.

Letrozole (Femara tradename)
Is an oral non steroidal aromatase inhibitor drug for the treatment of hormonally-responsive breast cancer after surgery.

3-Diminished ovarian reserve:-
In women with diminished reserve, there is a poor response to ovulation induction medications.In some, it is due to lack of oocytes; in others however, it is due to a decrease in follicular FSH receptors.With the use of letrozole, an increase in intrafollicular androgen is known to increase follicular FSH receptors (20).

Dosing
Letrozole is typically administered on day 3-7 of the menstrual cycle at doses of 2.5-7.5 mg/day in 2.5 mg increments.Little investigation has been attempt to define optimal dosing in infertile women, most studies utilizing letrozole at 2.5mg daily for 5 days show between one and two mature follicles drown at this dose.Available evidence suggests a dose-response with letrozole, with higher doses (5mg) producing more mature follicles and higher ovulation rates and pregnancy rates as well (21).The 1 st randomized controlled trial addressing letrozole dosing was performed in 2007.Badawy et al. utilized either 2.5, 5, or 7.5 mg daily although they found no differences in pregnancy rates, the number of mature follicles was significantly higher in the 7.5 mg group versus 5 or 2.5mg (22).The optimum length of treatment in a cycle is also unknown at this time.In a recent study letrozole used for 5 days and 10 days, ovulation rates were similar but the number of mature follicles was high in 10 days treatment (23).

Contraindications
Letrozole is contraindication in pregnancy, and lactation .

Adverse effects
Approximately 10-20% of women will experience side effects but because the medication does not directly fit into the estrogen receptor, these effects are usually mild:  Sweating. Hot flashes. Blurred vision. Headache. Arthralgia (joint pain ) and fatigue. Nausea. Ovarian cyst. Multiple pregnancy 3-5% risk of twins which is little than clomid. No differences in birth defects observed in children who were conceived using either CC or letrozole (2).

Interactions
Letrozole inhibits the liver enzyme cyp2a6 , and to a lesser extent cyp2cla in vitro, but no relevant interactions with drugs like cimetidine and warfarin have been observed.

Patients And Methods
Setting:-This is a prospective randomized study conducted in the fertility center in Al-Sader medical city between March 2014 and September 2014.The study protocol was approved by the institutional ethics committee, and informed consent was obtained from all study participants.Patients:-( 115) infertile PCOS women were recruited into the study.PCOS diagnosed according to the Rotterdam revised 2003 criteria for polycystic ovary syndrome , patients had a history of oligomenorrhea or amenorrhea, ovaries with at least 12 sub-capsular cysts 2-8mm in diameter, and /or elevated serum testosterone and Hirsutism.Our inclusion criteria included patients in the age of 18-35 years, having infertility for more than one year, and patients of anovulatory PCOS.Patients with hyperprolactinemia, thyroid disorder, male factor, BMI >35, suspected tubal factor, unexplained infertility were not included in the study.Methods:-Patients were randomized to receive either 100 mg CC (n=60) or 5 mg letrozole (n=55) daily for 5 days beginning on day 3 of the menstrual cycle.Clomiphene citrate (Clomid; Sanofi Aventis, France) and Letrozole ( Femara; Novartis pharma AG, Basle, Switzerland).Transvaginal ultrasound examination was performed on day 2 of the menstrual cycle before treatment was commenced, also hormonal studies like FSH, LH, prolactin, TSH, and testosterone were performed .Follicular development was monitored using transvaginal ultrasound from day 10 of menstrual cycle till a follicle attained >16mm diameter.The endometrial thickness was measured at the greatest diameter perpendicular to the midsagital plane in the fundus region including both layers of the endometrial cavity .The number of follicles and endometrial thickness in patients of both groups were documented.When at least one mature follicle with a mean diameter ≥17 mm was observed, HCG (pregnyl) at dose of 10.000 IU was used to trigger ovulation and the ovulation was confirmed by seeing follicle collapse on subsequent ultrasound 48 hrs after the injection.Each woman was asked to have timed intercourse 24 hrs to 36 hrs after HCG injection.Ovulation was diagnosed when the mature DF was approximately 17 to 22 mm followed by evidence of rupture approximately 3 to 4 days later.If the dominant follicle (DF) was absent (DF < 17 mm), a repeat TVS was performed every 3 -4 days later.The absence of a dominant follicle (DF < 17 mm) by Day 20 was considered as a nonresponse or anovulation.Serum E2 levels on day of HCG was done for all patients.Pregnancy was diagnosed by serum level of β-HCG performed once the patient missed her period.Outcome measures:-Primary outcome measures were the mean number of follicles, endometrial thickness and ovulation rate, while secondary outcome measure was pregnancy rate compared in both the groups.The patients were given only one cycle of treatment for the study.

Statistical Analysis
The number of mature follicles, the endometrial thickness, the ovulation and pregnancy rates were compared in the two groups after one cycle of stimulation.A group t-test or the Student's t-test was used to compare data as appropriate.A P-value < 0.05 was considered to be statistically significant.

Results
During the study period, a total of 115 patients were analyzed for recruitment, 60 women were in 100mg Clomiphene citrate group and 55 women received 5mg letrozole group.Table 1 summarizes the demographic profile of patients.There was no statistically significant difference in the mean age, BMI, basal FSH, type and duration of infertility in both groups of patients.Although LH and prolactin levels were significantly higher in Letrozole group but still within normal levels.Comparison between responders and non-responders within each group regarding baseline endocrine and demographic variables did not find significant difference in any of them (Table 3).Pregnancy rate per responder was also higher in letrozole group (26.09%) in comparison with CC group (18.60%), although this difference also was not statistically significant (P=0.398).E2 level at day of HCG of CC group was (380.79+268.29pg/ml)which was significantly higher (P= 0.000) in comparison with the level of E2 of letrozole group (166.216+84.54pg/ml).Letrozole, an aromatase inhibitor, has shown to be effective in inducing ovulation and pregnancies in women with anovulatory PCOS and in patients with CC resistance or failure (18).It also improves the ovarian response to FSH in poor responders.(28)Aromatase inhibitors are non-steroidal compounds that suppress estrogen biosynthesis by blocking the action of the enzyme, aromatase, which converts androstenedione and testosterone to estrogens.The efficient estrogen-lowering property of letrozole could be utilized to temporarily release the hypothalamus from negative feedback effect of estrogen and thereby inducing an increased discharge of FSH.(18).In the ovary, aromatase inhibitors increase follicular sensitivity to FSH by the accumulation of intraovarian androgens.As the dominant follicle grows and estrogen levels rise, normal negative feedback occurs centrally, resulting in suppression of FSH secretion and atresia of the smaller growing follicles.A single dominant follicle and monoovulation should occur in most cases (29).In the endometrium, estrogen receptors may be upregulated, resulting in rapid endometrial growth once estrogen secretion is restored following clearance of Letrozole because its short half life (45h) (25,30).Response: In our study the response in Letrozole group was better than CC group, as the anovulation (no follicle >16mm) in 1 st group was 16.36% while in CC group was 28.33% (  4).This difference was statistically insignificant.But another studies are reported that using 2.5-5 mg/day of letrozole has a better endometrial response compared with endometrial response using CC in the dose of 50-100 mg/day.(27,Dicky 1996).Similar findings were reported in the study of Baruah et al. 2009, which demonstrated that endometrial thickness and sub-endometrial blood flow were significantly better in cases receiving induction with letrozole than CC despite comparable follicular response (37).This has also been reported with Al-Fozan et al. ( 33,2004).Other studies reporting that most patients taking Letrozole had a thicker endometrium compared to those taking Clomiphene citrate(38).Similar findings showed that Letrozole had an overall greater beneficial effect on the endometrium (29,39).This is possibly due to an increase in the number of growing follicles and thus a higher level of estrogen and progesterone in CC group in our study, although endometrial thickness in both study groups was >7 mm.

Number of Pregnancies
In our study, consistent with the number of successful ovulatory cycles, pregnancy rate was notably higher in the Letrozole treatment group compared to the Clomiphene citrate group with 19 (25.3%) and 12 (16.0%)pregnancies, respectively; however, this was not significantly different.The pregnancy rate observed in our study was consistent with other reported studies such as that by Mitwally (18), in which a pregnancy rate of 25% was observed for PCOS patients treated with 2.5 mg Letrozole.Additionally, Atay (38) also found a pregnancy rate of 21.6% after treatment with 2.5 mg Letrozole and 9.1% after treatment with 100 mg Clomiphene citrate, which was statistically significantly different.There are other studies that showed significantly higher pregnancy rates with Letrozole than with Clomiphene citrate (23,40,41).With CC, supraphysiologic levels of estrogen can occur without control suppression of FSH because the normal estrogen receptor-mediated feedback mechanisms are blocked.This results in multiple follicular growth and higher multiple pregnancy rates with CC than are found in letrozole cycles (42).This is corresponding with our result, Table 5.
It has been reported that serum E2 level on the day of hCG administration was statistically significantly lower in the letrozole group than the CC group (36,43).In our study, we have also found that the mean total E2 was significantly higher in CC group as compared with letrozole group Table 4.This also indicates the multifollicular response in CC group.

Conclusion:
Letrozole in patients with PCOS is as effective as clomiphene citrate in inducing ovulation, ovulation rate and the incidence of pregnancy was higher with letrozole than that with clomiphene citrate.Apart from that, Letrozole treatment is prone to production of monofollicles and hence leads to reduced incidences of the adverse pregnancy outcome of multiple fetuses as compared to treatment with Clomiphene citrate.

Recommendations:
 Further studies are needed to determine optimal dosing and the long term safety for women treated with the drug.


Decrease of uterine blood flow.

Table ( 1) Patient characteristics of both CC and Letrozole groups (mean±SD)
(2)m 60 patients on CC 17 (28%) ended by no response (no follicle >16mm) and 43 patients continue the program, while 9(16%) from letrozole group not responding to stimulation induction and 46 continue the study.Table(2)below showed that the group of letrozole was higher in number of responsive when compared with CC group but still not significant.