Letrozole and Clomiphene Citrate Effect on Endometrial and Subendometrial Vascularity in Treating Infertility in Women with Polycystic Ovary Syndrome

Abstract

Objective: Letrozole appears not to have any of the adverse effects on the endometrium that are frequently associated with clomiphene citrate (CC) during ovulation induction. The aim of the study is to compare the effect of letrozole compared to CC on endometrial thickness and endometrial and subendometrial blood flow. Materials and methods: Infertile women with polycystic ovary syndrome (PCOS) were allocated for ovulation induction; 102 women were treated with Letrozole and 99 women with CC. Thirty-five patients from the CC group showed a resistance to or failure of CC with a thin endometrium, and were re-treated with letrozole. The main outcome measures were ovulation and pregnancy rates/cycle, endometrial thickness, detection rates, and resistance index (RI) and pulsatility index (PI) of subendometrial and endometrial blood flow. Results: In spite of a nonsignificant difference in ovulation rate between letrozole and CC groups (70.6% vs. 64.6%), a clinical pregnancy rate was achieved in 29/102 (28.4%) women with letrozole and 20/99 (20.2%) women with CC treatment (p<0.05) and in 6/35 (17.1%) in women re-treated with letrozole after CC resistance or failure. There were significant differences in endometrial thickness, detection rates, and RI and PI of subendometrial and endometrial blood flow between the two groups and between 35 women re-treated with letrozole after CC resistance or failure when compared with CC treatment alone. Conclusions: Letrozole may increase fertility by improving endometrial receptivity compared with CC alone. (J GYNECOL SURG 28:405)

Introduction

Since the 1960s, the first line of treatment for anovulation in infertile women has been clomiphene citrate (CC). The choice of CC was appropriate because the drug was highly effective in inducing ovulation in selected patients, with the advantages that it was orally administered, relatively safe, and inexpensive. However, CC was also found to have adverse effects, especially in the form of antiestrogenic endometrial and cervical mucous changes that could prevent pregnancy in the face of successfully induced ovulation.^1,2

The endometrium has adhesive qualities that allow the implantation of gestational sacs; this feature is referred to as endometrial receptivity.³ An endometrium that is thinner than 6 mm is usually associated with a significant likelihood of failure to conceive.^3,4 A reduction in endometrial thickness below the level thought to be needed to sustain implantation was found in up to 30% of women receiving CC.³ In addition, successful implantation requires a receptive endometrium, with synchronous development of glands and stroma. Decreased uterine blood flow during the periimplantation stage may also explain the poor outcome of CC treatment.^5,6

Newer options for ovulation induction are the third-generation aromatase inhibitors (AIs), the most commonly used being letrozole. AIs for ovulation induction are orally administered and are relatively inexpensive, with minor side effects such as very infrequent headaches and leg cramps.⁷ Importantly, unlike CC, letrozole is devoid of any antiestrogenic peripheral action and does not deplete estrogen receptors. Letrozole is also cleared from the circulation more rapidly due to a shorter half-life compared to CC.^1,8 Letrozole has been associated with higher pregnancy rates compared to CC.⁹

With the advance of diagnostic ultrasonography, the clinical use of ultrasonic technology has increased as a way to track changes in the uterine wall thickness in the assessment of endometrial blood flow for predicting endometrial receptivity.¹⁰ Uterine artery Doppler indices—resistance index (RI) and pulsatility index (PI)—are limited in assessing the endometrium because most of the blood passing through the uterine arteries supplies the myometrium not the endometrium. So it is more logical to evaluate the vascularization around the endometrium in order to assess endometrial receptivity.¹¹ As the spiral arteries are located just beneath the endometrium, a Doppler study of the spiral arteries can be a useful marker for endometrial response.¹² Moreover, there is no significant difference between conventional 2D and 3D power Doppler sonography for the assessment of endometrial blood flow.¹¹

The aim of this study was to compare the effect of letrozole and CC on endometrial thickness and endometrial and subendometrial blood flow as detected by color Doppler ultrasonography in infertile women with polycystic ovary syndrome (PCOS).

Materials and Methods

This study started after approval was obtained from the Institutional Review Board of Jeddah Clinic Hospital, Jeddah, Saudi Arabia. All participants gave verbal and written informed consent. The procedures used were in accordance with the guidelines of the Helsinki Declaration on human experimentation.

From November 2008 to September 2011, 220 infertile women with PCOS were randomized to one of two treatment conditions: letrozole or CC. All participants had been diagnosed with PCOS. The major criteria for diagnosis of PCOS were oligo- and/or anovulation, clinical or biochemical signs of hyperandrogenism, and polycystic ovaries with ultrasound, which is in accordance with the revised 2003 Rotterdam criteria of PCOS.² Exclusion criteria included hyperprolactinemia, congenital adrenal hyperplasia, thyroid disease, other causes of amenorrhea such as premature ovarian failure, and clinically suspected Cushing's syndrome or androgen-secreting neoplasm. Exclusion criteria also included all women who had received metformin or ovarian drilling in the previous 6 months. Other causes of infertility were excluded by documentation of a normal uterine cavity and at least one patent fallopian tube, and each woman's current partner had a semen concentration of at least 2×10⁷/mL. All subjects were in good health with no major medical disorders.

Patients were randomly allocated to the letrozole group or CC group by means of a series of blind envelopes numbered from 1 to 220. Each patient was invited to choose an envelope and was placed by the clinic secretary in either the letrozole group or the CC group.

The letrozole group included 110 patients who were treated with 5 mg/day of letrozole (Femara; Novartis, Switzerland) in two divided doses from cycle day 3 to 7. The CC group included 110 patients who were treated with 100 mg/day of CC (Clomid; Sanofi Aventis, France) in two divided doses from cycle day 3 to 7. The patients were not blinded about the treating drug in either group.

Monitoring was done by transvaginal ultrasound (TVS) starting from day 10 and continuing every alternate day until the day of ovulation or day 20; if ovulation did not occur, the cycle was considered resistant to induction. The endometrial pattern and thickness and the number of follicles in each group were documented. To remove any interobservational bias, ultrasound in all patients was demonstrated by a single observer (MF Selim) who was blinded to the treating drug. An injection of 10,000 IU/mL of human chorionic gonadotropin (hCG; Choriomon; IBSA, Switzerland) was given to the patients intramuscularly when a dominant follicle ≥18 mm and triple-line endometrial pattern ≥5 mm thickness were observed. Timed intercourse was advised after the hCG injection.

On the day of the hCG injection, TVS examination was performed with the patient in the lithotomy position using a 8-MHz transvaginal transducer with color Doppler facility (Logiq^® 9; GE Healthcare, Waukesha, WI). Just before TVS examination, systolic and diastolic blood pressures and heart rate were taken, and, in the cases of abnormal values, the examination was delayed until these parameters were normalized. Subjects completely emptied the bladder before TVS examination to minimize any external effects on blood flow.

The follicular diameter was measured in two perpendicular planes and the mean was calculated. The endometrial echogenicity pattern was described as either an “homogeneous pattern” or a “triple-line pattern.” The “homogeneous pattern” was defined as a single hyperechoic, hypoechoic, or isoechoic layer with absent or a poorly defined central echogenic line, whereas the “triple-line pattern” was defined as a multilayered endometrium consisting of a prominent outer and inner hyperechoic line and inner hypoechoic regions.³ Due to poor pregnancy rates, cases of an homogenous endometrial pattern were excluded from the study.³ After a true longitudinal view of the uterus had been obtained, the endometrial thickness was measured as the maximum thickness between the highly reflective interfaces of the endometrial–myometrial junction. The measurement included both layers of the endometrium.

Subendometrial and endometrial vascularity Doppler measurements images (from uterine fundus to the cervico-uterine junction at the level of the internal os in a longitudinal plane) with color signals were obtained by power Doppler. A power flow map was superimposed on the subendometrial and endometrial regions. The Doppler flow indices of vessels seen at the subendometrial and endometrial regions were studied at random on the areas of maximum color intensity, and the lowest values for resistance to flow were recorded. The blood flow velocity waveforms from the subendometrial vessels were obtained by placing the Doppler gate over the color area and activating the pulsed Doppler function. A recording was considered satisfactory when at least five consecutive waveforms were obtained, each demonstrating the maximum Doppler shift. Blood flow impedance was evaluated and expressed as PI and RI on three consecutive uniform waveforms, and these two parameters were calculated electronically by the machine.

The presence of subendometrial and endometrial vascularity was noted, and their detection rates were calculated in each group as the number of positive examinations for subendometrial and endometrial vascularization divided by the total number of power Doppler assessments performed. Endometrial borders were set as the outer limits of the hyperechogenic myometrium–endometrium interface, whereas subendometrial areas were considered arbitrarily 1 cm around the endometrial borders.

At 1 week post hCG injection, the occurrence of ovulation was documented by one or more of the following criteria:

• Development of a dominant follicle ≥18 mm, followed by the disappearance or reduction in size of the dominant follicle by more than 5 mm.

• A change in the shape and appearance of internal echoes within the follicle.

• Appearance of free fluid in the Pouch of Douglas.

A serum pregnancy test was done 20 days post hCG injection and, if positive, ultrasound examination was performed 2 weeks later to confirm intrauterine pregnancy and to determine the presence of gestational sacs. Only clinical pregnancies were included, defined by the presence of one or more gestational sacs.

Patients who did not ovulate (resistance) or failed to conceive (failure) after 100 mg/day of CC and who showed thin endometrial development (endometrial thickness ≤6 mm on the day of hCG administration) were re-treated with 5 mg/day of letrozole in two divided doses in next cycle of CC resistance or failure, and the same study protocol methodology was repeated.

The mean number of follicles, endometrial thickness, the Doppler study of endometrial and subendometrial vasculatures, ovulation rate, and pregnancy rate were compared in both groups. A comparison was also made in the group of CC resistance or failure with thin endometrium between treatment with CC and after re-treatment with letrazole.

The data were obtained from a population of 102 women in the letrozole group and 99 in the CC group. In the letrozole group, eight women were excluded because of missed follow-up visits (three women), treatment suspension (two women), and homogenous not triple-line endometrial pattern (three women). In the CC group, 11 women were excluded because of missed follow-up visits (four women), treatment suspension (two women), and homogenous not triple-line endometrial pattern (five women). Endometrial thickness was ≤6 mm in 35/79 (44.3%) women who did not ovulate or conceive in the CC group who were then re-treated in the next cycle with letrozole (Fig. 1).

Fig. 1.

Flow chart of the participants. PCOS, polycystic ovary syndrome; CC, clomiphene citrate.

Analysis

Statistical analysis was performed using SPSS V13.0 (SPSS Inc., Chicago, IL). Normally distributed continuous variables were compared with the independent sample test. We used the nonparametric Mann–Whitney U-test to analyze continuous variables and the Fisher's exact test and chi square test for categorical variables. The correlations between pregnancy rates, endometrial thickness, and endometrial and subendometrial blood flow impedance (RI and PI) were studied with Pearson correlation test. Statistical significance was set at p<0.05.

Results

A total of 201 cycles of ovulation induction were carried out, of which 102 cycles were in the letrozole group and 99 cycles were in the CC group. There were no statistically significant differences in demographic characteristics of both groups of patients (Table 1). The mean age, body mass index (BMI), the mean duration, and whether the patients had primary or secondary infertility were comparable between the two groups.

Table 1.

Demographic Characteristics of Patients Treated with Letrozole and Clomiphene Citrate (CC)

Variable	Letrozole group (n=102)	CC group (n=99)	p value
Age (years)	26.0±2.7	25.1±3.1	NS
BMI (kg/m²)	24.4±4.3	23.8±3.7	NS
Duration of infertility (years)	2.9±0.6	2.6±0.7	NS
Primary infertility (n)	78/102	79/99	NS
Secondary infertility (n)	24/102	20/99	NS

Data expressed as mean±standard deviation.

BMI, body mass index; NS, not significant.

The two studied groups were comparable with regard to the duration until hCG administration. No cancelled cycles occurred due to excessive stimulation or the occurrence of ovarian hyperstimulation syndrome. Table 2 shows that, there were no statistically significant differences in the mean number of follicles (≥ 18 mm) in the letrozole group and CC group 1.36±1.0 vs. 1.27±1.1 respectively, p>0.05). The mean trilaminar endometrial thickness in the letrozole group was 9.9±1.8 mm compared to 7.7±1.6 mm in the CC group, which was statistically significant (p<0.05).

Table 2.

Comparison of the Different Variables in the Letrozole and Clomiphene Citrate (CC) Treated Groups

Variable	Letrozole group (n=102)	CC group (n=99)	p value
Days until hCG administration (n)	12.6±1.4	12.9±1.3	NS
No. of follicles ≥18 mm	1.36±1.0	1.27±1.1	NS
Endometrial thickness (mm)	9.9±1.8	7.7±1.6	<0.05^a
Detection rate of endometrial and subendometrial blood flow (n, %)	86/102 (84.3%)	40/99 (40.4%)	<0.05^a
Subendometrial artery
• Pulsatility index(PI)	1.27 (0.82–3.23)	1.67 (0.94–4.02)	<0.05^a
• Resistant index (RI)	0.75 (0.61–0.99)	0.87 (0.62–1.00)	<0.05^a
Ovulation rate/cycle (n, %)	72/102 (70.5%)	64/99 (64.6%)	NS
Pregnancy rate/cycle (n, %)	29/102 (28.4%)	20/99 (20.2%)	<0.05^a

Data expressed as mean±standard deviation; but PI and RI expressed as median (range). NS, not significant; hCG, human chorionic gonadotropin.

Statistically significant.

The detection rate for subendometrial and endometrial blood flow was significantly (p<0.05) lower in CC patients than in letrozole patients (Table 2). The RI and PI values of the subendometrial arteries showed significantly lower impedance in the letrozole group compared to the CC group. There was a positive correlation between endometrial thickness and endometrial and subendometrial blood flow in the letrozole group, and there was a negative correlation between endometrial thickness and endometrial and subendometrial blood flow in the CC group. There was no significant difference in the ovulation rate between the two groups (70.5% vs. 64.6%). As regard pregnancy rate, 29 of 102 letrozole cycles (28.4%) and 20 of 99 CC cycles (20.2%) resulted in pregnancy (Table 2). The pregnancy rates were positively correlated with endometrial thickness (r=0.71). Also the pregnancy rates were positively correlated with PI/RI of endometrial and subendometrial blood flow (r=0.62). These correlations were statistically significant (p<0.05). No multiple pregnancy or side effects were noted in either the letrozole or CC groups.

Table 3 shows that in the study of participants with CC resistance or CC failure with endometrial thickness ≤6 mm, letrozole induction of ovulation was associated with an ovulation rate of 45.7% (16/35 women) and a pregnancy rate of 17.1% (6/35 women). Also, the detection rate for subendometrial and endometrial blood flow and RI and PI values on subendometrial vessels were significantly (p<0.05) higher with CC treatment than after treatment with letrozole. There was a significant (p<0.05) improvement in mean endometrial thickness of CC-treated women after receiving letrozole (5.4±0.9 mm 8.4±2.5 mm).

Table 3.

Comparison of the Different Variables in Patients with Clomiphene Citrate (CC) Resistance or Failure with Thin Endometrium After Letrozole Administration

Variable	Letrozole after CC (n=35)	CC group before letrozole (n=35)	p value
Days until hCG administration (n)	13.1±1.2	12.8±1.9	NS
No. of follicles ≥18 mm	1.18±1.3	1.03±1.4	NS
Endometrial thickness (mm)	8.4±2.5	5.4±0.9	<0.05^a
Detection rate of endometrial and subendometrial blood flow (n, %)	19/35 (54.2%)	8/35 (22.8%)	<0.05^a
Subendometrial artery
•Pulsatility index (PI)	1.43 (0.98–3.73)	1.93 (1.24–4.82)	<0.05^a
•Resistant index (RI)	0.98 (0.69–1.39)	1.02 (0.89–1.24)	<0.05^a
Ovulation rate/cycle (n, %)	16/35 (45.7%)	6/35 (17.1%)	<0.05^a
Pregnancy rate/cycle (n, %)	6/35 (17.1%)	0/35 (0%)	<0.05^a

Data expressed as mean±standard deviation but resistant index and pulsatility index expressed as median (range). NS, not significant.

Statistically significant.

Discussion

Endometrial receptivity is crucial for the implantation of an embryo. Both endometrial thickness and adequate endometrial perfusion are vital to implantation. A minimum endometrial thickness of 6 mm is acceptable for implatation.^3,13 The coexistence of a thinner endometrium in association with no triple-line pattern reflects a diminished endometrial responsiveness to ovarian hormones leading to a low clinical pregnancy rate.¹⁴ CC is a nonsteroidal selective estrogen receptor modulator that has predominant antiestrogenic action resulting in long-lasting estrogen receptor depletion. Letrozole is a potent, nonsteroidal AI. It can mimic the action of CC without depletion of estrogen receptors. Hence, cervical mucus and a thin endometrium are not expected with treatment with AIs.¹³ The high supraphysiological levels of estrogen attained during ovarian stimulation with CC may explain some of the adverse effects of CC, although reducing estrogen synthesis with AIs may ameliorate such deleterious effects.¹⁵

The results of this study indicate that infertile women with PCOS experience no significant changes in the number of mature ovarian follicles (diameter ≥18 mm) and ovulation rate, although a significant increase in pregnancy rates was observed in patients who received letrozole in comparison to CC. This study also indicates a significant higher ovulation rate and pregnancy rate after letrozole in cases of CC resistance or failure with a thin endometrium compared with CC therapy. As regard the endometrial thickness, it is reported that the use of letrozole results in a better endometrial response (9.9±1.8 mm) compared to the endometrial response obtained using CC (7.7±1.6 mm). Also, in the letrozole re-treatment group after CC resistance or failure with a thin endometrium, a thicker endometrium was demonstrated (8.4±2.5 mm) compared to the previous treatment with CC (5.4±0.9 mm). Letrozole improved endometrial and subendometrial vascularity detection rates and decreased the impedance of the subendometrial arteries compared to CC and also after letrazole treatment in CC resistance or failure with a thin endometrium. The evaluation of endometrial and subendometrial vascularity showed that the pregnancy rates were related to PI and RI indices.

These results are similar to those that showed that CC can cause inadequate endometrial thickness in 15–50% of patients. This is probably due to estrogen receptor depletion.^15–17 It is probable that the cause of endometrial thickening in patients receiving letrozole is because of improved endometrial vascularization.^18,19 Baruah et al.¹² concluded that the effect of letrozole on endometrial thickness in patients with anovulatory PCOS was significantly better compared to CC. Both the RI and PI of the subendometrial spiral arteries showed significantly lower impedance compared to the CC group. Although there was a positive correlation between endometrial thickness and spiral artery blood flow impedance in the letrozole group, the correlation was not statistically significant. Yet Doppler velocimetry of the spiral arteries can be a useful supportive tool for evaluating endometrial response in the treatment of anovulatory PCOS patients with infertility.

According to results derived from the current study as well as previous ones,^20–22 it seems that pregnancy rates are higher in patients receiving letrozole than those receiving CC. In a clinical study of 22 women who failed to respond to CC and who were re-treated with letrozole, ovulation occurred in 75% of patients and pregnancy was achieved in 25%.²¹ In another study of CC-resistant women with PCOS, letrozole induction of ovulation was associated with an ovulation rate of 54.6% and a pregnancy rate of 25%.²² In a study by Rahmani et al.,²³ CC-resistant patients had a different response to letrozole in that 44.2% of the cycles had normal follicles of which 23.9% resulted in pregnancy. Increasing the dosage can improve the chance of ovulation and pregnancy. Only seven (15.9%) patients taking 2.5 mg of letrozole daily became pregnant, while 11 patients became pregnant by increasing the dosage to 5 mg. Kamath and George²⁴ reported that more than 60% of women who are resistant to CC ovulate after treatment with letrozole. Discrepancies between ovulation and pregnancy rates may be attributed to different studies' participants and protocols. Also intra- and interobserver variability of the technique may account for the differences.

Kupesic et al.²⁵ found that the conventional 2D color Doppler and the 3D power Doppler ultrasonography have similar efficiencies in predicting endometrial receptivity by combined the parameters of endometrial thickness and subendometrial blood flow. They also reported that the resistance is more indicative than presence or absence of blood flow alone in the subendometrial area. Yet Edi-Osagie et al.²⁶ reported that the presence of blood flow in the subendometrial area as well as the low Doppler indices of its arties correlated well with endometrial receptivity and pregnancy rate.

Endometrial blood flow has been correlated with increased expression of angiogenic factors, predominately of vascular endothelial growth factors (VEGF) that have a vasodilatation effect mediated by the release of prostacyclin and nitric oxide by endometrial cells.²⁷ There is a positive correlation between the estradiol level and VEGF in the endometrial cell,^27,28 As CC causes the depletion of estrogen receptors, so CC can negatively affect endometrial blood flow and receptivity.

Conclusions

Subendometrial and endometrial blood flow is significantly impaired with CC treatment.^15,18,19 This study indicates that letrozole may have a significant positive impact on endometrial perfusion and increase uterine receptivity through improving the endometrial thickness and blood flow by sparing estrogen receptors from the depletion effect caused by CC. Letrozole can be used as the first choice for induction of ovulation in cases of PCOS or it may be used in cases of CC resistance or failure. However, larger randomized, controlled studies are required to confirm the effectiveness of the use of AIs for ovulation induction. These advantages, if confirmed, suggest that AIs may be a valuable option to replace CC in the future as the new primary treatment for ovulation induction.

Disclosure Statement

No competing financial interests exist.

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