Progesterone administration for luteal phase deficiency in human reproduction: an old or new issue?

The uterus differentiates into an altered state when implantation-competent blastocysts are ready to initiate implantation. This state is called uterine receptivity for implantation, i.e. the implantation window, and lasts for a limited time. At this stage, the uterine environment is conducive to blastocyst growth, attachment and the subsequent events of implantation. The main hormones involved in uterine receptivity are the ovarian steroids, progesterone and estrogen. Uterine sensitivity to implantation is classified into pre-receptive, receptive and non-receptive (refractory) phases. In humans, the endometrium is classified histologically and functionally into proliferative and secretory phases during the average 28–30 day menstrual cycle. During the secretory phase, the uterus is considered pre-receptive for the first ~7 days following ovulation; it then becomes receptive during the mid-secretory phase, which spans 7–10 days after ovulation; the non-receptive (refractory) phase comprises the rest of the secretory phase [7].

Molecular and genetic evidence indicates that locally produced signaling molecules, including cytokines, growth factors, homeobox transcription factors, lipid mediators and morphogens, together with ovarian hormones serve as autocrine, paracrine and juxtacrine factors to obtain uterine receptivity [8]. Therefore, the implantation mechanism includes the cooperation of several factors, such as the integrins on both blastocyst and receptive endometrium surfaces, progesterone which stimulates the endometrium receptivity and the decidulization of stroma and a series of cytokines, such as IL-1 and EGF, which participate in the regulation of endometrial immune tolerance, expression of integrin nd prostaglandins production [7, 8].

The corpus luteum is derived from the transformation of granulosa and theca cells into luteal cells in response to the mid-cycle surge of gonadotropins or to an exogenous human chorionic gonadotropin (hCG) bolus administration. The most important function of the corpus luteum is progesterone secretion which is necessary to obtain a secretory transformation of the endometrium and to sustain the early pregnancy. Progesterone prepares the endometrium for pregnancy by stimulating proliferation in response to hCG. This occurs in the luteal phase of the menstrual cycle.

During the midluteal phase of a natural cycle, progesterone is involved in the modulation process of the expression of ultrastructural hallmarks of secretory transformation, such as giant mitochondria, subnuclear glycogen deposits, pinopodes and nucleolar channel system (NCS) [9]. With the aim of inducing endometrial competence/receptivity, progesterone can also act by stimulating the immune system to produce non-inflammatory T-helper 2 cytokines and C3-C4, as shown in patients with autoimmune diseases [10, 11], increasing nitric oxide production, with improvement of the blood flow and oxygen to the endometrium [12]. Progesterone is also able to reduce the contractility of the myometrium at the time of the implantation [13].

The crucial role of the corpus luteum in human reproduction in the maintenance of early pregnancy is demonstrated by the harmful effects of a lutectomy during the first weeks of a pregnancy. Initial studies on corpus luteum function demonstrated that, in patients at 7 weeks of pregnancy, after a tubal ligation, the plasma progesterone levels were normal and pregnancy continued but, after a tubal ligation plus lutectomy, the plasma progesterone levels dropped to near zero within 5 days and the pregnancy was aborted [14]. In this week of gestation, after tubal ligation and lutectomy, progesterone replacement was effective in maintaining the pregnancy. At and after 8 weeks of amenorrhea, tubal ligation plus lutectomy decreased plasma progesterone levels until day 4 but these then increased to pre-treatment concentrations with successful continuation of pregnancy [14].

This “genuine” endometrial competence, however, can be negatively affected in clinical practice by several functional diseases, such as polycystic ovarian syndrome (PCOS) and obesity [15] and anatomic abnormalities, such as congenital uterine anomalies [16], endometrial polyps [17], myomas (submucosal) [18], hydrosalpinx [19] and endometriosis [20]. In fact, the treatments of these diseases have been discussed as potential therapies to improve fertility and clinical pregnancy rate in spontaneous and/or in in-vitro fertilization (IVF) cycles. The pregnancy rate was found to be two-fold higher [relative risk (RR) 2.0, 95 % confidence interval (CI) 1.1 to 3.9] after myomectomy of submucosal myomas [18] or after polipectomy of 3–24 mm endometrial polyps (RR 2.1, 95 % CI 1.5 to 2.9) [21]. Laparoscopic salpingectomy for hydrosalpinges versus no-surgical treatments improved the clinical pregnancy rate by more than two-fold [odds ratio (OR) 2.49, 95 % CI 1.60 to 3.86) [19]. Finally, compared with diagnostic laparoscopy, laparoscopic surgery for endometriosis was associated with a significant improvement of the clinical pregnancy rates (OR 1.89, 95 % CI 1.25 to 2.86) [20].

Endometrial competence can be assessed using different methods, such as histology, timing, ultrasound and biomarkers.

In consideration of the LPD definition, histological evaluation of the endometrium has been considered the gold standard procedure to study endometrial competence. The histological method is based on a careful examination of an endometrial biopsy, and on the definition of the histologic characteristics of a secretory endometrium and describing the temporal responses to progesterone, i.e. endometrial dating. An endometrium is considered out of phase when there is a lag of more than 2 days [22]. Over the years, the histological method has developed more specific instruments, such as scanning electron microscopy [23, 24] and the immunohistochemical analysis of estrogen and progesterone receptors [25]. However, randomized clinical trials (RCTs) suggest that endometrial biopsy is an inaccurate tool for differentiating fertile women from women with LPD and infertility [1].

The timing as criteria of endometrial competence is based on LH peak, ultrasound demonstration of ovulation, basal body temperature shift and menstrual flow after the biopsy but it also presents a wide inter- and intra-subject variability [1, 26].

Ultrasound assessment is still used to evaluate endometrial competence though no significant correlation has been seen between endometrial measurement (>7 mm) and the pregnancy rate; moreover, the triple-layered pattern is visible in the same rate in infertile and fertile women (91 % vs. 90 %, respectively) [27]. In order to increase accuracy of the ultrasound method and to find a predictor sign of pregnancy during IVF-treatment, the power-Doppler was proposed as the instrument for evaluating endometrial competence, but no statistically significant differences in the vascularization indexes of endometrial and sub-endometrial blood flows were found between pregnant and non-pregnant groups [28].

Several molecular markers of endometrial competence have been discovered in blood and uterine fluid analysis and endometrial biopsy, such as estradiol, progesterone, pinopodes, glycodelin, IL-1 system, cytokines, integrins and HOXA genes [29]. Serum progesterone assay has been proposed as surrogate of endometrial competence but it has been observed that no minimum progesterone levels can define a “fertile” luteal phase [30] because progesterone concentrations may fluctuate up to eightfold within 90 min [31] and its levels peak 6 to 8 days after the ovulation [32].

Among the over one hundred different genes identified, all potential biomarkers of the implantation window, few studies exist with adequate power and validity based on study design to help determine which biomarkers have the greatest value and consistency; therefore, no reliable methods to assess “receptivity” have been established or adequately validated [33]. Nevertheless, the transcriptomic approaches and microarray technology make it possible to identify biomarkers of endometrial receptivity and to report modifications related to the gene expression profile associated with the transition of the human endometrium from a pre-receptive to a receptive state during a natural cycle and, most of all, to reveal either moderate or strong alterations of endometrial receptivity under controlled ovarian stimulation (COS) protocols [34]. In fact, a study analyzed the endometrial gene expression profiles by paired samples from the same patients during the pre-receptive to the receptive transition both in a natural and a subsequent stimulated cycle [34]. This study has shown that COS regimens affect the transcriptomic pattern of endometrial cells in comparison with natural cycles; in particular, there were numerous differences in the main systems involved in the implantation process, such as the TGFb signaling pathway, the complement and coagulation cascades and leukocyte transendothelial migration [34]. This information could open new perspectives, particularly in patients with multiple implantation failures [34]. A recent study revealed that some cases of repeated implantation failure could be associated with an aberrant gene expression profile, particularly of transcripts related to the immune function and complement activation, and altered progesterone signaling might be an underlying mechanism for such endometrial gene expression deregulation in women with repeated implantation failure [35]. Other research has been focused on the family of fibroblast growth factors (FGFs), which affects embryo implantation and supports improved endometrial trophoblastic interaction [36]. Compared with the fertile group, FGF-1 is not expressed strongly enough in the failed IVF patients, which may have caused a lack of endothelial cell migration, important for implantation, stopped the process of blood vessel formation or induced early vascularization of implantation problems [37].

However, in the current state, no type-IA evidence has been proposed by the main scientific societies for assessing endometrial competence in infertile couples. In fact, the Royal College of Obstetricians & Gynecologists (RCOG) guidelines for infertility do not recommend the use of basal body temperature charts nor an endometrial biopsy to evaluate the luteal phase in routine infertility investigation [38]. The blood test measurement of serum progesterone in the mid-luteal phase of the cycle is recommended but with type-B evidence [38]. At the same time, according to the recommendation of the American Society for Reproductive Medicine (ASRM), no diagnostic test for luteal phase insufficiency has been proven to be reliable in a clinical setting [1].

The discussion regarding the clinical role of LPD is mainly due to the lack of reproducible and reliable diagnostic tests. Nevertheless, LPD is a common condition, that occurs during a series of clinical patterns characterized by low follicular-stimulating hormone (FSH) levels, altered follicular FSH/luteinizing hormone (LH) ratio and/or abnormal FSH and LH pulsatility, such as functional hypothalamic amenorrhea, thyroid and prolactin disorders [39], obesity and PCOS [15] and during COS for IVF cycles [40]. Therefore, the correct approach to the treatment of LPD is the identification and correction of any underlying condition. On the other hand, in case of no underlying dysfunction, the treatment becomes empiric [1].

Notwithstanding the empiricism of the LPD treatment, there are different clinical conditions where luteal phase support may be more or less useful, and can drive the choice for a specific luteal phase support.

In spontaneous cycles there are no direct data on the efficacy of luteal phase support for improving fertility. A recent Cochrane meta-analysis on miscarriage prevention showed no statistically significant difference between women receiving progestogens (either natural and synthetic) and those receiving only placebo or no treatment (OR 0.99, 95 % CI 0.78 to 1.24) [41]. The same meta-analysis found a statistically significant reduction (OR 0.39, 95 % CI 0.21 to 0.72) in miscarriage rate after progestogen administration only in women with recurrent miscarriage, i.e. three or more consecutive miscarriages. Moreover, these findings deserve further studies because the trials included were of poorer methodological quality [41]. Also for the treatment of threatened miscarriage, there is insufficient evidence to support the routine use of progestogens (whether natural or synthetic) [42]. In conclusion, in natural and unstimulated cycles, no treatment for LPD has been shown to improve pregnancy outcomes [1].

The non-gonadotropin induced ovulatory cycles mainly use clomiphene citrate (CC), while aromatase inhibitors (AIs) and metformin are still considered experimental treatments and to be employed in selected cases, respectively.

CC is a nonsteroidal selective estrogen modulator with both estrogenic and anti-estrogenic properties that interferes with the normal feedback mechanisms and leads to increased and prolonged FSH and LH secretion, which in turn stimulates follicular development. Cumulative CC administrations induce weak and prevalent anti-estrogenic effects on sensitive tissues, such as endometrium and/or ovary-related luteal phase defect and/or folliculogenesis alterations [43]. The competitive binding of the CC with estrogen receptors makes the estrogenic sensitive organs less-sensitive to endogenous and exogenous steroid [43]. Moreover, CC administration appears to be related to a reduced perifollicular vascularization and to a rate of high grade follicles significantly lower than in healthy controls [44]. Also the vascularization of corpus luteum is significantly lower and with higher resistance in CC cycles, considering this hypo-vascularization of corpus luteum as a possible LPD cause [44].

To this regard, several data are available in literature about luteal phase progesterone support after ovulation induction in intrauterine insemination (IUI) cycles. A recent meta-analysis concluded that progesterone support did not benefit the clinical pregnancy rate in patients undergoing ovulation induction with CC (RR 0.89, 95 % CI 0.47 to 1.67) nor a significant difference in miscarriage per cycle between the two groups (OR 1.03, 95 % CI 0.52 to 2.04) [13]. Of note, no heterogeneity in the findings obtained was detected and there were no data on live birth to perform data synthesis. The same results were obtained in another recent meta-analysis [45]. In CC-stimulated cycles, progesterone administration did not improve the reproductive outcomes, such as the live birth rate (RR 1.30, 95 % CI 0.68 to 2.50) and the clinical pregnancy rate (RR 1.17, 95 % CI 0.82 to 1.65) and no differences were observed regarding miscarriage rate between the progesterone-treated and not-treated groups in the CC stimulation protocol (RR 1.14, 95% CI 0.63 to 2.06) [45].

In CC-stimulated cycles, the combined administration of estrogens and progesterone was tested in clinical studies showing not univocal results. In fact, endometrial estrogen receptors are blocked by competitive binding of the CC. Thus, they could be not stimulating by estrogens. Moreover, a RCT demonstrated that ethinylestradiol administration from day 8 for 5 days (0.05 mg daily) significantly improves the efficacy of progesterone support administered intramuscularly [46]. Similarly, another RCT showed a significant reproductive benefit of oral estradiol administration (3 mg daily in two administrations from cycle day 8 until ovulation) followed by vaginal progesterone in CC cycles [47]. Finally, a very recent randomized, double-blind, placebo-controlled trial compared in PCOS women the clinical pregnancy rate between two groups treated with CC plus ethinyl estradiol and CC plus placebo, respectively. The study resulted in an increase of clinical pregnancy rate in the CC + EE group (29 % vs 10 %, p = 0.02) even if there was no statistically significant difference in the ongoing pregnancy rate between the two groups [48]. However, an interesting recent retrospective cohort analysis revealed that luteal phase progesterone supplementation in CC-IUI cycles can improve endometrial receptivity with an effect closely related to the endometrial thickness [49]. Patients who appeared to receive the greatest benefit of progesterone supplementation had an endometrial thickness of 6–8 mm; their clinical pregnancy rate was found to be improved two-fold (OR 2.04; 95 % CI 1.01 to 4.14) [49]. These patients seem to have an endometrium still receptive to progesterone administration, whereas patients with an endometrial thickness less than 6 mm are not responsive to progesterone supplementation for CC-related receptors depletion/inhibition and patients with an endometrial thickness greater than 8 mm represent a group with good reproductive prognosis in which progesterone supplementation is unable to provide further reproductive improvement.

Metformin improves the regular menstrual cycles and increases ovulation rate in patients with PCOS, although the efficacy of the drug is extremely variable both between different PCOS populations and within the same population [50]. The efficacy of metformin in inducing ovulation in patients with PCOS is probably due to a direct action of the drug on the ovary; in fact, the ovulatory response to the drug seems to be related more to local drug sensitivity or resistance than to improvements in the systemic hormonal and/or metabolic environments [51], as shown by the analysis of follicular fluid that seemed to confirm that metformin acts directly on the ovary, improving local levels of androgens, ovarian insulin resistance and the levels of several growth factors [50]. As regards the use of metformin as ovulation inductor, unfortunately few and no direct data exist concerning the need of a luteal phase support. Under metformin treatment, the hormonal pattern and the ovarian dynamics of the ovulatory cycles were found to be similar to spontaneous cycles as observed in normo-ovulating controls [52]. In addition, the preovulatory follicles obtained under metformin treatment had a vascularization similar to that observed during natural cycles of healthy women, and the rate of high grade preovulatory follicles observed in women with PCOS who ovulated with metformin was not significantly different from controls [44]. Finally the vascularization around the corpus luteum was found to be similar between metformin and spontaneous cycles, confirming the beneficial effects of metformin on the corpus luteum function [44].

Letrozole is an aromatase inhibitor recently employed as ovulation inducer. A recent large multicenter randomized double-blind parallel controlled trial, published in 2014, demonstrated the superiority of letrozole as first-line therapy for anovulatory infertility in women with PCOS when compared with CC [53]. A systematic review of RCTs with meta-analysis concluded that letrozole is associated with significantly higher live birth rates than with CC (OR 1.64, 95 % CI 1.32 to 2.04) and with significantly higher clinical pregnancy rate compared to CC (OR 1.40, 95 % CI 1.18 to 1.65) even if the quality of the evidence was rated as low [54]. Letrozole acts by inhibiting the aromatase, a cytochrome P450 enzyme complex which is responsible for androgen to estrogen conversion, so it induces a hypoestrogenic state that stimulates, through activation of hypothalamic-pituitary axis, increased FSH secretion and ovarian follicle development [43]. It does not exert an antagonist effect on endometrial estrogen receptors; on the other hand suppression of the systemic estrogen levels and in peripheral tissues can result in up-regulation of the estrogen receptors in the endometrium, leading to rapid endometrial growth once follicle development starts and estrogen secretion is restored [43].

In non-IVF cycles with gonadotropins COS, two recent meta-analyses [45, 46] have demonstrated the benefit in improving reproductive outcomes with vaginal progesterone use as luteal phase support. Specifically, in the first meta-analysis, the clinical pregnancy rate was increased to 70 % (OR 1.77, 95 % CI 1.20 to 2.60), as well as the likelihood of live birth per cycle (OR 2.63, 95 % CI 1.42 to 4.80), in patients receiving progesterone support [45]. In the second meta-analysis, patients treated with FSH showed higher biochemical pregnancy (RR 1.81, 95 % CI 1.36 to 2.43), clinical pregnancy (RR 1.57, 95 % CI 1.15 to 2.15) and live birth (RR 2.28, 95 % CI 1.49 to 3.51) rates on receiving progesterone supplementation [46]. No differences were observed regarding miscarriage rate between the two study groups (progesterone treated and not-treated groups) [46]. In both meta-analyses [45, 46], data heterogeneity was low and not significant.

To the regard of the optimal progesterone dosage to use in non-IVF COS cycles with gonadotropins, very few clinical evidences are available in literature. A very recent RCT, evaluating two doses of vaginal progesterone for IUI cycles in terms of pregnancy rates, demonstrated that 300 mg of intravaginal micronized progesterone should be the maximum dosage for luteal phase when compared with 600 mg [55].

To explain in detail the role of progesterone and/or progestogens for the luteal phase support in gonadotropins COH for IVF cycles, the issue should be approached considering conventional and new protocols of gonadotropins COH separately.

Due to increased mean age of maternity in developed countries, IVF using donor oocytes is an increasingly used infertility treatment option for women with irreversible ovarian function loss or primary ovarian failure [132] showing a pregnancy and implantation rates in oocyte donation cycles higher than in standard IVF/ICSI cycles [133]. In these cases, the support of the luteal is an essential prerequisite. In addition, as explained above, more and more frozen-thawed embryo transfers (FET) are employed with the aim of avoiding OHSS in high-risk patients. FET makes it possible for the embryos generated by IVF and ICSI to be stored and utilized later, with a segmentation of the IVF cycle, separating the ovarian stimulation and trigger from the fresh transfer, and vitrifying all embryos for subsequent transfer in frozen thawed cycles.

Moreover, not only for reducing the OHSS risk, in recent years the amount of FET cycles has dramatically increased due to the trend towards transferring fewer embryos after a fresh IVF cycle, and as a result of improved laboratory techniques [134, 135] until to the emerging “freeze-all” policy as an alternative to fresh embryo transfer (ET) to improve IVF outcomes [106]. A recent prospective, observational, cohort study has suggested that the “freeze-all” policy is advantageous when compared with fresh ETs [136]. Clinical (RR 1.29, 95 % CI 1.05 to 1.59) and ongoing pregnancy (RR 1.28, 95 % CI 1.01 to 1.62) rates were significantly better after FET [136]. In addition, a recent meta-analysis of observational studies has shown that singleton pregnancies after FET are associated with a lower risk of obstetric outcomes, such as small for gestational age baby (RR 0.45, 95 % CI 0.30 to 0.66) and preterm birth (RR 0.84, 95 % CI 0.78 to 0.90) [137].

Unlike the complex ovarian stimulation protocols of IVF and ICSI cycles, in the FET cycles, the primary target is limited to adequate preparation of the endometrium to receive the thawed embryos. The endometrial preparation can be achieved by means of two different methods, a natural cycle (NC) which consists of using endogenous sex steroids production from developing follicles in patients with regular ovulatory cycles and the artificial cycles (AC), where the endocrine preparation of the endometrium is achieved by sequential administration of estrogens and progestogens to mimic a normal cycle, more suitable for women without regular ovulatory cycles. The entire success of the NC-FET cycle depends on the correct identification of ovulation and calculation of the likely subsequent period of optimal endometrial receptivity [138, 139]. The identification of ovulation is based on detecting the spontaneous LH surge, therefore LH levels need to be regularly (daily) monitored in blood or urine samples, because it is assumed that ovulation will occur 36 – 40 h after LH peak [140]. Unfortunately, the LH surges in urine lag up to 21 h behind the appearance of the blood surge, creating difficulties in correct assessing of data. To overcome these problems, a modified-NC (mNC) has been proposed, which consists of hCG triggering when dominant follicle is of optimal size (>17 mm), after an ultrasound evaluation to ensure the appropriate timing of hCG administration.

In the AC-FET the timing of embryo thawing and transfer is planned according to the moment of progesterone supplementation but the sequential administration of exogenous sex steroids does not guarantee complete pituitary suppression (at least 5 % of early luteinizations, [141]), putting the endometrium at risk of early exposure to progesterone. Also for the AC cycle, a modified AC-FET (mAC) has been proposed consisting in the co-treatment with GnRH agonist in order to down-regulate the pituitary and prevent early follicular luteinizations [142].

A recent meta-analysis regarding the optimal endometrial preparation method in FET revealed no significant advantage of one specific approach to prepare the endometrium for FET in terms of clinical pregnancy rates or live birth rates [142]. In particular, no significant benefit of true NC-FET versus modified NC-FET with regard to clinical pregnancy (OR 0.91, 95 % CI 0.74 to 1.10), ongoing pregnancy (OR 1.0, 95 % CI 0.66 to 1.60) or live birth (OR 1.0, 95 % CI 0.63 to 1.60) was observed. Also luteal phase support was evaluated as potential influencing factor on pregnancy rate but the meta-analysis concludes that currently there is too little evidence supporting a positive effect of luteal phase support in patients undergoing NC-FET [132]. More recently, a retrospective study evaluating 228 patients showed that progesterone for luteal phase supplementation decreases miscarriage rate and improves live birth rate in NC-FET cycles [143] but, due to different study designs, the overall conflicting results of the available studies, do not make it possible to conclude a positive effect of luteal phase support in patients undergoing NC-FET [144].

Other issues of discussion concerned the different timing of progesterone starting and routes of administration in donor and/or FET cycles. A Cochrane review [145] analyzed the most effective endometrial preparation for women undergoing embryo transfer with frozen embryos and with embryos derived from donated oocytes. Starting progesterone on the day of oocyte aspiration (OR 1.92, 95 % CI 1.08 to 3.42) or on the day after (OR 1.81, 95 % CI 1.01 to 3.24) led to higher pregnancy rate than when progesterone was started the day before oocyte retrieval [145]. However, no difference was found between starting progesterone the day of oocyte aspiration or the day after (OR 0.94, 95 % CI 0.53 to 1.68) [95, 145]. Finally, no differences were found (OR 0.75, 95 % CI 0.24 to 2.34) between starting one or two days before the oocyte retrieval when the transfer is performed on blastocyst stage [145].

As regards progesterone route of administration, no statistical significant difference was detected in live-birth and clinical pregnancy rate between vaginal and intramuscular administration [145]. On the contrary, in a retrospective cohort study [146] women supplemented with vaginal progesterone gel had significantly lower rate of clinical pregnancy (36.9 % vs. 51.1 %, respectively) and live birth (24.4 % vs. 39.1 %, respectively) compared with intramuscular progesterone. The benefit of intramuscular route could be explained with the higher and continuous serum progesterone levels that act relaxing uterus and reducing the frequency of endometrial waves in the luteal phase that are associated with lower pregnancy rates [73]. Moreover, the booster injection of intramuscular progesterone (50 mg IM, once every 3 days) did not improved the pregnancy rates of patients who received vaginal progesterone (100 mg, three times daily) [147].

The worst results in vaginal progesterone group probably depend on the dosage of vaginal progesterone. In a retrospective study [148] patients treated with vaginal progesterone gel twice daily had a lower risk of pregnancy loss compared with women treated once a day (43.7 % vs. 67.4 %, respectively) resulting in a delivery rate (20.5 % vs. 8.7 %, respectively) more than two-fold higher [148]. However, considering recipients who received vitrified blastocysts on day 6, no statistical differences were seen in implantation and pregnancy rate between vaginal and intramuscular progesterone luteal phase support [149].

Very few data are available regarding the best dosage of progesterone administration. A recent study seems to demonstrate that the optimal dose of intramuscular progesterone to for luteal phase support ranges between 50 and 100 mg/day [150]. In fact, that dose lets to reach progesterone blood levels similar to those observed during the mid-luteal phase of a spontaneous ovulatory cycle. Moreover, an increase in progesterone dosage could be useful in donor and/or frozen-thawed AC cycles when the endometrial pattern at ultrasound is non-homogeneous hyperechoic in mid-luteal phase [151].