Scholarly article on topic 'Modern management of thin lining'

Modern management of thin lining Academic research paper on "Basic medicine"

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{"Thin endometrium" / "Endometrial thickness" / "Endometrial pattern" / "Pregnancy rates" / G-CSF / "Stem cell therapy"}

Abstract of research paper on Basic medicine, author of scientific article — Youssef Mouhayar, Fady I. Sharara

Abstract Objective To define “thin” endometrium in fertility treatment, and to critically explore the available treatment options. Design A review of the scientific literature. Setting N/A. Methods An electronic literature search pertaining to patients with “thin” endometrium undergoing fertility treatment was performed through April 2016. Results Adequate endometrial growth is an integral step in endometrial receptivity and embryo implantation. Whether idiopathic or resulting from an underlying pathology, a thin endometrium of <7mm is linked to a lower probability of pregnancy; however, no reported thickness excludes the occurrence of pregnancy. Several treatment modalities have been studied and include extended estrogen, gonadotropin therapy, low-dose hCG, tamoxifen, pentoxifylline, tocopherol, l-arginine, low-dose aspirin, vaginal sildenafil, acupuncture and neuromuscular electric stimulation, intrauterine G-CSF, and stem cell therapy. All treatment modalities except vaginal sildenafil, intrauterine GCF, and stem cell therapy were inconsistent in showing significant improvement in pregnancy rates. Early results of stem cell therapy trials seem promising. Conclusions EMT <7mm is associated with lower probability of pregnancy in ART. Vaginal sildenafil appears to be a reasonable first line therapy option, and G-CSF appears to be a potential second option, while stem cell therapy seems to be a promising new treatment modality.

Academic research paper on topic "Modern management of thin lining"

Middle East Fertility Society Journal (2016) xxx, xxx-xxx

Middle East Fertility Society Middle East Fertility Society Journal

www.mefsjournal.org www.sciencedirect.com

REVIEW

Modern management of thin lining

Youssef Mouhayar a, Fady I. Sharara bc*

aDept of OB/GYN, University of Miami/Jackson Memorial Hospital, Miami, FL, United States b Virginia Center for Reproductive Medicine, Reston, VA, United States cDept of OB/GYN, George Washington University, Washington, DC, United States

Received 31 May 2016; revised 23 June 2016; accepted 4 September 2016

KEYWORDS

Thin endometrium; Endometrial thickness; Endometrial pattern; Pregnancy rates; G-CSF;

Stem cell therapy

Abstract Objective: To define "thin" endometrium in fertility treatment, and to critically explore the available treatment options.

Design: A review of the scientific literature.

Setting: N/A.

Methods: An electronic literature search pertaining to patients with "thin" endometrium undergoing fertility treatment was performed through April 2016.

Results: Adequate endometrial growth is an integral step in endometrial receptivity and embryo implantation. Whether idiopathic or resulting from an underlying pathology, a thin endometrium of <7 mm is linked to a lower probability of pregnancy; however, no reported thickness excludes the occurrence of pregnancy. Several treatment modalities have been studied and include extended estrogen, gonadotropin therapy, low-dose hCG, tamoxifen, pentoxifylline, tocopherol, l-arginine, low-dose aspirin, vaginal sildenafil, acupuncture and neuromuscular electric stimulation, intrauter-ine G-CSF, and stem cell therapy. All treatment modalities except vaginal sildenafil, intrauterine GCF, and stem cell therapy were inconsistent in showing significant improvement in pregnancy rates. Early results of stem cell therapy trials seem promising.

Conclusions: EMT <7 mm is associated with lower probability of pregnancy in ART. Vaginal sildenafil appears to be a reasonable first line therapy option, and G-CSF appears to be a potential second option, while stem cell therapy seems to be a promising new treatment modality. © 2016 Middle East Fertility Society. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Corresponding author at: Virginia Center for Reproductive Medicine, 11150 Sunset Hills Rd, Suite 100, Reston, VA, United States. E-mail address: fsharara@vcrmed.com (F.I. Sharara). Peer review under responsibility of Middle East Fertility Society.

http://dx.doi.Org/10.1016/j.mefs.2016.09.001

1110-5690 © 2016 Middle East Fertility Society. Production and hosting by Elsevier B.V.

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Contents

1. Introduction........................................................................................................................................................00

2. Materials and methods........................................................................................................................................00

3. Results................................................................................................................................................................00

3.1. Hormonal adjustment....................................................................................................................................00

3.2. Pentoxifylline and tocopherol..........................................................................................................................00

3.3. Low-dose aspirin..........................................................................................................................................00

3.4. Acupuncture, neuromuscular electric stimulation and electro-acupuncture..........................................................00

3.5. Vaginal sildenafil..........................................................................................................................................00

3.6. Granulocyte colony-stimulating factor (G-CSF)................................................................................................00

3.7. Stem cell therapy..........................................................................................................................................00

4. Discussion..........................................................................................................................................................00

Conflict of interest..................................................................................................................................................00

References ..............................................................................................................................................................00

1. Introduction

Embryo implantation is a very delicate and well-orchestrated process that is governed by the interaction between several maternal and embryonic factors, ultimately resulting in adherence of the blastocyst to the endometrium (1-5). For a short period of time during the normal menstrual cycle, the endome-trium represents the fertile "soil" for the implanting embryo (6). The human endometrium undergoes complex changes, in response to circulating estrogen and progesterone, which culminate at the mid-luteal phase of the menstrual cycle when it becomes suitable to host the blastocyst (7-9). These changes occur at the morphological, biochemical, and molecular levels; any faux pas may result in failed implantation (10-13). Identifying a receptive endometrium is essential; however, an ideal method that can reliably predict endometrial receptivity has yet to be determined. In clinical practice, high-resolution ultra-sonography is routinely used to monitor follicular development and endometrial responsiveness during controlled ovarian stimulation. Several ultrasound markers, such as endometrial thickness (EMT), pattern, blood flow impedance, and uterine volume have been evaluated for their predictive role of endometrial receptivity; however, they had low specificity and positive predictive value for detecting a receptive endometrium (14,15). Endometrial patterns, such as a triple-layer endometrium on day of human chorionic gonadotropin (hCG) trigger or oocyte retrieval and embryo transfer might be a better predictor of implantation than endometrial thickness (16-25). A certain endometrial development however, is an integral part of a receptive endometrium. Despite that a minimal endometrial thickness of 6 mm has been reported to be essential for achieving implantation in assisted reproductive technologies (ART), successful pregnancies were documented with a minimum EMT of 4 mm (26-29). In fact and up to this date, there is no consensus on a cutoff value of an EMT below which implantation rates even decline in ART. Using receiver operating characteristics (ROC) area under the curve, a cutoff limit of endometrial thickness (on day of hCG trigger) above which implantation could be predicted was not detected by three reports, whereas two studies reported a threshold thickness of 8 mm (30-34). While several reports showed that endometrial thickness has a predictive value for successful pregnancies in ART, others have demonstrated the opposite

(18,22,35-58). Moreover, most of the reports used a cutoff EMT of 7 mm while others used 6 mm, 8 mm, 9 mm, or 10 mm (18,32-34,38,40,44,46-52,56-60).

Several mechanisms have been proposed to explain the underlying pathophysiology of thin endometrium. Intrauterine adhesions, ovarian stimulation with clomiphene citrate (CC), as well as prolonged use of progesterone, or combined oral contraceptive pills have been associated with thin endome-trium (61-63). Vascular epithelial growth factor (VEGF), which plays a critical role in angiogenesis, appears to be an important factor in the pathophysiology of thin endometrium. Adequate endometrial development, which is hormonally mediated, is highly dependent on adequate blood supply. Miwa et al. elegantly described that with increased impedance across the radial uterine arteries, there is resultant decrease in VEGF expression and subsequent poor vascular development, resulting in thin endometrium (64).

In 2008, Senturk et al. reviewed thin endometrium in ART and the available treatment modalities (extended estrogen administration, vaginal sildenafil, vitamin E, pentoxifylline, and luteal phase GnRH-a supplementation), and concluded that these were ineffective (65). More recently, Lebovitz et al. concluded that the treatment of "thin" endometrium remains a challenge, with only minor improvements achieved with the currently available treatment modalities (66). A recent systematic review and meta-analysis of 10,724 cases showed that EMT as an independent variable is not predictive for the occurrence of pregnancy (67). This meta-analysis however, found that the most commonly reported cutoff of 7 mm occurred in only 2.4% of the cases (260/10,724), and this cutoff was associated with a significant drop in the probability of pregnancy (67). We have elected to review this topic as new treatment modalities emerge. Here we provide a more thorough review of the available literature to date, aiming to define "thin" endometrium in infertile patients, and to critically analyze the proposed treatment modalities.

2. Materials and methods

We performed a review of the literature on thin endometrium, its pathophysiology, and treatment. Abstracts, case reports, original, and review articles were considered. A computer-based systematic literature review was performed through

April 2016. The Cochrane database, PubMed, Medline registries, and other online sources were searched using the broad terms: thin endometrium, thin endometrium and IVF, thin endometrium and ART, and treatment of thin endometrium. The titles and abstracts were screened for relevance, and relevant articles were analyzed in detail to determine which studies could be included in the review. Furthermore, the references cited in relevant studies and review articles were hand searched to identify further relevant studies. Studies were considered eligible if they were conducted to assess or compare different treatment modalities of thin endometrium. Changes in mean endometrial thickness and pregnancy rates were the two principle summary measures, and were independently extracted from individual studies.

3. Results

3.1. Hormonal adjustment

Endometrial proliferation is dependent on serum estrogen levels, which results in progressive growth of the functional endometrium in the proliferative phase. On this basis, patients with thin endometrium were offered supplemental exogenous estrogen. Studies were mostly in frozen-thawed embryo transfer (FET) cycles and in vitro maturation (IVM). In 2006, Chen et al. randomized 36 patients with endometrial thickness <8 mm to either extend estrogen treatment followed by FET, or to proceed directly with embryo transfer (68). The study group received oral estradiol valerate for 14-82 days, followed by embryo transfer when the EMT was greater than 8 mm. The endometrial thickness was significantly higher after treatment in patients who received the estradiol treatment (8.6 ± 0.7 mm vs. 6.7 ± 0.9 mm, P = 0.031), with a significantly higher pregnancy rate than in patients who proceeded with embryo transfer (38.5% vs. 4.3%, P = 0.016). In this study however, the EMT was similar in patients who conceived compared to those who did not, in both the study and control groups (68). Shen et al. presented a case of a 37 year-old woman with a refractory thin endometrium (<6 mm) who was treated with extended estrogen supplementation for 9 days followed by controlled ovarian hyperstimulation, and this combination resulted in a live twin birth at 36 weeks (69).

Poor endometrial development has also been suggested to be a reason behind the lower pregnancy rates in IVM cycles. To improve endometrial development in IVM cycles, Elizur et al. compared the administration of low-dose human meno-pausal gonadotropin (hMG) and micronized 17b-estradiol supplementation in IVM cycles with thin endometrium (70). The authors noted a significant improvement in endometrial thickness from <6 mm to 7.7 ± 1.8 mm and 7.3 ± 1.7 mm with low-dose hMG and 17b-estradiol, respectively, without a significant difference between the two groups. There was no difference in pregnancy rates between the two arms of the study, nor was there an untreated control group for comparison (70).

Luteal phase support with gonadotropin-releasing hormone agonists (GnRH-a) is another form of hormonal manipulation that was employed in patients with thin endometrium undergoing IVF. Qublan et al. prospectively randomized 120 patients undergoing IVF with thin endometrium (67 mm), to either GnRH-a or placebo during luteal support (71). Sixty patients

received 0.1 mg of Triptorelin on days of egg retrieval, embryo transfer, and 3 days after embryo transfer, while the remaining 60 patients served as the control group. EMT increased from 6.89 ± 0.24 mm to 8.92 ± 1.6 mm in the study group which was significantly higher than the increase noted in the control group which was from 6.83 ± 0.26 to 7.12 ± 0.45 mm (8.92 ± 1.6 mm vs. 7.12 ± 0.45 mm, P < 0.05). Pregnancy rate was also significantly higher in the study group vs. the control group (36.6% vs. 13.7%, P < 0.01) (71).

Adding low-dose human chorionic gonadotropin (hCG) during endometrial preparation with estradiol was investigated as well. In a pilot study by Papanikolaou et al. seventeen patients with resistant thin endometrium (<7 mm) during fresh or frozen donor-embryo cycles were recruited (72). 150 IUs of hCG were administered daily for seven days starting on days 8 or 9 of estrogen administration. The mean EMT increased from an average of 5.18 mm to 6.01mm (P = 0.008); however, 5 of 17 patients (29.4%) did not experience any improvement in EMT. The pregnancy rate following treatment was 52.9% (9/17 patients), including pregnancies in two patients who had not improved their EMT after treatment (72).

Clomiphene citrate (CC) has been widely utilized for ovulation induction. Similar to other selective estrogen receptor modulators (SERMs), CC has mixed estrogenic and anti-estrogenic effects along the hypothalamic-pituitary-gonadal-u terine axis (73). In fact, CC's positive effects in treating infertility by inducing ovulation are counteracted by its negative effects on endometrial proliferation (74-76). Tamoxifen (TMX) on the other hand is another SERM that is similar to CC but has an estrogen agonist effect at the level of the endometrium, and on this basis it was suggested that TMX could be used for ovulation induction (77). In a prospective trial, Wang et al. had 131 patients with various infertility diagnoses who had failed to develop an EMT of 8 mm (78). Patients were allocated to either TMX or CC with alternating gonadotropins. Sixty-one patients (81 cycles) received 40 mg of tamoxifen daily on days 3-9 and 70 patients (82 cycles) received 100 mg CC daily on days 3-7 with alternating hMG (150 IUs). The TMX group had a significantly thicker EMT on day of hCG trigger vs. CC with alternating hMG (10.8 ± 2.3 mm vs. 6.7 ±1.3 mm, P < 0.001), as well as a higher clinical pregnancy rate following intrauterine insemination (32.1% vs. 15.9%, P = 0.015) (78). In 2010, Reynolds and colleagues retrospectively reviewed 19 women who had failed to develop an EMT of P 7 mm during OI with CC and who were switched to OI with TMX (79). The mean EMT, measured on either day 12 or 14 of the cycle, increased from 5.5 ± 0.8 mm to 8.8 ± 1.3 mm, P < 0.001 in the TMX treated cycles with a pregnancy rate of 42.1% (79). A recent prospective randomized trial by Morad et al. compared OI with TMX versus CC plus vaginal sildenafil in 65 patients with EMT < 7 mm in a previous OI cycle with CC only (80). Of those, 34 were randomized to receive TMX 40 mg daily on days 3-7 of the cycle, and 31 patients to receive 100 mg of CC daily with 25 mg of vaginal sildenafil four times per day on days 3-7 of the cycle. EMT, measured on day of hCG trigger, significantly increased in both groups in comparison with the previous CC only cycles: from 5.7 ± 1.2 to 8.81 ± 1.32, P < 0.05 in TMX group and from 5.5 ± 1.4 mm to 9.3 ± 1.2 mm; however, there was no difference in pregnancy rates between the two groups after IUI (18.75% vs. 27.59%, P > 0.05) (80). Most

recently Chen et al. reported three cases of recurrent unresponsive thin endometrium (<6 mm) who were successfully treated with TMX to increase their EMT (81). Patients were undergoing programmed FET, and TMX was used to simulate endometrial proliferation. Patients were given 20 mg of TMX daily for 5 days. The endometrial thickness increased to 7.7, 7.8, and 8.1 mm in the first, second, and third patient respectively, 7-11 days after TMX withdrawal with three clinical pregnancies and two healthy deliveries (81).

3.2. Pentoxifylline and tocopherol

Pentoxifylline (PTX) is a methylxanthine derivative used to treat vascular diseases. In vivo, it has been reported to increase erythrocyte flexibility, to vasodilate, and to inhibit inflammatory reactions and tumor necrosis factor (TNF) (82). PTX and tocopherol (Vitamin E) combination has been reported to improve endometrial thickness in patients with radiation-induced thin endometrium, and was therefore tried in oocyte recipients with thin endometrium unresponsive to estradiol therapy. In 2002, Ledee-Bataille et al. administered PTX and tocopherol (400 mg and 500 IU respectively twice daily) to 18 oocyte-donor recipient patients with thin endometria (66 mm) who had failed to respond to micronized vaginal estradiol (83). After six months of treatment, the EMT significantly increased by 1.3 mm ± 1mm. The pregnancy and delivery rates were 33% and 27%, respectively. However, there was no significant difference in the endometrial thickness between those who conceived and those who did not, before or after the treatment (83). The mechanism of action of this combined treatment on endometrial thickness and pregnancy outcome is unclear; however, it is hypothesized that it inhibits inflammatory reactions and decreases TNF-alpha levels. In a case series by Letur-Konirsch and Delanian, three oocyte-donor recipients with estrogen-resistant thin endometria (mean EMT 4.9 mm) were treated with combined PTX and toco-pherol (800 mg and 1000 IU respectively) for 9 months (84). There was an increase in the endometrial thickness to a mean of 7.4 mm, and 2 of 3 patients conceived (84). Acharya et al. also administered combined PTX and tocopherol (800 mg and 1000 IU respectively) to 20 infertile patients with thin endometrium over an average duration of 8.1 months (85). There was a significant increase in EMT at the end of the treatment (4.9 ± 1.5 mm vs. 7.4 ± 0.9 mm, P = 0.001) resulting in a 40% pregnancy rate (85).

The effects of administering vitamin E alone were also studied. In a prospective observational trial, 600 IU of Vitamin E daily significantly increased the endometrial thickness in 52% of patients with an EMT < 8 mm (7.2 mm vs. 8.3 mm, P < 0.05) (86). In that same trial, L-Arginine (6 grams/day) treatment was also successful in six out of 9 (67%) patients (7.4 mm vs. 8.0 mm, P < 0.05). In a prospective randomized controlled trial by Cicek et al. 400 IU of vitamin E was administered to patients with unexplained infertility during controlled ovarian hyperstimulation (COH) (87). There was a significant increase in EMT to 9.6 ±2.1 mm vs. 8.2 ± 2.0 mm in patients who received vitamin E with clomiphene citrate for ovulation induction compared to those who did not (P = 0.001). However, there were no significant differences in implantation rates and ongoing pregnancy rates between the two groups (87).

3.3. Low-dose aspirin

Low-dose aspirin is hypothesized to increase endometrial blood flow by decreasing impedance across the uterine artery. It has been previously reported that low-dose aspirin results in lower pulsatility index of the uterine artery and subsequently improves pregnancy rates (88). In their report on low-dose aspirin use in frozen-thawed embryo transfers Check and colleagues noted an increase in mid-luteal phase EMT when compared to controls (89). Weckstein et al. randomized 28 patients with thin endometrium (<8 mm) in an oocyte donation program to either low-dose aspirin (81 mg) with estrogen or estrogen only (90). There was no significant difference between the aspirin group and non-aspirin group (1.6 ± 1.5 mm vs. 0.9 ± 0.8, respectively). Despite the increased implantation rate in the aspirin group, there was only a trend for improved clinical pregnancy rate in the aspirin treatment group (60% vs. 31%). Interestingly, in those patients whose endometrial thickness remained less than 8 mm in the two groups, the clinical pregnancy rate was significantly higher for those who received aspirin (83% vs. 25%, P < 0.05) (90). In another prospective randomized trial, patients with thin endometrium (<8 mm) undergoing COH and intrauterine insemination (IUI) were randomized to either receive 100 mg of aspirin daily starting day 1 of the cycle until the pregnancy test, or no aspirin therapy (91). There were no significant differences in the mean EMT after treatment between the aspirin group and the nonaspirin group (7.2 ±1.8 vs. 5.8 ± 1.4, respectively). The clinical pregnancy rate however, was significantly higher in the aspirin group vs. non-aspirin group (18.4% vs. 9.0%, P = 0.036) (91).

3.4. Acupuncture, neuromuscular electric stimulation and electro-acupuncture

Acupuncture, one of the oldest interventions of traditional Chinese medicine, has been utilized in several obstetrical and gynecologic conditions, including infertility. Several reports were able to show that acupuncture may improve pregnancy rates in ART (92,93). Two other reports were able to show that electro-acupuncture was able to reduce blood flow impedance across the uterine artery in infertile patients undergoing IVF (94,95). In a prospective, non-randomized trial, Stener-Victorin was able to demonstrate that electro-acupuncture reduced uterine artery blood flow impedance in 10 infertile but otherwise healthy patients with baseline impedance (94). In a randomized controlled trial (RCT), Ho et al. treated thirty infertile patients with electro-acupuncture twice weekly for 4 weeks, whereas 14 other patients served as a control group (95). There was a significant reduction in the uterine artery blood flow impedance in the study compared to the control group, without a significant difference in pregnancy rates between the two groups (95). In another recent single-blinded randomized controlled trial, Shuai et al. treated patients undergoing FET, with either transcutaneous electrical acupuncture point stimulation (TEAS) or mock TEAS. EMT on day of hCG trigger was similar between the two groups (11.26 ± 1.5 mm vs. 10.74 ± 1.54 mm), whereas the study group had a higher number of triple-line endometrium (91.2% vs. 41.2%, P = 0.002), and a higher clinical pregnancy rate (44.1% vs. 20.6%, P = 0.038) (96). However, as far as we

know, there have not been any studies on improving endome-trial thickness using acupuncture.

Pelvic floor neuromuscular electrical stimulation (NMES) has been used in patients with thin lining. In their preliminary report, Bodombossou-Djobo et al. evaluated 41 subjects with an EMT 6 7 mm who failed to conceive in two prior ART cycles (97). Twenty had NMES in their subsequent FET cycle, whereas 21 proceeded directly with FET. NMES was applied daily for a total of 3-4 days starting on day 9 of the stimulation. EMT significantly increased in the treatment group compared to the control group (7.93 ± 1.42 mm vs. 6.78 ± 0.47 mm, P = 0.002) but without a significant difference in the clinical pregnancy rates between the two groups (97).

3.5. Vaginal sildenafil

Sildenafil citrate is phosphodiesterase-5 inhibitor that enhances the vasodilatory effects of nitric oxide, which in turn increases subendometrial blood flow. In 2000, Sher et al. reported four patients who had previously had an EMT between 5-7 mm in a prior failed IVF or IUI cycles, and who were all given vaginal sildenafil 25 mg four times per day for 8-12 days during COH (98). The EMT measured on day of hCG administration and was found to be between 8 and 12 mm (98). The same four patients had also undergone a prior mock cycle with leuprolide acetate and received the same daily dose of vaginal sildenafil for 7 days, then 7 days of placebo followed by vaginal sildenafil and estradiol for another 7 days. The pulsatility index decreased after treatment with sildenafil, but was back to baseline after 7 days of placebo, indicating increased diastolic blood flow after sildenafil administration (98). Two years later the same group administered vaginal sildenafil (25 mg, 4 times daily) on days 3 through 10 in 105 patients undergoing IVF who had failed to develop an EMT of 9 mm in prior failed IVF cycles (99). The patients had various causes leading to poor endometrial development, including pregnancy-related endometritis, DES anomalies, fibroids, adenomyosis, and idiopathic. Seventy-three patients (70%) experienced an increase in their EMT to greater than 9 mm, and had a significantly higher implantation and ongoing pregnancy rates when compared to those who failed to respond (29% vs. 2%, P < 0.01, and 45% vs. 0%, P < 0.01 respectively). The overall ongoing pregnancy rate in that cohort was 31.4% (33/105) (99). In a smaller trial by Check et al. 16 patients who had failed to attain an EMT of 8 mm during FET cycles were assigned to either vaginal silde-nafil or vaginal estradiol in addition to oral estradiol (100). Nine patients received 25 mg of vaginal sildenafil four times daily from days 3 to 9 with no significant change in their EMT after treatment (6.3-6.4 mm). Of the remaining seven patients who received vaginal estradiol, 6 were previously given vaginal sildenafil without improvement in EMT. The pregnancy rate was only 16.6% (1 out of 6 attempted FET) (100).

Asherman's syndrome (AS) is another cause of suboptimal endometrial development that can lead to subfertility. Zinger et al. successfully treated two cases with subfertility and thin endometrium (<7 mm) during previous stimulated cycles (oral estradiol or clomiphene citrate) (101). Vaginal sildenafil was administered for 8-15 days during the treatment cycles. One patient experienced an increase in EMT from 6.5 mm to

8.9 mm, and the second patient from 5.0 mm to 6.6 mm. Both patients had a full term healthy singleton deliveries (101). In another trial by Takasaki et al. vaginal sildenafil significantly increased the EMT in 11 out of 12 patients (92%) from 7.1 mm to 9.4 mm (P < 0.01), with a 50% pregnancy rate after 12 IVF cycles (102). Similar positive effects on improving EMT were also noted by Morad and colleagues where 31 patients experienced significant improvement in EMT from 5.5 ± 1.4 mm to 9.3 ± 1.2 mm (80). Most recently, Eid et al. administered vaginal sildenafil to 22 patients with an EMT of 7 mm and elevated pulsatility index (PI >0.3) for a total of 7 days between ovulation trigger and embryo transfer

(103). Sixty-eight percent (15/22) of patients experienced improvement in EMT and a decrease in their PI. Implantation rates and pregnancy rates were higher in patients who responded to treatment (26% vs. 7%, and 40% vs. 14%, respectively) (103).

3.6. Granulocyte colony-stimulating factor (G-CSF)

G-CSF, initially described as a hematopoietic growth factor, has been shown to have important functions in nonhematopoi-etic cells, including the endometrium, and has a potential role in promoting early endometriotic lesions in a murine model

(104). Gleicher et al. hypothesized that G-CSF might have a direct role promoting endometrial growth, and reported a case series of 4 patients with thin endometria between 3 and 6.5 mm who failed to improve with oral and vaginal estrogen as well as with vaginal sildenafil (in one of the patients) (105). All four patients had intrauterine G-CSF infusion (300 ig) 2-9 days before ET, and had a significant increase to at least 7 mm within 48 h. All four patients conceived, one of which had an ectopic (105). Two years later, the same group performed a prospective pilot cohort study of 21 patients undergoing IVF who had EMT < 7 mm on the day of hCG trigger despite oral and vaginal estrogen as well as vaginal sildenafil (106). All patients received 300 ig of G-CSF intrauterine infusion 612 h prior to hCG trigger. A second infusion was needed in only 3 of 21 patients (14.3%). Overall, EMT increased from an average of 6.4 ± 1.4 mm to 9.3 ± 2.1 mm (P < 0.001) after G-CSF treatment, but did not differ between those who conceived and those who did not. The overall clinical pregnancy rate in the cohort was 19.1% (106).

Lucena et al. reported a patient with thin endometrium (<5.7 mm) during an in vitro maturation (IVM) cycle who was given 300 ig of intrauterine G-CSF on day of oocyte retrieval (107). The EMT subsequently improved to 8.9 mm, and embryo transfer resulted in a full term delivery (107). Check and colleagues reported a patient who had multiple IVF-ET and FET cycle failures due to "thin" endometrium of 6 mm despite treatment with oral and vaginal estrogen as well as vaginal sildenafil (108). The patient was given intrauter-ine G-CSF as described by Gleicher et al. (105); however, her EMT was only 5 mm and had a negative pregnancy test after embryo transfer (108). Li et al. then reported on 69 patients who failed to develop an EMT greater than 7 mm in FET cycles despite escalating endometrial preparation protocols (109). Fifty-nine patients were included in this retrospective analysis, of which 34 opted for G-CSF treatment (28 refused), and received intrauterine G-CSF (100 ig) on the day of ovulation, or day of progesterone start, or day of hCG administra-

tion (109). The cycle cancelation rate due to thin endometrium was lowest in the G-CSF treatment group (69.39% vs. 48.75% vs. 17.5%, P < 0.05 self-controlled vs. control vs. treatment groups, respectively), and there was a trend toward better implantation (15.8% vs. 7.89%), and clinical pregnancy rates (30.30% vs. 20.0%) in the G-CSF group compared to both control groups (109). Kunicki et al. administered intrauterine G-CSF (100 ig) to 37 patients who failed to develop an EMT of 7 mm on day of hCG trigger during prior IVF cycles. In this prospective study, intrauterine G-CSF was given 6-12 h prior to hCG trigger (based on Gleicher's earlier report), which resulted in a significant increase in EMT from 6.74 ± 1.75 mm, to 8.42 ± 1.73 (P < 0.001) within 72 h (110). The clinical pregnancy rate was 18.9%; however, there was no difference in the EMT between patients who conceived and those who did not (110).

Recently, Barad et al. tried to expand the use of G-CSF on endometrial thickness for all patients undergoing IVF or FET, regardless of endometrial thickness. The study group received 300 ig/cc G-CSF (on the day of HCG trigger), while the control group received placebo (saline) (111). The EMT increased significantly in the entire cohort by approximately 1.36 mm without a difference between the G-CSF and control groups, with similar clinical pregnancy rates (111).

In the most recent trial to date, Xu et al. prospectively randomized 30 patients with EMT < 7 mm during frozen-thawed embryo transfer cycles to either intrauterine G-CSF or G-CSF with endometrial scratch, with 52 patients serving as the control group (112). G-CSF was administered ''on the day that one follicle became dominant (12 mm)". The EMT increased significantly after treatment (d 3.9 ± 2.0 mm, P < 0.001) without a difference between the two subgroups (G-CSF vs. G-CSF + scratch), and the implantation and clinical pregnancy rates were significantly higher in both treatment subgroups compared to the control group (31.5% vs. 13.9%, P < 0.01, and 48.1% vs. 25.0%, P = 0.038 respectively) (112).

3.7. Stem cell therapy

Multiple evidence, some dating back to the early 1980s, supports the presence of endometrial stem/progenitor cells in the basalis and functionalis layers of the human endometrium (113-116). With the tremendous regenerative capacity of the endometrial lining during each estrous cycle, it was hypothesized that these stem/progenitor cells play an important role in endometrial regeneration. Despite their discovery over 30 years ago, the regenerative capacity of endometrial stem cells was just recently proven when Cervello and colleagues demonstrated that human endometrial adult stem cells are able to generate human endometrium after transplantation in NOD-SCID mice renal capsules (117). The role of stem cells in endometrial regeneration is not limited to local endometrial progenitor cells; in fact, hematopoietic and non-hematopoietic bone marrow-derived stem cells (BMDSCs) are recruited to the endometrium in response to injury. Taylor showed that BMDSCs play a role in the regeneration of endometrial stro-mal and epithelial layers of bone marrow transplant patients (118). These findings were further affirmed by other investigators, who showed that CD 45 + hematopoietic progenitor cells are able to colonize the uterine epithelium and play an important role in uterine epithelial regeneration; even more

interestingly, male origin BMDSCs were capable of composing the endometria of female bone marrow transplant recipients, further indicating their role in endometrial injury repair (119,120). In their elegant study Cervello et al. recently demonstrated that BMDSCs do not associate with endometrial side cell population, but rather they exert their regenerative potential in a paracrine fashion, ultimately stimulating endometrial side cell population (121). The role of BMDSC in endometrial regeneration was further demonstrated in several murine models (122-124). In fact, uterine ischemia/reperfusion injury results in a 2-fold increase in bone marrow-derived stem cell recruitment to the endometrium (125). This recruitment seems to be independent of G-CSF and only serves in uterine repair after injury rather than monthly cyclic regeneration of the endometrium (125).

The popularity of the regenerative potential of bone marrow stem cells led researchers to investigate their effects in the treatment of Asherman's syndrome and thin endometrium. Alwadhi and colleagues administered BMDSCs to female mice (via tail veins) with AS which were later bred after 3 estrous cycles (126). 9 of 10 treated mice conceived whereas only 3 of 10 non-treated mice conceived in the non-transplanted mice vs. 10 of 10 in the control group (126). In a similar study, Kilic et al. induced AS in Wistar albino rats and later treated them with either mesenchymal stem cells (MSCs) or oral estrogen or combined MSC and oral estrogen (127). All treatment groups demonstrated a decrease in fibrosis when compared to control, mostly noted with combined MSC and estrogen treatment (127).

To further elucidate the role of BMDSCs in regenerating thin endometrium, Zhao et al. compared thin endometria infused with bone marrow mesenchymal stem cells (BMSCs) to controls and normal rats (128,129). The treatment groups showed significant increase in EMT compared to the control groups in both trials respectively (325.35 ± 75.51 im vs. 187.53 ± 34.38 im, P <0.05 and 359.13 ± 49.70 im vs. 187.53 ± 34.38 im, P < 0.05) (128,129).

A recent comparative study analyzed the role of different BM-derived cell subtypes in endometrial regeneration (130). Various subtypes of BM-derived cell were injected into tail veins of a total body irradiated murine model. Freshly isolated unfractionated BM cells, hematopoietic progenitor cells, endothelial progenitor cells (EPCs), mesenchymal stem cells, and in vitro cultured mouse Oct4+ BM-derived hypoplast-like stem cells supported endometrial regeneration (130).

The first human application was in 2011, in a case of thin endometrium (3.6 mm) secondary to AS refractory to estradiol treatment (131). Autologous endometrial angiogenic stem cells were infused into the uterine cavity and was followed by high dose estradiol valerate, aspirin (75 mg PO daily), and four cycles of cyclical estrogen and progesterone therapy to finally reach an EMT of 7.1 mm. Three donor oocyte embryos were transferred resulting in a single viable intrauterine pregnancy at 8 weeks as detected by ultrasonography (131). This was followed by a case series of 6 patients with refractory AS who were treated with autologous mononuclear stem cells implantation. The mean EMT significantly increased from a pretreat-ment measurement of 1.38 ± 0.39 mm to 4.05 ± 1.4, 5.46 ± 1.36, and 5.48 ± 1.14 mm at 3, 6, and 9 months respectively (P < 0.05) (132). Cervello et al. investigated whether human CD 133 + BMDSCs would promote endometrial growth in a murine model of Asherman's syndrome (133). The authors

found that the BMDSCs engrafted around small endometrial vessels in all damage horns and resulted in a significant increase in epithelial gland cells proliferation via paracrine fashion by up-regulation of thrombospondin 1 and down-regulation of insulin-like growth factor 1 (133).

In a recent human pilot trial, Santamaria and colleagues infused autologous CD 133 + bone marrow-derived stem cells (BMDSCs) into the spiral arterioles of 11 patients with refractory Asherman's syndrome and 5 patients with refractory endometrial atrophy (134). An increase in endometrial thickness lasting up to six months was noted in patients with AS (4.3-6.7 mm) and those with refractory atrophic endometrium (4.2-5.7 mm), with subsequent conception attempts resulting in three spontaneous pregnancies (2, 4, and 19 months after treatment) and seven positive pregnancies after 14 embryo transfers (134).

4. Discussion

"Thin endometrium" occurs not uncommonly in clinical practice and poses a frustrating challenge to both patients and physicians. It is not exactly clear how thin endometrium results in lower chances of pregnancy; however, hypotheses such the harmful role of reactive oxygen species (ROS) or possibly not enough "soil" to sustain the "seed" have been suggested. The functional layer of the endometrium undergoes several changes during the menstrual cycle relative to the basal layer, which homes the larger-caliber oxygen-rich spiral arteries. It is hypothesized that, with thin endometrium, the proximity to the ROS-rich basal layer is detrimental for embryo development and implantation (66). Clinicians are faced with the challenge of where to draw the cutoff, and how and whether to treat it. The most widely accepted measurement is 7 mm; however, this cutoff is not absolute, as pregnancies have been reported at much lower values (27-29). More importantly, endometrial thickness alone does not seem to have a strong predictive capacity for the occurrence of pregnancies in IVF cycles, but more so a factor for the assessment of conception probability which significantly drops below a thickness of 7 mm (63). Other key factors such as endometrial pattern play a vital role in successful implantation as well, and may be more important than thickness. In fact, in the setting of CCS-tested euploid embryos, different endometrial patterns appear to have a better correlation with the likelihood of pregnancy than thin endometrium (25). Specifically, a mid to late secretory stage endometrial type (type 3 endometrium) at time of ovulation trigger, had a negative correlation with implantation rates (25). Furthermore, a trilaminar endometrial pattern on embryo transfer day might be a better prognostic factor of cycle outcomes than endometrial thickness in IVF/ICSI cycles (24). New diagnostic tools are emerging to aid in objectively assessing endometrial receptivity. According to the initial findings by Mahajan, the endometrial receptivity array (ERA) is an accurate test in determining the window of implantation. In fact 75% of patients (n = 13) with an EMT 6 6 mm in that study were found to have a receptive endometrium according to the customized endometrial receptivity microarray test (135). ERA however is an invasive and costly test with early results that must be validated with larger studies.

Over the years, multiple treatment modalities have been studied in hopes of improving refractory thin endometrium

with or without Asherman's syndrome. Extended estrogen was beneficial in improving EMT and implantation rates in some, but not all, patients with thin endometrium due to specific etiologies such as endometritis and multiple curettages. This was particularly helpful for patients undergoing IVM with thin endometrium, where extended vaginal estrogen and low dose hMG were beneficial in improving endometrial thickness or pregnancy rates. However, these studies were limited by small sample size and the fact that EMT was similar between patients who conceived and those who did not; thus, it is difficult to draw a positive conclusion regarding the benefits of the prolonged estrogen treatment in the absence of further supporting results. Two lone studies, one using luteal GnRH-a support and another using low-dose HCG in programmed FET cycles seem promising; however, data are lacking to draw solid recommendations (71,72). Tamoxifen citrate, as an ovulation induction agent, seems to be a good alternative to clomi-phene citrate in patients who have poor endometrial development during COH with IUI or FET (78-81). Treatment with Tamoxifen however may be accompanied with undesired side effects such as hot flashes, and the possibility of increased risk of endometrial cancer and adenomyosis (78). Long courses of PTX and tocopherol or tocopherol-only were used in oocyte-donor recipient patients, unexplained infertility, and patients who received radiotherapy. Although some trials noted a significant improvement in EMT, there was no evidence of a significant improvement in pregnancy rates, therefore not justifying the lengthy treatment protocol. Aspirin supplementation on the other hand had no beneficial role in oocyte-donor recipient patients, but it showed improvement in pregnancy rates without improvement in EMT in patients with thin endometrium undergoing IUI. This improvement in pregnancy rates however, did not reach normal expected levels for COH and IUI. Electro-acupuncture significantly decreased blood flow impedance across the uterine arteries but had no effects on pregnancy rates in IVF cycles, and none of the conducted trials specifically addressed patients with thin lining (94-96). While pelvic floor NMES with biofeedback significantly improved EMT, it showed no benefit in improving pregnancy rates (97).

Vaginal sildenafil administration was beneficial for a significant percentage of patients with various infertility diagnoses who had two prior failed IVF cycles or of those who are undergoing ICSI. The implantation and pregnancy rates were significantly better in those patients who responded, indicating that vaginal sildenafil could be a reasonable first line treatment option. However these findings could be limited by the retrospective nature of the largest study supporting them.

Treatment with intrauterine G-CSF also received its share of interest despite its significant cost, but the majority of trials showed beneficial effects for G-CSF in patients with thin endo-metrium. In fact, out of the seven reviewed papers, a case report and one retrospective analysis in FET cycles did not demonstrate a positive effect of G-CSF on improving EMT in patients with thin lining (105-110,112). On the other hand, there is no evidence to support G-CSF administration to all patients undergoing IVF or FET (111). While the use of GCSF seems promising in increasing EMT and possibly pregnancy rates, most studies suffer from small sample sizes, and different studies used different doses and time points when G-CSF was administered, making interpretation rather difficult. Larger

prospective, randomized, placebo-controlled, trials are therefore sorely needed.

The most promising evolving treatment modality in refractory cases appears to be stem cell therapy, as the administration of intrauterine angiogenic endometrial cells improved the endometrial lining of patients with Asherman's syndrome or refractory thin lining. Murine models showed promising results when bone marrow mesenchymal stem cells administration restored endometrial thickness to normal after total body irradiation. Research is also developing to determine what specific bone marrow-derived stem cell subtypes will have an impact on endometrial regeneration. In an early human trials, autologous bone marrow-derived stem cells gave promising results in restoring endometrial thickness for a period of 6 months in patients with Asherman's syndrome or refractory thin endometrium, with excellent subsequent pregnancy rates, making stem therapy a potentially valuable option for patients who fail traditional treatment options (132,134). It is important to note however, that stem cell therapy treatment is invasive requiring a bone marrow biopsy and interventional radiology assistance for injection into the uterine arterioles.

In conclusion, a receptive endometrium plays a critical role in embryo implantation, and adequate endometrial growth is essential to this process. Poor endometrial development is associated with a decreased probability of pregnancy; yet, it is not the sole predictor of pregnancy occurrence, and endometrial pattern may be more critical. Among multiple available treatment options, vaginal sildenafil during the stimulation cycle appears to be a reasonable first line treatment option, whereas intrauterine G-CSF infusion before ovulation trigger could be a second line treatment option, provided large randomized studies evaluating outcomes including when is the best time point in a cycle (and at what dose) to administer G-CSF are performed. Stem cell therapy appears to be promising in refractory cases; however, significantly more research on safety, effectiveness, and cost, is needed before this modality becomes adopted in the treatment of this frustrating condition.

Conflict of interest

The authors declared that there is no conflict of interest. References

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