Significant reproductive medicine success, achieved within recent decades, haven’t diminished the demographic problems of population decline in our country. According to the World Health Organization, the infertile couple prevalence in Russia exceeds 15%, which is considered critical for the reproduction of the nation [1, 2]. Despite the thirty-year history of assisted reproductive technologies (ART) existence, the effectiveness of in vitro fertilization (IVF) stays unchanged and is only 30–40%. The Russian Association of Human Reproduction (HRE) reported the efficiency of the IVF program fresh cycle to be 34.8% per cycle in 2016 (33.9% in 2015). The frozen embryo transfer effectiveness was virtually unchanged compared to 2015. The pregnancy rate in this group was 38.5% per cycle (compared to 38.6% in 2015) [3]. Uterine infertility factor is the main cause of IVF failure after the transfer of good quality embryos [4]. Two main factors of pregnancy achievement are the normal embryo and receptive endometrium, capable of perceiving it. This is the reason why "thin" endometrium significantly decreases the chance of pregnancy. It is generally accepted that the endometrial thickness <7 mm reduces chances for a productive conception to minimum. Endometrial hyporeceptiveness plays the leading role in the implantation failure and may be caused by chronic inflammatory diseases, hormonal dysfunction, impaired receptor expression, and other factors [5]. The correlation between receptivity and endometrial thickness as an implantation failure factor has been observed in many studies [6–9]. Moreover, the repeated ART failure rate in patients with thin endometrium reaches 45% [10].

The recent retrospective UK study analyzed the data from more than 25000 fresh IVF cycles from 2006 to 2016. A correlation between the endometrial thickness, the onset of pregnancy and the birth rate was determined. The birth rate was 15.6% in women with endometrial thickness less than 5 mm and it gradually increased to 33.1% when endometrial thickness was 10 mm. Statistical modeling identified the optimal threshold for endometrial thickness to be ≥10 mm [11].

The current problem is inability to influence the non-receptive endometrium. Women with regenerative-plastic endometrial insufficiency are the most difficult to treat group in terms of endometrial functional activity restoration.

Treatment methods to improve the endometrial thickness in IVF programs involve intensive courses of estrogen and progesterone, aspirin, surgical interventions, and physiotherapeutic procedures. However, in clinical setting these methods do not always render the desired results, and further investigations are essential to develop new approaches to “thin” endometrium management [12]. The researchers and clinicians all over the World continue to search for the “thin” endometrium problem solvation to increase the effectiveness of ART programs.

Growth hormone influences the endometrial thickness

Growth hormone (GH) receptors belong to the cytokine receptor family, which includes more than 30 receptors, such as prolactin, erythropoietin, thrombopoietin, granulocyte colony stimulating factor (GCSF) and interleukin (IL) 3, 6 and 7 receptors [13].

GH is a single polypeptide chain consisting of 191 amino acids, produced by somatotropic cells in the anterior pituitary gland [14]. GH is involved in gonadal steroidogenesis, gametogenesis and ovulation. Exogenously administered GH stimulates endometrial proliferation, indirectly induces decidualization via estradiol receptors, increasing the amount of uterine insulin-like growth factor binding proteins (IGFBPs) and prostaglandins [15].

N. Cui et al. (2019) compared the pregnancy rate in women, receiving GH or estrogens to prepare the endometrium for the thawed embryo transfer. The successful pregnancy in GH group patients was achieved in 42.5% compared to 18.9% in women, receiving estrogens to treat the “thin” endometrium (p <0.05). The authors concluded that GH can improve pregnancy rates in women undergoing embryo transfer in the cryocycle, increasing endometrial cell proliferation and vascularization [16].

Granulocyte colony-stimulating factor and its role in endometrial thickness regulation

Granulocyte colony-stimulating factor (GCSF) is a glycoprotein, affecting cytokines and growth factors. Immunological mechanisms that occur in the endometrium are involved in the process of implantation. GCSF stimulates the secretion of endogenous cytokines [17]. Tanaka et al. (2000) conveyed the study, which played an important role in GCSF implementation into IVF programs. The authors concluded that GCSF influences the proliferation and differentiation of normal endometrial stromal cells and decidual stromal cells [18]. GCSF stimulates the neutrophilic granulocyte proliferation and improves the embryo implantation efficiency via the macrophage activation in decidual cells. GCSF also increases secretion of cytokines by Th2 cells and causes the regulatory stimulation of T cells [19].

N. Gleicher et al. (2011) studied the action of GCSF on the endometrial thickness in women with embryo transfer interruption in the previous IVF cycle due to the “thin” endometrium [19]. The other study (2013), which included larger group of patients, reported that intrauterine infusion of GCSF significantly increased the thickness of the endometrium and the frequency of pregnancy, and all examined women with thin endometrium became pregnant [20]. In 21 women with “thin” (less than 7 mm) endometrium and previous unsuccessful attempts to increase its thickness, using citradiol, sildenafil citrate and/or beta-blockers, underwent intrauterine infusion of GCSF on the day of chorionic gonadotropin (hCG) injection. If the endometrium did not reach 7 mm in 48 hours repeated intrauterine infusion of GCSF was performed prior to transvaginal ovarian puncture. According to the authors, the thickness of the endometrium in patients increased from 6.4±1.4 to 9.3±2.1 mm (p<0.001). Clinical pregnancy was achieved in 19% of women, with 1 case of ectopic pregnancy.

A study by Sarvi et al. (2017) included the patients with endometrial thickness less than 6 mm on the hCG injection day, who did not respond to sildenafil and estradiol in previous cycles [21]. Patients were randomly assigned into 2 groups: the main (n=15) and control group (n=13). The main group patients underwent continuous 5-minute infusion of GCSF (300 micrograms in 1 cm³) into the uterine cavity via a catheter for embryo transfer. Controls received 1 cm³ of normal saline solution with the same type of catheter. The same day, all participants were intramuscularly injected with hCG 10,000 units. On the day of the puncture, the endometrial thickness was measured using transvaginal ultrasound (US) in all participants. 2-3 days after transvaginal puncture, a second dose of GCSF was administered in women with the thickness of the endometrium <6 mm. 2-3 embryos were transferred on the same day after GCSF infusion. 2 weeks after embryo transfer, serum β-hCG was obtained, and the fetal heart rate was evaluated by a transvaginal ultrasound in 4 weeks. The authors concluded that GCSF infusion increased the thickness of the endometrium. Patients of the main group had a significantly higher endometrial thickness (8.0±1.0 mm) compared to the control group (6.3±1 mm) on the days of transvaginal ovarian puncture and embryo transfer (9.1±1.5 mm vs. 6.9±1.1; p<0.001). In patients of the main group, the average rate of endometrial thickness increase was also significantly higher. The biochemical pregnancy rate was higher in patients of the main group (10.3%) compared to the controls (5.4%; p<0.001). However, no significant difference in the clinical pregnancy rate was found between two groups (the main group — 15.3%, the control group — 20%; (p=0.7).

The effect of tamoxifen on the endometrial thickness

Tamoxifen is an antitumor drug that inhibits the estrogen action and is one of the most important medications used for estrogen receptor positive breast cancer. Ke et al. (2017) studied tamoxifen action on the “thin” endometrium in patients preparing for embryo transfer in the cryocycle [22]. All patients with the endometrial thickness of less than 7.5 mm in the previous cycle were divided into groups by etiology of the “thin” endometrium: group 1 — patients with a history of uterine synechia, group 2 — patients, who had uterine curettage, group 3 — patients with polycystic ovary syndrome (PCOS). In all groups tamoxifen caused an endometrial thickness increase. The greatest increase in endometrial thickness was observed in PCOS women — up to 9.31±1.55 mm. They also showed the highest clinical pregnancy rate (60%) and live birth rate (55.56%) per embryo transfer (p<0.001).

A prospective study by Sharma et al. (2018) describes the use of clomiphene citrate, tamoxifen, and gonadotropins for intrauterine insemination in women with endometrial thickness less than 7 mm [23]. Clinical pregnancy rates in tamoxifen and gonadotropin groups were comparable (14.52 and 14.89%), whereas it was lower in patients taking clomiphene citrate (4.94%, p<0.002). The birth rate in these groups was 12.2, 12.7, 3.2%, respectively (p<0.004). The authors concluded that tamoxifen can increase the endometrial thickness and the live birth rate in women with "thin" endometrium, a history of previous clomiphene citrate use and unsuccessful attempts in ART programs.

Sildenafil effect on the endometrial thickness

Sildenafil is a potent selective inhibitor of cyclic guanosine monophosphate (cGMP) of the specific type 5 phosphodiesterase (PDE5). It inhibits the breakdown of cGMP and, thus, enhances the effect of nitric oxide on the vascular wall smooth myocytes, leading to uterine blood flow improvement. The combination of sildenafil with estrogens promotes estrogen-induced proliferation of the endometrium [24].

Vascular endothelial growth factor (VEGF) and transcriptional malignant tumor suppression factor (p53) genes control cell growth and promote angiogenesis. These processes are essential for adequate endometrial blood supply and subsequent implantation of the embryo [7, 25]. Sildenafil causes a significant increase in p53 activity and VEGF levels, promoting angiogenesis [26, 27].

Dehghani Firouzabadi et al. (2013) conducted a randomized controlled trial, which showed that sildenafil citrate was able to increase endometrial thickness and pregnancy rate in patients with previous unsuccessful embryo transfer attempts and poor endometrial proliferation [28]. The authors evaluated the effect of sildenafil citrate on the endometrial thickness (determined by ultrasound exam), structure and the frequency of implantation in the frozen embryo transfer cycles. Patients were divided into equal groups, each comprising 40 women. Study group patients were prescribed 50 mg sildenafil citrate daily in addition to estradiol starting on the 1st day of the cycle up to the day of progesterone preparations’ administration. Women in the control group received estradiol to prepare the endometrium for transfer of cryopreserved embryos. The study reported a significantly higher endometrial thickness in the study group compared to the controls (9.2 mm vs. 8 mm) (p<0.0001). The implantation rate was statistically higher in patients of the sildenafil group (14.16%) compared to the control group — 8.75% (p=0.22). The authors concluded that the use of sildenafil citrate improves endometrial receptivity and recommend the use of this drug for the “thin” endometrium preparation in patients with previous unsuccessful attempts in ART programs.

Fetih et al. (2017) studied the effect of vaginal gel, containing sildenafil and clomiphene citrate, on endometrial thickness in patients with a history of failed stimulation attempts [29]. Patients were divided into 2 groups: group 1 — women who used clomiphene citrate alone, group 2 — patients who used clomiphene citrate gel with sildenafil starting from the day 7 of the menstrual cycle and until the day of hCG administration. According to the study results, the endometrial thickness in the first group was 6.6±1.4 mm, compared to 9.3±3.1 mm in the study group (p<0.001), an increase in uterine blood flow according to Doppler ultrasound exam in women of the second group was also observed. The clinical pregnancy rate in the first group was 2.3% compared to 8.4% in patients of the second group. Thus, researchers from Egypt concluded that intravaginal sildenafil increases the endometrial thickness and enhances uterine blood flow, resulting in pregnancy rate increase in patients with “thin” endometrium.

Physiotherapy influence on the endometrial thickness

Physiotherapeutic treatment is included in pregravid endometrial preparation guidelines worldwide. The later causes a positive effect on uterine hemodynamics and leads to an increase in the pregnancy rate.

Neuromuscular electrical stimulation (NMS) is one of these physiotherapeutic methods. It is performed using electrodes located on the skin. Researchers from China analyzed the efficiency of defrosted embryo transfer in patients with “thin” endometrium and history of IVF failure after NMS treatment [30]. Patients were divided into 2 groups: the study group included women, who received NMS together with the hormonal treatment, and the controls, receiving only hormonal drugs. In 60% of patients who underwent NMS, the thickness of the endometrium after therapy was ≥8 mm. The average thickness of the endometrium before and after treatment was 5.6±0.82 mm and 7.93±1.42 mm, respectively, in patients of the main group compared to 5.5±1 mm and 6.78±0.47 mm in the control group. The difference was statistically significant (p=0.002). The pregnancy rate in the study group was higher (42% compared to 35%), but the difference was not statistically significant.

Russian researchers Schneiderman et al. (2014) treated endometrium with a mixture of gases (CO₂ and N₂) – the method, invented in National Medical Research Center of Obstetrics, Gynecology and Perinatology named after Kulakov (Ministry of Health of Russia), to prepare “thin” endometrium for IVF procedure [12]. The result of such exposure was the uterine mucous membrane blood circulation improvement with subsequent gradual increase in the thickness of the basal and functional layers of the endometrium. A gynecological massage was used also to enhance the effect. The study included 65 patients of reproductive age (25–40 years old), suffering from primary or secondary infertility and having “thin” endometrium, refractory to the other treatment methods. Group A included 20 infertile patients with thin endometrium, who received endometrial irrigation with a mixture of CO₂ and N₂ gases on the 7th, 9th and 11th days of the menstrual cycle; group B — 15 women, receiving also a course of gynecological massage (5 procedures) in addition to endometrial gas irrigation; group C – control group, including 30 patients. The study reported the effectiveness of combined treatment of women with thin endometrium (endometrial irrigation with a mixture of CO₂ and N₂ and gynecological massage) in preparation for the IVF procedure. It significantly increased the thickness of the endometrium: 11.1 mm in group B women compared to 5.7 mm in group C controls. Endometrial gas irrigation, combined with gynecological massage, may be used to prepare the patient for subsequent embryo transfer, thereby increasing the chances of a positive outcomes of the procedure.

Melkozerova et al. describe a complex of therapeutic and diagnostic measures within the developed concept using intrauterine irrigation with cavitation solutions [10, 31, 32]. The use of this complex helped to restore the endometrial functioning and receptivity in 85.9% of women, reduce early pregnancy loss rate by 3.3 times, contributed to the fertility restoration in 69.34% of women and live birth rate in 50.53% of patients with previous reproductive failures due to impaired endometrial receptivity.

Platelet-enriched plasma influences the endometrial thickness

Autologous plasma therapy is a little-studied method for increasing the receptivity and thickness of the endometrium. Autologous plasma application in medical practice started with the introduction of autologous blood in 19th century, when Max Schede injected the patient’s blood into the wound during surgical debridement, resulting in improved regeneration. In the 20th century, it was first applied in gynecology, when Bakshev developed the treatment method for the pelvic inflammatory disease using the autologous blood. In the 21st century, autologous blood was replaced by autologous plasma, which contains growth factors, allowing local restoration of normal autocrine regulation in the endometrium [33]. Platelet alpha-granules contain abundant proteins: platelet-derived growth factor (PDGF), transforming growth factor (TGF), platelet-derived angiogenic factor (PDAF), VEGF, epidermal growth factor (EGF), insulin-like growth factor (IGF), fibronectin, cytokines. The plasma also contains a number of biologically active proteins, such as IGF-I and hepatocyte growth factor (HGF). Therefore, autologous plasma modulates and regulates the function of primary growth factors. The properties of the growth factor combination, contained in platelet-enriched autologous plasma differ from those of recombinant growth factors, each of which is responsible for a specific regenerative mechanism: for example, PDGF stimulates the mesenchymal cell proliferation and migration and activates angiogenesis, while IGF promotes the differentiation of new cells [ 34].

Growth factors are delivered to tissues via injection or application (irrigation) of autologous plasma and are concentrated by the use of higher amounts of platelets — this stimulates the formation of fibroblasts (connective tissue cells). Fibroblasts produce collagen, hyaluronic acid and elastin. This process leads to connective tissue production and synthesis of capillaries. As a result, metabolic processes are restored, tissue microcirculation and respiration are improved, local immunity is activated, triggering natural regeneration processes and acting on them synergistically [35]. Angiogenesis, macrophage chemotaxis, fibroblast proliferation and migration, production of collagen, elastin, osteoblasts, intercellular matrix and endothelial cells — are all biological effects, that stimulate cellular mechanisms, and are enhanced due to plasma components. [36]. Analgesic, anti-inflammatory, immunomodulating, immunostimulating, antibacterial effects of autoplasma are all clinically proven [37].

Some authors reported that autologous plasma therapy in patients with refractory and suboptimal endometrium promoted a dynamic increase in its thickness and vascularization improvement [38, 39].

Chang et al. (2015) evaluated platelet-rich plasma effectiveness combined with standard hormone replacement therapy (HRT) in patients with thin endometrium (less than 7 mm) who had a history of IVF program embryo transfer canceling due to poor endometrial response to the standard preparation [38]. Patients (n=5) undergone an intrauterine infusion of autologous platelet-rich plasma on the 10th day of HRT; when the thickness of the endometrium did not increase after 72 hours, repeated intrauterine infusion was performed. Embryo transfer was performed when endometrial thickness reached ≥7 mm. As a result, all patients had a clinical pregnancy.

In 2016 authors from Iran published the results of a pilot study evaluating the efficacy of platelet-enriched plasma use in patients in a cryocycle [39]. The observation group included 20 women, undergoing the intrauterine infusion of 0.5 ml of autologous platelet-rich plasma. Intrauterine infusion was performed 48 hours prior to defrosted blastocyst transfer. According to the study results, a clinical pregnancy was registered in 16 patients and 18 women developed a biochemical pregnancy. One patient developed an early spontaneous abortion and one women developed a hydatid mole. Platelet-rich autologous plasma has been shown to improve IVF results in cryocycles.

Conclusion

The presence of receptive endometrium is an essential part of the embryo implantation process, whereas an insufficient endometrial growth reduces the effectiveness of assisted reproductive technologies. Many treatments for thin endometrium are targeted to improve its responsiveness, but the effectiveness of most methods remains questionable. The search for effective methods to influence the “thin” endometrium in order to improve the results of assisted reproductive technology programs still continues.

The authors declare no conflict of interest.