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Reproductive Biology
After more than four decades of assisted reproductive technology (ART) practice worldwide, today more than 60% of women undergoing in vitro fertilization (IVF) treatments fail to become pregnant after the first embryo transfer and nearly 20% of patients are suffering from unexplained recurrent implantation failures (RIFs) and repeated pregnancy loss (RPL). The literature reported different causes of RIF–RPL, mainly multifactorial, endometrial and idiopathic. RIF remains a black box because of the complicated categorization and causes of this physio-pathological dysregulation of implantation and pregnancy process after ovarian stimulation. Many options were suggested as solutions to treat RIF–RPL with controversial results on their usefulness.
- infertility
- assisted reproductive technology
- implantation failure
- endometrium immunomodulation
1. Introduction
In assisted reproductive technology (ART) programs, 60–70% of women fail to become pregnant after embryo transfer. Repeated implantation failure (RIF) remains a black box in daily practice due to the complicated categorization and causes of this physio-pathological dysregulation [1]. Different causes of RIF were reported, mainly multifactorial, endometrial and idiopathic. Multifactorial RIF can be caused by maternal and paternal factors, gamete and embryo quality, infections and lifestyle changes in combination with psychological status and oxidative stress [1,2]. Impaired endometrium function such as abnormal growth or loss of vascularization can account for endometrial RIF, but idiopathic RIF, caused mainly by abnormal cross-talk between the embryo and endometrium, remains the principal question and needs to be elucidated [1].
RIF may be defined as a failure to obtain a pregnancy after multiple viable embryo transfers during IVF treatment [3], but its definition is inconsistent between studies. The most common definition was portrayed by Bashiri and colleagues [4] who describe RIF as three or more pregnancy failures following the transfer of at least three good-quality embryos [4]. However, other authors such as Coughlan and colleagues [5] suggest including maternal age, number of embryos transferred and number of previous cycles to the definition of RIF [5]. Interestingly, a consensus is emerging thanks to a recent extensive survey. It was proposed to define RIF as the failure to achieve a clinical pregnancy after 2–3 IVF cycles with 1–4 good-quality embryos [6]. RIF is a challenge for clinicians as its etiology includes various possible causes [2].
The causes of RIF can be divided into two categories: maternal (uterine anatomic abnormalities, chronic endometritis, non-receptive endometrium, antiphospholipid antibody syndrome and immunological factors) and embryonic (genetic defects and other factors specific to embryonic development) causes [3]. In the absence of male factors, oxidative stress, bad-quality embryos and anatomical abnormalities such as hydro-salpinx and thrombophilia, RIF seems to be caused by impaired endometrial function such as abnormal endometrial growth or loss of vascularization [4]. However, RIF caused by immunological factors could be manageable using several innovative therapeutic options. Among them, intrauterine administration of human chorionic gonadotropin (HCG), granulocyte colony-stimulating factor (G-CSF) or autologous peripheral blood mononuclear cells (PBMCs) has been suggested as a treatment for patients suffering from RIF [4,7,8,9,10,11,12,13,14,15,16].
2. Endometrium Immunomodulation via Intrauterine Insemination of Activated Autologous Peripheral Blood Mononuclear Cells (PBMCs)
PBMCs from patients with RIF are usually isolated during the ovulation period using a lymphocyte separation medium composed of an iso-osmotic poly-sucrose and sodium diatrizoate solution to separate mononuclear cells (including B-lymphocytes, T-lymphocytes and monocytes) from the other blood cells. After separation, PBMCs are generally activated with hCG or corticotropin-releasing hormone (CRH) and cultured in vitro for 24–72 h in a humidified incubator with 5% CO2 at 37 °C (Figure 1).
Figure 1. PBMC isolation technique and in vitro culture (PBS: phosphate-buffered saline; PBMC: peripheral blood mononuclear cell).
After culture, PBMCs are administered in utero using a catheter [4,7,8,9,10,11,12,13,14,15]. However, the number of cells administered in utero is not homogeneous among all studies investigating the use of PBMC in the treatment of RIF (Table 1). Although there were some methodological variations between studies in terms of the number of previous cycles, cycle type, and number and quality of transferred embryos, patients were generally administered with 10 to 30 million PBMCs [7,8,9,10,11,12,13,14,15,16]. Madkour and colleagues showed a significant increase in clinical pregnancy rate (CPR) with only 1 million cells [10]. Furthermore, in a recent meta-analysis, Qin and colleagues have demonstrated that CPR was higher when less than 100 million PBMCs/mL were administered in utero, suggesting that although the quantity of cells inseminated is not homogeneous, intrauterine administration of PBMC does appear to be an effective treatment for patients suffering from RIF [17].
Table 1. Main studies using PBMCs to treat RIF.
| Study | Number of Previous Failed IVF Cycles | Sample Size | Day of Blood Collection | PBMCs Co-cultured with | Duration of PBMC Culture | Number of PBMCs Administered In Utero | Transfer Type | Stage of Embryo | Implantation Rate (Control vs. Case) | Clinical Pregnancy Rate (Control vs. Case) | Miscarriage Rate (Control vs. Case) | Live Birth Rate (Control vs. Case) | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Control | Case | ||||||||||||
| Yoshioka et al., 2006 [7] | ≥4 | 18 | 17 | On the day of oocyte retrieval | hCG: 5 IU/mL | 48 h | 20 × 106 | Fresh | 1, 2 or 3 blastocysts | 4.1% vs. 23.4% (p = 0.0034) | 11.1% vs. 41.2% (p = 0.042) | Not specified | 7.6% vs. 55.6% (p = 0.013) |
| Okitsu et al., 2011 [16] | ≥1 | 170 | 83 | On the day following ovulation or the day after | Not activated | No culture | 30 × 106 | Frozen/ thawed |
early cleavage embryo or blastocyst | ≥1 RIF: 21.1% vs. 21.6% (ns); 3 RIF: 9.38% vs. 25.0% (p = 0.041) | ≥1 RIF: 32.9% vs. 34.9% (ns); ≥3 RIF: 16.7% vs. 42.1% (p = 0.039) | Not specified | ≥ 1 RIF: 21.8% vs. 21.7% (ns); ≥3 RIF: 11.1% vs. 21.2% (ns) |
| Makrigiannakis et al., 2015 [9] | ≥3 | 45 | 45 | On the day of oocyte retrieval | CRH: 107 M/1.106 cells/mL | 48 h | 20 × 106 + 107 CRH | Fresh | 2 or 3 blastocysts (grade 3BB and above) | Not specified | 0% vs. 44.44% (p < 0.001) | Not specified | Not specified |
| Madkour et al., 2016 [10] | ≥2 | 27 | 27 | On the day of ovulation induction | Complete culture medium + 75 IU of hMG | 72 h | 1 × 106 | Fresh | 1, 2 or 3 early cleavage embryos | ≥2 RIF: 9% vs. 22% (p = 0.02); 2 RIF vs. ≥ 3 RIF: 15% vs. 35% (p = 0.09) | ≥2 RIF: 15% vs. 44% (p = 0.045); 2 RIF vs. ≥ 3 RIF: 29% vs. 70% (p = 0.04) | ≥2 RIF: 17% vs. 75% (p = 0.08) 2 RIF vs. ≥ 3 RIF: 20% vs. 14% (p = 0.8) | Not specified |
| Yu et al., 2016 [11] | ≥3 | 105 | 93 | On the day following ovulation | hCG: 10 IU/mL | 24 h | 10–20 × 106 | Frozen/ thawed |
early cleavage embryo | 11.43% vs. 23.66% (p < 0.05) | 20.95% vs. 46.24% (p < 0.05) | 31.8% vs. 20.9% (ns) | 14.28% vs. 34.41% (p < 0.05) |
| Li et al., 2017 [12] | ≥1 | 339 | 294 | Two days before embryo transfer | hCG: 10 IU/mL | 24 h | 10–20 × 106 | Fresh and frozen/ thawed |
2 or 3 early cleavage embryos or 2 or 3 grade 2 blastocysts at day 5 and 3BB and above at day 6 | 1 RIF: 32.33% vs. 29.35% (ns); 2 RIF: 27.74% vs. 35.98% (p = 0.048); 3 RIF: 26.23% vs. 23.20% (ns); ≥4 RIF: 4.88% vs. 22.00% (p = 0.014) | 1 RIF: 41.23% vs. 43.75% (ns); 2 RIF: 42.18% vs. 48.15% (p = 0.016); 3 RIF: 36.84% vs. 42.22% (ns); ≥4 RIF: 14.29% vs. 39.58% (p = 0.038) | Not specified | 1 RIF: 36.84% vs. 37.5% (ns); 2 RIF: 33.33% vs. 34.26% (ns); 3 RIF: 24.56% vs. 28.89% (ns); ≥4 RIF: 9.58% vs. 33.33% (p = 0.038) |
| Makrigiannakis et al., 2019 [13] | ≥3 | 26 | 26 | On the day of oocyte retrieval | CRH: 107 M/1.106 cells/mL | 48 h | 20 × 106 + 107 M CRH | Fresh | 2 or 3 grade 1 or 2 early cleavage embryos | Not specified | 0% vs. 57,69% (p < 0.01) | Not specified | Not specified |
| Nobijari et al., 2019 [15] | ≥1 | 128 | 122 | 5 days before the frozen/thawed embryo transfer | CRH (concentration not specified) | 48–72 h | 20 × 106 + 107 M CRH | Frozen/ thawed |
early cleavage embryo or blastocyst | Not specified | <3 RIF: 30.4% vs. 30.8% (p = 0.91); ≥3 RIF: 19,7% vs. 38,6% (p = 0.01) | Not specified | Not specified |
| Pourmoghadam et al., 2020 [14] | ≥3 | 50 | 50 | On the day of ovulation induction | hCG: 10 IU/mL daily | 48 h | 15–20 × 106 | Frozen/ thawed |
early cleavage embryo or blastocyst | Not specified | 22% vs. 42% (p = 0.032) | 24% vs. 8% (p = 0.029) | 20% vs. 38% (p = 0.047) |
CRH: corticotropin-releasing hormone; hCG: human chorionic gonadotropin; RIF: recurrent implantation failure.
3. Immunoregulation of the Endometrium during Embryo Implantation: Biological Function and Molecular Pathway
To achieve successful embryo implantation and pregnancy, an appropriate dialogue between the embryo and the endometrium must take place [18].
In the uterine environment, a particular form of natural killer (NK) cells with a unique transcriptional profile, the uterine NK (uNK) cells, represents the most abundant lymphocyte population, especially in the endometrium [19,20,21]. In fact, most of the immune cells present in the uterus usually display a unique phenotype [18]. Peripheral blood NK cells express CD56+CD16+ at their membrane surface and are characterized by a highly cytotoxic profile [22]. However, uNK cells are less toxic since they do not express CD16 on their membrane surface [23]. During the menstrual cycle, levels of uNK cells start to increase in the mid-secretory phase, which could explain their importance in embryo implantation [24,25,26,27].
Dendritic cells (DCs), another type of innate immune cells, have a crucial role in the site of embryo implantation and maternal–fetal interface. DCs act as antigen-presenting cells to T cells and have the unique ability to induce a primary immune response, a phenomenon crucial for successful pregnancy [28]. In addition, DCs can influence trophoblast invasion by regulating the secretion of cytokines and the production of endometrial cell-surface proteins. Through the regulation of immune cell functions and actions, DCs have a major role in the establishment of a special local immune environment essential for embryo implantation and placental development [29]. Human decidual DCs, however, seem to have an immature phenotype characterized by a low expression of CD40, CD80, CD86 and CD205 [30,31]. DCs seem to be involved in the immune tolerance of the implantation site through the regulation of T-cell proliferation and the elimination of antigen-specific T cells. In the decidua, uterine dendritic cells (uDCs) are also crucial in maintaining pregnancy [32]. Since the 1990s, it has been known that maternal T cells are essential to the complex mechanisms of immune tolerance, a phenomenon critical to the invasion of the endometrium by the blastocyst [33].
T-cell interactions can be performed directly by cell–cell contact or indirectly through the secretion of pro-inflammatory or anti-inflammatory cytokines [34]. Pro-inflammatory cytokines such as interleukin (IL)-1β, -6, -12, -2 and -18; tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) are mainly produced by T helper (Th) 1 cells, while anti-inflammatory cytokines such as IL-4, IL-10, IL-13 and TGF-β1 are mostly secreted by Th2 cells [35]. The pro-inflammatory Th1 profile was shown to be associated with successful and normal pregnancy at early and late pregnancy stages. In the midgestation stage, however, a shift to an anti-inflammatory Th2 profile must take place to establish tolerance to the foreign fetal antigens [36]. An imbalance in these cytokine profiles has been associated with spontaneous abortion and common complications of pregnancy [37,38,39]. Moreover, it has been shown that levels of pro-inflammatory cytokines (such as IL-2 and IFN-γ) decreased while levels of anti-inflammatory cytokines (such as IL-4 and IL-10) increased in the induction of immune tolerance to allografts [40,41]. The implication of T cells, especially CD4+ CD25+ Foxp3+ Treg cells, in the initial stages of pregnancy is therefore needed for the prevention of an alloreactivity action by the endometrium against the fetus through cascades of immunoregulation actions [42,43].
Treg, Th1 and Th2 cells are, however, not the only T-cell subtypes known to be crucial for successful embryo implantation. Th17 cells, a subset of T cells showing remarkable plasticity, are also indispensable in the immunoregulation of embryo implantation as well as in maintaining normal pregnancy [44].
Monocytes and macrophages also play an important role during the menstrual cycle and pregnancy [14,45,46]. Macrophages regulate trophoblast activity by promoting endometrial tissue remodeling and angiogenesis [47]. Pregnancy hormones directly and indirectly modulate the recruitment of monocytes in the uterus and participate in their differentiation and stimulation into functional macrophages [48]. Intrauterine administration of PBMCs could also be a source of hCG-activated macrophages and regulate the uterine environment at the embryo implantation site [14].
This entry is adapted from the peer-reviewed paper 10.3390/ijms232112787
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