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THE IMPORTANCE OF THE ENDOMETRIAL LAYER IN PREVENTING OF
FAILURE IN VITRO FERTIZILATION
Abdujapparova Naziraxon Erikli qizi
Obstetrician-gynecologist at
Republican Specialized Scientific and Practical Medical Center for Mother and Child
Health of Fergana Branch
https://doi.org/10.5281/zenodo.13896941
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. 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. 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. RIF may be defined as a failure to obtain a pregnancy after multiple
viable embryo transfers during IVF treatment, but its definition is inconsistent between studies.
The most common definition was portrayed by Bashiri and colleagues who describe RIF as
three or more pregnancy failures following the transfer of at least three good-quality embryos.
However, other authors such as Coughlan and colleagues suggest including maternal age,
number of embryos transferred and number of previous cycles to thedefinition of RIF.
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. RIF is a challenge for clinicians as its etiology includes various possible causes.
The causes of RIF can be divided into two categories: maternal (uterine anatomic abnormalities,
chronic endometritis, non-receptive endometrium, antiphospholipid antidiv syndrome and
immunological factors) and embryonic (genetic defects and other factors specific to embryonic
development) causes. 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.
However, RIF caused by immunological factors could be manageable using several innovative
therapeutic options. Among them, intrauterine administration of human chorionic
gonadotropin (HCG), granulocyte colonystimulating factor (G-CSF) or autologous peripheral
blood mononuclear cells (PBMCs) has been suggested as a treatment for patients suffering from
RIF. Intrauterine administration of autologous PBMC prior to embryo transfer was proposed to
regulate the immune environment of the endometrial tissue. Yoshioka and colleagues were the
first to propose this immunotherapy to help RIF patients. Since then, this therapeutic option
was recommended as an effective treatment for RIF according to numerous studies. The
present study is a review aiming at summarizing studies that used this immunotherapy to
evaluate its benefit regarding RIF patients.
Materials and methods.
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-
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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). After
culture, PBMCs are administered in utero using a catheter. However, the number of cells
administered in utero is not homogeneous among all studies investigating the use of PBMC in
the treatment of RIF. 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. Madkour and
colleagues showed a significant increase in clinical pregnancy rate (CPR) with only 1 million
cells. 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 frm RIF.
Intrauterine administration of PBMCs in patients suffering from RIF aims to improve
endometrial receptivity by regulating the Th1/Th2 cytokine ratio and growth factors to
stimulate many cascades of cytokines and matrix metalloproteinase actions. Increased
peripheral blood Th1/Th2 ratio was shown to be detrimental to embryo implantation.
However, PBMCs produce many cytokines that can regulate Th1/Th2 imbalance in women
suffering from RIF. Furthermore, PBMCs are known to increase the secretion of growth factors
and Th1 pro-inflammatory and anti-inflammatory cytokines at the time of embryo implantation
to boost endometrial receptivity. This immunotherapy was shown to improve progesterone
(P4) production in cultured human granulosa luteal cells. Ovarian steroids such as P4 and β-
hCG are among the most crucial factors needed in the immunoregulation of embryo
implantation. Luteinizing hormone (LH) and hCG have also an important role in establishing
the immune tolerance mechanisms of embryo implantation. These two gonadotropins were
shown to affect immune cells by binding to the LH/hCG receptors present at the surface of
several immune cell types. Furthermore, it has been shown that hCG has the capacity to
downregulate pro-inflammatory immune responses during pregnancy. During the embryo
implantation window, β-hCG seems to play a role in the immunoregulation of the endometrium
in increasing Fas ligand expression (APO-1, CD95) in the endometrial cells to facilitate
trophoblast invasion. Increased peripheral blood Treg cell levels have also been shown to be
positively associated with higher pregnancy rates in IVF treatment. These cells being attracted
to trophoblasts by hCG supports the fact that the administration of hCG could be an effective
treatment for some infertile women. Moreover, it has been shown by Mansour and colleagues
that intrauterine hCG injection before embryo transfer could significantly improve
implantation and pregnancy rates. Intrauterine administration of PBMCs for patients suffering
from RIF has been shown to be specifically efficient for increasing implantation and pregnancy
rates in women with three or more previous implantation failures. Recently, Nobijari and
colleagues and Pourmoghadam and colleagues presented a different strategy to administer
PBMCs in RIF patients using frozen–thawed embryo transfers. Nobijari and colleagues
confirmed the effectiveness of this immunotherapy by showing an increase in CPR in patients
with three or more implantation failures undergoing frozen–thawed embryo transfer.
Pourmoghadam and colleagues only administrated PBMCs in utero in RIF patients with a low
Th-17/Treg cell ratio. Furthermore, in the study of Pourmoghadamand colleagues, PBMCs were
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activated in vitro with 10 IU/mL hCG for 48 h before the intrauterine administration, while
Nobijari and colleagues activated the PBMCs in vitro with CRH for 48–72 h. In RIF patients, it
has also been shown that levels of IL-1β, TNF-α and IFN-γ, three pro-inflammatory cytokines,
were increased in the PBMC culture medium, suggesting that PBMCs secrete these Th1
cytokines when treated with hCG. Moreover, Pourmoghadam and colleagues have shown that
CPR and live birth rates increased significantly and miscarriage rates decreased significantly in
RIF patients treated with PBMCs compared to control. In addition, Makrigiannakis and
colleagues have shown that the insemination of autologous PBMCs treated with CRH before
blastocyst or early cleaved embryo transfer presented better results than PBMCs without CRH
treatment in terms of CPR in women with RIF.
Therefore, these three studies supported the effectiveness of this immunotherapy for
patients suffering from RIF undergoing fresh or frozen–thawed embryo transfer, especially
when PBMCs are treated with CRH. However, these findings are still limited because, in the
study of Pourmoghadam and colleagues for example, the authors measured only three pro-
inflammatory cytokines, and they did not show anti-inflammatory cytokine levels with PBMC
administration for RIF women or in a control group. The increase in these cytokine levels
should be compared to a control, not treated cells, but the authors did not perform this
comparison.
Researchs.
Immunological therapy approaches other than intrauterine administration of
PBMCs for the management of RIF patients were reported in the literature. These
immunotherapies focus on elevated Th1/Th2 ratio, abnormal TNF-α/IL-10 ratio, elevated NK
cells and auto-antibodies. One of these immunomodulatory agents that have been described for
RIF patients is intravenous immunoglobulin IgG. Patients receiving this treatment have shown
significantly higher implantation and clinical pregnancy rates compared to non-treated
patients. This treatment has been extensively used, but the results are heterogeneous.
According to many studies, the application of IVIg has shown positive effects on RIF patient
pregnancy rates and in patients with increased immunological risk factors. Granulocyte colony-
stimulating factor (G-CSF) was also shown to have positive effects on embryo implantation in
women suffering from RIF, especially when endometrial thickness was insufficient.
Furthermore, a recent meta-analysis showed that G-CSF was an effective treatment for
women with thin endometrium or with recurrent IVF failures. G-CSF was originally used as a
treatment for thin endometrium to thicken it. Increased implantation rates were shown after
G-CSF treatment in patients with an endometrium thickness ≥7 mm on the day of embryo
transfer. These results were confirmed by another study conducted by Xu and colleagues in
which they showed a higher implantation rate in women treated with G-CSF compared to
controls. Furthermore, Kalem and colleagues have shown that the administration of G-CSF into
the uterine cavity in RIF patients with normal endometrium did not alter the endometrial
thickness, clinical pregnancy rates or live birth rates in comparison with a control group.
Vitamin E, which has been shown to improve capillary blood flow in different organs, and
sildenafil citrate, which improves uterine artery blood flow, were also proposed as a treatment
for thin endometrium. In the study of Miwa and colleagues, 23 out of 25 patients showed
improved radial artery, 17 patients had increased endometrial thickness and 13 patients
developed an endometrium thickness of more than 8 mm. Sher and Fisch were the first to
suggest the use of sildenafil during the follicular phase and until ovulation trigger as a treatment
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for thin endometrium of women undergoing IVF with fresh embryo transfer. They reported an
improvement in uterine blood flow and in endometrial thickness. These results were confirmed
in a larger cohort study showing a 45% pregnancy rate.
However, a randomized controlled trial study reported no significant difference in
endometrial thickness and pregnancy rateafter administration of sildenafil and valerate
estradiol during the luteal phase following fresh embryo transfer. Another randomized
controlled trial study did not show any improvement in uterine blood flow or in endometrial
thickness. Recently, a randomized placebo-controlled trial study reported that the use of
vaginal sildenafil on the hCG injection day did not present a statistically significant
improvement in endometrium thickness; however, the implantation was significantly higher in
women who received sildenafil with placebo compared to women who received only sildenafil
or only placebo. More trials are needed to confirm the effectiveness of these treatments on
endometrium thickness and/or RIF. Nakagawa and colleagues proposed a treatment using
immunosuppressive drugs such as tacrolimus, one of the major immune-suppressive agents
that have been used after allogeneic organ transplantation to reduce the alloreactivity of a
recipient’s immune system and to decrease the risk of the rejection. This treatment has shown
positive results on successful implantation and pregnancy outcome in RIF patients with
elevated Th1/Th2 ratios, suggesting that this immunological imbalance plays a crucial role in
causing RIF. However, the posology of this drug must be determined more accurately to
maintain the levels of the essential Th1 cytokines necessary for embryo implantation. Another
treatment using atosiban administration was proposed for RIF women. In fact, it is a receptor
of oxytocin and V1a vasopressin, proposed to avoid uterine contractions during embryo
transfer, which could be detrimental in embryonic apposition. However, according to the
review of Makrigiannakis and colleagues, various randomized controlled trial studies reported
a non-significant effect on reproductive outcomes, and only two non-randomized studies on
RIF patients report a significant benefit after atosiban treatment. Therefore, more randomized
studies are needed to verify the efficiency of atosiban as a benefic treatment for RIF women.
Chang and colleagues reported that autologous platelet-rich plasma (PRP) promotes
endometrial growth and improves pregnancy outcomes during IVF. After being collected from
the peripheral vein in acid citrate dextrose solution A (ACD-A) anticoagulant tubes, PRP was
prepared by separating the various components of the blood using multiple centrifugations.
This PRP, within 10 min after clotting, can activate cytokines and growth factors which become
bioactive and increasingly secreted. These factors include vascular endothelial growth factor
(VEGF), transforming growth factor (TGF), platelet-derived growth factor (PDGF) and
epidermal growth factor (EGF), which can regulate cell migration, attachment, proliferation and
differentiation, while promoting extracellular matrix accumulation. This could lead to
ameliorated implantation conditions and improved pregnancy, as was revealed by Chang and
collaborators. Other studies could confirm these results; for example, in 2019, Kim et al. showed
that autologous PRP treatment increases the activity of cytokines and growth factors compared
to that observed without the use of PRP, especially when combined with frozen–thawed
embryo transfer. These studies support the suggestion of PRP as a useful treatment for RIF.
However, in a recent study that used PRP treatment in patients with a history of failed
implantation before frozen–thawed embryo transfer, the authors did not find significant
differences in the pregnancy results in comparison with controls. A recent study by Ibañez-
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Perez and colleagues suggested a non-invasive method of microRNA based signatures obtained
from very small volumes of endometrial fluid collected just before day 5 frozen embryo
transfers to identify the competence of the endometrium in implantation. This technique could
help physicians to avoid RIF by changing the embryo transfer strategy when the results show
an unfavorable implantation pattern by using immunomodulation techniques from the first IVF
cycle.
Conclusions
. There is no scientific consensus about the best immunological treatment for
RIF patients presenting an imbalanced Th1/Th2 ratio or immune dysregulation. However,
recentstudies have shown the potential of the intrauterine administration of hCG-activated
PBMCs and activated PRP as a good way to modulate endometrial receptivity. The
immunotherapy field strategy needs to be further elucidated for a better understanding of
maternal immunotolerance to embryo implantation. Proteomic investigations of biomarkers
produced by immunological cells and their pathways should be continued to identify the exact
combination of immunological factors needed for successful implantation. Correcting
immunological dysregulations in embryo implantation by intrauterine administration of
PBMCs or treatment with activated PRP seems to be a promising solution in RIF. It is clear that
we need to know much more about maternal immune tolerance and the exact role of each
biomarker involved in embryo–endometrium cross-talk to improve implantation and reduce
repeated implantation failure and pregnancy loss.
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