- Academic Editor
Background: Repeated implantation failure (RIF) after in vitro
fertilization/intracytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET) can
be a devastating reality for some patients with infertility. Our objective was to
evaluate the potential role of the complete blood count (CBC)
parameters, on treatment outcome in patients with repeated IVF implantation
failure. Methods: This retrospective clinical study, involving a total
of 173 patients, consisted of 64 patients with RIF who underwent a fresh IVF-ET
cycle, underwent 3 or more IVF cycles, and 109 patients in the control group who
became pregnant in the first IVF-ET cycle. Results: Duration of
infertility, number of grade 2 embryos and red cell distribution width (RDW) were
significantly higher in RIF patients (p
Repeated implantation failure (RIF) after IVF/ICSI-ET (in vitro fertilization/intracytoplasmic sperm injection-embryo transfer) can be a devastating reality for some patients with infertility. It is a challenging topic for both clinicians and patients. The definition is inconsistent and differs from that of repeated IVF failure, which includes cases in which embryos cannot be transferred [1]. Reviewing the literature, one comes across different definitions [2]. They are as follows: (a) patients under 40 years of age with a negative pregnancy test after three consecutive embryo transfer (ETs) with good quality embryos, (b) patients under 40 years of age with at least 4 good-quality embryos in whom no clinical pregnancy (CP) has occurred despite three consecutive ETs, (c) patients without implantation despite two consecutive fresh or frozen transfers of at least 4 good quality cleavage stage embryos or at least 2 good quality blastocysts [3, 4, 5]. The definition includes both patients who do not show measurable signs of implantation, such as elevated human chorionic gonadotropin (hCG) levels and patients who later show positive hCG levels without showing a gestational sac on ultrasound [3].
In order to offer meaningful solutions to RIF patients, the etiology of RIF must
first be determined. RIF may be due to embryologic, maternal, or both causes.
Maternal factors include uterine anatomic problems, endometrial pathologies
affecting endometrial receptivity, hypercoagulability conditions, and immunologic
factors [6]. Various uterine pathologies, including uterine polyps, fibroids,
uterine septum, and adhesions, can interfere with embryo implantation [7].
Another important maternal condition is endometriosis. In endometriosis, the
quality, quantity, and implantation rate of oocytes and embryos are decreased;
the spontaneous abortion rate increases [8]. Older maternal age, smoking in both
parents, high body mass index (
In addition to the known factors underlying implantation, inflammatory, immunologic, and infectious causes as well as states of hypercoagulability are of interest and have been studied in detail. Natural killer (NK) cell and lymphocyte concentrations in the periphery are increased in RIF patients [11]. In addition, an increased Type 1 helper/Type 2 helper (Th1/Th2) ratio in peripheral blood has been associated with embryo rejection [12]. In the study of autoimmunity, there is no evidence that autoantibodies directly lead to implantation failure, although there is a strong association with RIF, particularly with antiphospholipid antibodies [9]. There are some data suggesting that hereditary thrombophilias may play a role in RIF, although this needs to be confirmed by further studies [13]. Some women with RIF have been found to have chronic endometritis due to bacterial colonization without clinical signs of infection [14].
In RIF of unexplained etiology, investigation of the above causes is both expensive and time-consuming. However, an ideal diagnostic marker for diagnosing many diseases should have high sensitivity and specificity, be rapidly accessible, inexpensive, and noninvasive [15]. Complete blood count (CBC) is also an ideal analysis for diagnosing many diseases. Some CBC parameters such as leukocytes, neutrophils, and neutrophil-to-lymphocyte ratio (NLR) are considered inflammatory markers [16, 17]. In recent years, platelet/lymphocyte ratio (PLR) and mean platelet volume (MPV) have also been increasingly used as markers of chronic inflammation. There are some reports of NLR, MPV, and PLR in infertile women with polycystic ovary syndrome (PCOS) [18]. Red blood cell distribution width (RDW), also examined in blood counts, reflects the degree of heterogeneity of red blood cell volume (anisocytosis) and has traditionally been used for differential diagnosis of anemia [19]. More recently, RDW has been recognized as an inflammation-related marker and it has been suggested that it may play a role in predicting mortality in inflammation-related diseases [20].
In this study, we aimed to investigate the potential role of markers in a much simpler CBC analysis compared with many more complex tests and analyzes for RIF, in which there are still unexplained conditions in the etiology despite many of the causes and risk factors mentioned above.
The retrospective clinical study presented was conducted in the Health Sciences University Hospital ART clinic. A computerized database reviewed one thousand five hundred thirty-five fresh IVF-ICSI cycles with good quality (grade 1 and 2) ET between September 2007 and June 2018. The study was approved by the institutional ethics committee.
Patients with 3 or more consecutive IVF cycles and a negative pregnancy test
(presence of a detectable beta subunit of hCG in serum) were included in the RIF
group. Some authors additionally used good ovarian reserve (follicle-stimulating
hormone (FSH)
Oocyte fertilization was assessed by observation of 2 pronucleus (PN) 18–20 hours after
ICSI. Fertilization rate (FR) was calculated as the ratio of 2 PN to mature
oocytes. At 42–44 hours after ICSI, day 2 embryos were classified based on
blastomere size, nucleation, and cytoplasmic morphology. At 61–65 hours after
ICSI, day 3 embryos were classified based on cell number, size, symmetry, and
degree of fragmentation using an embryo scoring system [23]. Grade 1 and 2 were
classified as good-quality embryos, whereas grade 3 and 4 were classified as
poor-quality embryos. Blastocysts with a day 5 (blastocyst) score
The primary outcome of this study was a correlation analysis between IVF and CBC parameters and a comparison of CBC parameters between RIF patients and the control group. A regression analysis was performed for the influence of CBC parameters on FR, and the secondary outcome was whether the obtained model was significant.
Blood tests, including CBC, are performed before all patients are enrolled in the IVF-ICSI treatment protocol. Venous blood samples were collected between 08:00 and 09:00 after a 12-hour fasting period in tubes containing ethylenediaminetetraacetic acid (EDTA). Complete blood count was performed using an automated blood analyzer (Cell-Dyn 3700, Abbott®, Abbott Park, IL, USA). Leukocyte, lymphocyte, neutrophil, monocyte, platelet, MPV and RDW values were recorded; NLR, PLR and platelet mass index (PMI) were calculated and recorded.
Statistical analysis was performed with the Statistical Program for the Social
Sciences version 20.0 (IBM SPSS Inc., Chicago, IL, USA). The distribution of continuous
variables was presented as mean and standard deviation (SD), whereas categorical
variables were presented as ratios and percentages of the total. Comparison of
continuous variables between groups was made with Student’s t-test or
Mann-Whitney U test, depending on the normality of the distribution, and
comparison of categorical variables was made with Pearson’s chi-square test or
Fisher’s exact test. Spearman correlation analysis was performed for the
relationship between CBC parameters and FR, with correction for body mass index
(BMI). Parameters with significant correlations were included in the model
established by regression analysis and evaluated. The significance level was
p
A total of 173 patients, including 64 RIFs and 109 controls, were included in
the study. Demographic characteristics and IVF cycle characteristics of the
included patients (mean
Parameter |
Group 1 (RIF) | Group 2 (control) | p |
Age (years) | 29.84 |
30.87 |
0.081 |
BMI (kg/m |
26.87 |
25.48 |
0.019 |
FSH (mIU/mL) | 6.78 |
6.72 |
0.768 |
LH (mIU/mL) | 6.09 |
5.63 |
0.153 |
E |
48.55 |
48.56 |
0.461 |
Duration of infertility (years) | 91.08 |
61.13 |
|
Number of antral follicles | 16.08 |
15.08 |
0.504 |
Initial stimulation dose at day 3 | 227.54 |
220.30 |
0.661 |
Days of stimulation | 10.02 |
10.04 |
0.606 |
Total gonadotrophin dose (IU) | 2203.71 |
2179.77 |
0.733 |
Estradiol on hCG day (pg/mL) | 2968.61 |
2864.04 |
0.790 |
Progesteron on hCG day (ng/mL) | 1.07 |
1.18 |
0.854 |
On the hCG day |
3.72 |
3.53 |
0.657 |
Endometrial thickness on hCG day (mm) | 10.30 |
9.89 |
0.226 |
Total number of retrieved oocytes | 13.80 |
12.69 |
0.735 |
Number of mature oocytes | 10.49 |
9.85 |
0.969 |
Oocyte quality index | 5.32 |
5.23 |
0.647 |
Number of 2 pronuclei | 5.47 |
5.54 |
0.499 |
Day 2 embryo scoring | 4.04 |
3.98 |
0.849 |
Cleavage stage embryo scoring | 4.07 |
4.05 |
0.698 |
Number of grade 1 embryos | 0.88 |
0.81 |
0.638 |
Number of grade 2 embryos | 0.78 |
0.27 |
hCG, Human chorionic gonadotropin; E
Parameters | Group 1 | Group 2 | Total | p value | |
Ovarian stimulation protocol | Micro-dose-flare up | 3 (4.7%) | 7 (6.4%) | 10 (5.8%) | p |
Long luteal agonist | 32 (50.0%) | 44 (40.4%) | 76 (43.9%) | ||
Antagonist | 24 (37.5%) | 52 (47.7%) | 76 (43.9%) | ||
Hypogonadotropic hypogonadism | 0 (0.0%) | 2 (1.8%) | 2 (1.2%) | ||
Luteal E |
4 (6.3%) | 4 (3.7%) | 8 (4.6%) | ||
Femara-Antagonist | 1 (1.6%) | 0 (0.0%) | 1 (0.6%) | ||
Total | 64 (100.0%) | 109 (100.0%) | 173 (100.0%) | ||
Type of drug used during induction | RecFSH + HMG | 28 (43.8%) | 41 (37.6%) | 69 (39.9%) | p |
RecFSH | 34 (53.1%) | 63 (57.8%) | 97 (56.1%) | ||
HMG | 2 (3.1%) | 5 (4.6%) | 7 (4.0%) | ||
Total | 64 (100.0%) | 109 (100.0%) | 173 (100.0%) | ||
Assisted hatching | No | 45 (70.3%) | 69 (63.9%) | 114 (66.3%) | p |
Yes | 19 (29.7%) | 39 (36.1%) | 58 (33.7%) | ||
Total | 64 (100.0%) | 108 (100.0%) | 172 (100.0%) | ||
Embryo transfer day | 2 | 2 (3.1%) | 4 (3.7%) | 6 (3.5%) | p |
3 | 38 (59.4%) | 44 (40.4%) | 82 (47.4%) | ||
4 | 0 (0.0%) | 3 (2.8%) | 3 (1.7%) | ||
5 | 24 (37.5%) | 56 (51.4%) | 80 (46.2%) | ||
6 | 0 (0.0%) | 2 (1.8%) | 2 (1.2%) | ||
Total | 64 (100.0%) | 109 (100.0%) | 173 (100.0%) | ||
Cause of infertility | Male factor | 26 (40.6%) | 52 (47.7%) | 78 (45.1%) | p |
Poor ovarian reserve | 4 (6.3%) | 7 (6.4%) | 11 (6.4%) | ||
Unexplained infertility | 27 (42.2%) | 47 (43.1%) | 74 (42.8%) | ||
Tubal factor | 7 (10.9%) | 3 (2.8%) | 10 (5.8%) | ||
Total | 64 (100.0%) | 109 (100.0%) | 173 (100.0%) |
RecFSH, recombinant follicle-stimulating hormone; HMG, human menopausal gonadotropin; IVF, in vitro fertilization.
CBC parameters | Group 1 | Group 2 | p |
WBC (10 |
7.51 |
7.14 |
0.704 |
Lymphocyte (10 |
1.98 |
1.97 |
0.639 |
Neutrophil (10 |
4.87 |
4.53 |
0.704 |
RDW (%) | 14.55 |
14.14 |
0.02 |
Monocytes | 0.39 |
0.38 |
0.988 |
Platelets (10 |
282.39 |
283.08 |
0.895 |
MPV (fL) | 8.17 |
8.39 |
0.156 |
PMI | 2294.94 |
2348 |
0.688 |
NLR | 2.44 |
2.45 |
0.853 |
PLR | 153.04 |
152.96 |
0.612 |
WBC, White blood cell; NLR, Neutrophil-to-lymphocyte ratio; PLR, Platelet-to-lymphocyte ratio; MPV, Mean platelet volüme; CBC, complete blood count; PMI, Platelet mass index; RDW, Red blood cell distribution width.
Independent variable | p |
Beta | 95% CI | VIF |
RDW | 0.946 | 0.005 | –0.021/0.023 | 1.020 |
Number of grade 2 embriyos | 0.430 | –0.077 | –0.125/0.054 | 2.082 |
Number of 2 pronuclei | 0.547 | 0.028/0.046 | 1.047 | |
Duration of infertility | 0.94 | –0.005 | –1 | 1.111 |
BMI | 0.439 | –0.053 | –2 | 1.034 |
Predictors: RDW, number of 2 pronuclei, number of grade 2 embryos, duration of infertility, BMI.
RDW, Red blood cell distribution width; VIF, Variance Inflation Factor; CI, confidence interval.
We could not find any study on RDW in the literature that included inflammation and blood count data on the etiology of RIF. In this regard, our study was the first in the literature to find that high RDW before IVF-ICSI treatment was associated with RIF.
Maternal and embryological factors play an important role in the etiology of RIF [6]. The most important maternal risk factors are advanced maternal age, high BMI, and smoking [9]. In addition, uterus abnormalities (uterine septum, etc.), uterine pathologies such as fibroids and polyps may interfere with implantation [7]. Endometriosis is known to decrease the implantation rate [8]. Therefore, in order not to compromise the results of our study, we excluded patients with uterine pathologies and patients with known endometriosis and endometrioma. Shapiro et al. [25] reported higher rates of embryo-endometrial mismatch, increased biochemical pregnancies, and lower live birth rates with increasing maternal age. We did not include patients older than 40 years in our study. It is also known that embryo quality is an essential factor affecting outcome, regardless of embryo developmental stage and number of embryos transferred [5]. Our study included only patients with good-quality (grade 1 and 2) embryo transfer.
There was no difference between the two groups regarding the cause of
infertility (tubal factor, male factor, unexplained infertility, and low ovarian
reserve). The low ovarian reserve was lower in group 1 (n: 4) than in group 2 (n:
7), and the difference was not significant. Patients with fewer than 4 follicles
on hCG day were used as an exclusion criterion in the definition of RIF because
the number of oocytes retrieved per cycle would decrease due to lower
implantation [21]. We also excluded patients with fewer than 4 follicles on hCG
day. The duration of infertility was significantly higher in RIF patients
(p
Routine genetic evaluation is not performed, while embryo morphology is assessed
for embryological factors. The prevalence of chromosomal aberrations, including
translocations, mosaicism, inversions, and deletions in RIF patients is 2%, with
the most common abnormality being a translocation [27]. Although the incidence is
low, preimplantation genetic diagnosis, embryo coculture, and preferential
blastocyst transfer are recommended in couples with RIF to minimize the embryonic
component [4]. Assisted hatching seems to slightly increase the achievement of
clinical pregnancy, but there is insufficient evidence for live birth rate [28].
In our study, there was no difference between the two groups in terms of ET day
and use of hatching assistance. Coculture was used in only 3 patients. Because no
information on preimplantation genetic diagnosis was available, patients with
chromosomal abnormalities (such as translocation carriers) in the parents were
excluded from the study. Our study found that RDW was significantly higher in the
RIF group than in the control group (p = 0.02) (Table 3). The difference
between the other CBC parameters was not significant (p
Considering all these mechanisms, high RDW may indicate insufficient trophoblast invasion due to increased inflammation, endothelial dysfunction, and thrombosis in spiral arterioles. In addition, shorter telomere length in the developing embryo could be a marker for short survival and biochemical abortion processes and predict RIF. Further studies are needed to understand whether there is a cause or effect here. Because the total number of patients with RIF per clinic in IVF clinics is small, multicenter and international research is needed to investigate the uncertainties surrounding this phenomenon in a good quality manner.
Our study was a retrospective study; no power analysis was performed and data from a single tertiary center were used. Despite evaluation over a period of more than 10 years, the number of patients was lower than expected because the group of RIF patients is relatively small and heterogeneous, and the cause was excluded. All patients included in the study did not have genetic analysis. This limits the ability of the results to reflect and generalize to the general population. Despite these limitations, this was the first study to examine the association between CBC parameters and RIF.
In our study, it was quite interesting that RDW stood out to be a significant negative predictive factor in RIF patients. This inexpensive and straightforward parameter provides valuable information for predicting subclinical and clinical diseases’ general health status, presence, and prognosis. Therefore, closer monitoring of RIF patients with elevated RDW values and future therapeutic interventions to lower RDW may be beneficial for RIF patients in desperate situations.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Conceptualization: ÖKG; Data Curation: ÖKG, OA, RÖ; Formal Analysis: ÖKG; Investigation: ÖKG, OA, RÖ; Methodology: ÖKG, OA, EB; Project Administration: ÖKG; Resources: ÖKG, OA, RÖ, EB, SD, ÖMT; Software: ÖKG, OA, RÖ, SD, ÖMT; Supervision: ÖKG, SD, ÖMT; Validation: ÖKG, OA; Visualization: ÖKG, OA, RÖ, SD; Writing – Original Draft Preparation: ÖKG; Writing – Review & Editing: ÖKG, SD, ÖMT. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work to take public responsibility for appropriate portions of the content and agreed to be accountable for all aspects of the work in ensuring that questions related to its accuracy or integrity.
The research project and protocols were approved by the Etlik Zubeyde Hanim Research and Training Hospital Institutional Review Board (21/12/2018/ issue 90057706-799). Since it was a retrospective study, the consent form was waived by the ethics committee.
Thanks to all the peer reviewers and editors for their opinions and suggestions.
This research received no external funding.
The authors declare no conflict of interest.
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