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Recurrent Pregnancy Loss Unit, The Capital Region, Copenhagen University Hospitals, Hvidovre Hospital, DK-2650, Hvidovre, Denmark and Rigshospitalet, Copenhagen, DenmarkDepartment of Obstetrics and Gynecology, The Fertility Clinic, Copenhagen University Hospital Hvidovre, DK-2650, Denmark
Recurrent Pregnancy Loss Unit, The Capital Region, Copenhagen University Hospitals, Hvidovre Hospital, DK-2650, Hvidovre, Denmark and Rigshospitalet, Copenhagen, Denmark
Recurrent Pregnancy Loss Unit, The Capital Region, Copenhagen University Hospitals, Hvidovre Hospital, DK-2650, Hvidovre, Denmark and Rigshospitalet, Copenhagen, Denmark
Recurrent Pregnancy Loss Unit, The Capital Region, Copenhagen University Hospitals, Hvidovre Hospital, DK-2650, Hvidovre, Denmark and Rigshospitalet, Copenhagen, DenmarkDepartment of Obstetrics and Gynecology, The Fertility Clinic, Copenhagen University Hospital Hvidovre, DK-2650, Denmark
Recurrent Pregnancy Loss Unit, The Capital Region, Copenhagen University Hospitals, Hvidovre Hospital, DK-2650, Hvidovre, Denmark and Rigshospitalet, Copenhagen, Denmark
Recurrent Pregnancy Loss Unit, The Capital Region, Copenhagen University Hospitals, Hvidovre Hospital, DK-2650, Hvidovre, Denmark and Rigshospitalet, Copenhagen, Denmark
Recurrent Pregnancy Loss Unit, The Capital Region, Copenhagen University Hospitals, Hvidovre Hospital, DK-2650, Hvidovre, Denmark and Rigshospitalet, Copenhagen, Denmark
Recurrent Pregnancy Loss Unit, The Capital Region, Copenhagen University Hospitals, Hvidovre Hospital, DK-2650, Hvidovre, Denmark and Rigshospitalet, Copenhagen, Denmark
Department of Obstetrics and Gynecology, The Fertility Clinic, Copenhagen University Hospital Hvidovre, DK-2650, DenmarkDepartment of Clinical Medicine, University of Copenhagen, Denmark
Recurrent Pregnancy Loss Unit, The Capital Region, Copenhagen University Hospitals, Hvidovre Hospital, DK-2650, Hvidovre, Denmark and Rigshospitalet, Copenhagen, DenmarkDepartment of Obstetrics and Gynecology, The Fertility Clinic, Copenhagen University Hospital Hvidovre, DK-2650, DenmarkDepartment of Clinical Medicine, University of Copenhagen, Denmark
What are the differences in menstrual blood lymphocytes between controls, patients with recurrent pregnancy loss (RPL) and patients with unexplained infertility (uINF)?
Design
Prospective study including 46 healthy controls, 28 RPL and 11 uINF patients. A feasibility study compared lymphocyte compositions of endometrial biopsies and menstrual blood collected during the first 48 h of menstruation in seven controls. In all patients, peripheral and menstrual blood from the first and subsequent 24 h were analysed separately by flow cytometry, focusing on the main lymphocyte populations and natural killer (NK) cell subsets.
Results
The first 24 h of menstrual blood resembles the uterine immune milieu as tested by endometrial biopsy. RPL patients showed significantly higher menstrual blood CD56+ NK cell numbers than controls (mean ± SD: 31.13 ± 7.52% versus 36.73 ± 5.4%, P = 0.002). Menstrual blood CD56dimCD16bright NK cells within the CD56+ NK cell population were decreased in RPL (16.34 ± 14.65%, P = 0.011) and uINF (15.7 ± 5.91%, P = 0.02) patients versus control (20.42 ± 11.53%). uINF patients had the lowest menstrual blood CD3+ T cell counts (38.81 ± 5.04%, control versus uINF: P = 0.01) and cytotoxicity receptors NKp46 and NKG2D on CD56brightCD16dim cells were higher in uINF (68.12 ± 11.84%, P = 0.006; 45.99 ± 13.83%, P = 0.01, respectively) and RPL (NKp46: 66.21 ± 15.36%, P = 0.009) patients versus controls. RPL and uINF patients had higher peripheral CD56+ NK cell counts versus controls (11.42 ± 4.05%, P = 0.021; 12.86 ± 4.29%, P = 0.009 versus 8.4 ± 3.5%).
Conclusions
Compared with controls, RPL and uINF patients had a different menstrual blood-NK-subtype profile, indicating an altered cytotoxicity. In future studies, this non-invasive analysis might enable identification and monitoring of patients receiving immunomodulatory medications.
). Patients with recurrent pregnancy loss (RPL) make up a subgroup of couples that experience infertility. Depending on guidelines, RPL is defined as at least two or more (consecutive) pregnancy losses from the time of conception until 24 weeks of gestation, and it affects 1–3% of couples trying to get pregnant (
). Oligo-/azoospermia, tubal factors, parental genetic abnormalities, uterine abnormalities and endocrine dysfunction are recognized risk factors for infertility, as well as RPL (
Diagnosis and Therapy Before Assisted Reproductive Treatments. Guideline of the DGGG, OEGGG and SGGG (S2k Level, AWMF Register Number 015-085, February 2019) - Part 1, Basic Assessment of the Woman.
Diagnosis and Treatment Before Assisted Reproductive Treatments. Guideline of the DGGG, OEGGG and SGGG (S2k Level, AWMF Register Number 015-085, February 2019) - Part 2, Hemostaseology, Andrology, Genetics and History of Malignant Disease.
). Yet, even with a comprehensive diagnostic work-up covering all the mentioned risk factors, the causes of around 50% of RPL and 30% of infertility cases (unexplained infertility, uINF) remain elusive (
Natural conception rates in couples with unexplained or mild male subfertility scheduled for fertility treatment: a secondary analysis of a randomized controlled trial.
). Yet T cells, particularly regulatory T cells (Treg), peripheral natural killer cells (pNK) and uterine natural killer cells (uNK) play a vital role as they are the main immunological regulators at the feto–maternal interface. Within recent years, the impact of pNK and uNK cells in infertility and RPL patients has been the focus of research (
). However, an endometrial biopsy is required for the examination of uNK cells, necessitating an invasive and uncomfortable procedure. More importantly, the small tissue samples are mainly analysed by immunohistochemistry, which limits the number of analysable markers and ultimately results in controversial data (
No difference in natural killer or natural killer T-cell population, but aberrant T-helper cell population in the endometrium of women with repeated miscarriage.
). Thus, it is not only necessary to improve the current approach to uNK cell profiling, but also to broaden the spectrum of immune markers in order to understand the endometrial immune function. Menstrual blood offers a non-invasive and convenient source of endometrial lymphocytes and may alleviate these issues (
A combination of immune cell types identified through ensemble machine learning strategy detects altered profile in recurrent pregnancy loss: a pilot study.
). However, published studies have used a variety of methods and rarely compare menstrual blood composition to endometrial biopsies. This study assesses lymphocyte subpopulations in endometrial biopsies, peripheral and menstrual blood samples of healthy individuals. Using this technique, it was possible to compare the peripheral and menstrual lymphocyte subpopulations in RPL and infertile patients to healthy controls, to clarify whether the uterine immune cell composition contributes to the RPL and uINF pathogenesis.
Materials and methods
Study population
In total, 46 healthy controls, 28 RPL patients and 11 uINF patients were included in this prospective study between March 2021 and February 2022. The controls consisted of nulliparous and parous women who had never experienced a pregnancy loss. RPL was defined as three or more consecutive pregnancy losses. Unexplained infertility was diagnosed when all the following criteria were met: unsuccessfully trying to conceive for at least 12 months; normal ovulatory cycles (24–35 days); bilateral tubal patency confirmed by either sono-hysterosalpingography or laparoscopy; a normal semen analysis according to the WHO criteria (
). In both patient groups, exclusion criteria were an abnormal 3D scan of the uterine cavity, chronic diseases (e.g. diabetes, chronic inflammatory bowel disease), endometriosis (grade 3 or 4), and in the uINF group: previous pregnancies (with another partner), a history of miscarriages or prior fertility treatment.
In controls, RPL and uINF patients, obstetric and medical histories as well as sociodemographic and lifestyle factors were obtained. These included age, body mass index (BMI), smoking, gravidity, parity and number of pregnancy losses. At least 3 months had to have passed since the last pregnancy loss before inclusion in the study. The sampling was performed in non-pregnant RPL or infertility patients and controls.
Sample collection
Peripheral blood
In tubes containing EDTA (Vacuette®, Greiner Bio-One, Kremsmünster, Austria), 36 ml of peripheral blood were collected on cycle day 2. Peripheral blood samples were delivered to the laboratory for rapid processing no later than 4 h after sampling.
Menstrual blood
During the first 48 h of menstruation, women used a menstrual cup (AllMatters ApS, Copenhagen, Denmark) to collect menstrual blood, which was then decanted into a Falcon tube. To be able to analyse the first 24 h of menstrual blood separately from the next 24 h, the blood was stored in two different Falcon tubes for each 24 h period. For storage at room temperature prior to processing, the Falcon tubes in which the menstrual effluent was stored contained 10 ml of RPMI-1640 Medium with 10% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA); and 0.3% sodium citrate (Merck, Darmstadt, Germany), to which pyruvate (1 mmol/l), penicillin (100 U/ml) and streptomycin (100 g/ml) were added (all from Thermo Fisher Scientific, Waltham, MA, USA). Samples were delivered to the laboratory for quick processing no later than 4 h after the last collection.
Endometrial biopsies
To compare endometrial biopsies with menstrual blood, in seven controls, menstrual blood was collected in two consecutive menstrual cycles, with an endometrial biopsy on cycle day 19–21 in the cycle following the first menstrual blood collection. Endometrial biopsies were performed using an endometrial suction cannula (Nexodis; Meringer, Kalisz, Poland). The specimen was then stored in 5 ml phosphate-buffered saline (PBS) (Gibco, Thermo Fisher Scientific) supplemented with 4% FBS (Sigma-Aldrich). Samples were delivered to the laboratory immediately after sampling.
Cell isolation
Peripheral blood
Centrifugation with Lymphoprep™ Density Gradient Medium (STEMCELL Technologies, Vancouver, BC, Canada) was used to isolate peripheral blood mononuclear cells (PMC), following the manufacturer's instructions using Greiner Bio-One Leucosep Centrifuge Tubes (GreinerBio-One).
Menstrual blood
The volume, viscosity, coagulation and signs of apparent cell lysis of each menstrual blood sample was registered. Menstrual blood mononuclear cells (MMC) were isolated using density gradient centrifugation. In brief, menstrual blood was washed and strained once, to remove mucus and blood clots using a 150 μm cell strainer (pluriStrainer®, pluriSelect Life Science, Leipzig, Germany). MMC were isolated following centrifugation with Lymphoprep™ Density Gradient Medium (STEMCELL Technologies) and washed according to the manufacturer's instructions.
Endometrial biopsies
In the laboratory, endometrial biopsies were washed in PBS–FBS 4%, weighed and cut into small pieces using a sterile scalpel. After being diluted to a final volume of 5 ml PBS–FBS 2%, the tissue solution was digested with 0.25 mg/ml Collagenase D (Merck, Darmstadt, Germany). To stop the digestion, 200 μl of EDTA (Invitrogen, Waltham, MA, USA) were added, and the digested tissue was then run through a 150 μm and a 50 μm cell strainer (pluriStrainer®, pluriSelect Life Science).
Analysis of isolated lymphocytes and NK cell subsets
Peripheral blood, menstrual blood and endometrial biopsy cells were counted in a Neubauer chamber and washed in DPBS–FBS 1%, using 5 million cells per sample (300 μl). Samples were analysed using a previously established antibody panel with minor modifications (
). After blocking with FcR Blocking Reagent (Miltenyi Biotec, Bergisch Gladbach, Germany), the cells were stained with the antibody master mixes containing the following antibodies in various combinations: CD16, CD45, CD3, CD4, CD8, CD64, CD14, CD19, CD57, CD62L, CD335 (NKp46), CD127, CD25, CD56, CD314 (NKG2D) at room temperature for 20 min (see Supplementary Table 1 for antibody details). After washing twice with Gibco™ PBS (Thermo Fisher Scientific), samples were stained with 7AAD (BD Biosciences, San Jose, CA, USA). Finally, all samples were analysed using a BD LSRFortessa (BD Biosciences) flow cytometer. Data acquisition was stopped at 150,000 CD45+ cells and this criterium was met for all samples. The procedure was standardized with daily calibration using CST beads (BD Biosciences) to minimize inter-experimental differences. Data were analysed using FlowLogic (Inivai Technologies, Mentone, Victoria, Australia); the gating strategy is shown in Supplementary Figure 1. NK cell receptors were analysed based on the population of CD3–CD56dimCD16bright and CD3–CD56brightCD16dim NK cells.
Ethical approval
Signed informed consent was obtained from all participants, allowing analysis of all clinical and laboratory data presented in this paper. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the Declaration of Helsinki 1964 and its later amendments or comparable ethical standards. The study was approved by the local institutional review board (protocol number: H-20024175, 25 August 2020) as well as the Danish Data Protection Agency (P-2020-988).
Statistics
SPSS Statistics for Windows, Version 26 (IBM Corp., Armonk, NY, USA) was used for statistical analysis. Student's t-test was used to compare two groups if the raw data were normally distributed based on the Shapiro–Wilk normality test. If the variables were not normally distributed, the Mann–Whitney U-test was used instead. For multiple group comparisons, one-way analysis of variance (ANOVA) was used in case of homogeneity of variance tested by Levene test, and Kruskal–Wallis non-parametric test was applied if homogeneity of variance was missing. For linked samples (menstrual blood versus peripheral blood versus endometrial biopsy), the non-parametric Friedman test was used. If significant results were obtained, post-hoc analysis was performed using Gabriel and Dunn–Bonferroni to correct for multiple comparisons. GraphPad Prism, Version 8.2.1 for Windows (GraphPad Software Inc., San Diego, CA, USA) was used to create the figures. A significance level of P < 0.05 was considered statistically significant for all statistical tests conducted.
Results
Study population
Characteristics of controls, RPL and uINF patients are shown in detail in Table 1. Controls were significantly younger than RPL and uINF patients (mean ± SD: 27.8 ± 4.3 versus 32.8 ± 3.3 versus 30.6 ± 5.2 years, respectively, P < 0.001). Cycle length and parity did not differ between groups, while – as expected by inclusion criteria – RPL patients had more pregnancies than both uINF patients and controls (P < 0.001). RPL patients had a higher BMI than controls and uINF patients (control versus RPL versus uINF: 21.7 ± 3.4 versus 25.6 ± 8.6 versus 22.2 ± 2.0 kg/m2, P = 0.012).
Table 1Characteristics of RPL and uINF patients and controls
Comparison of lymphocytes and NK cell subsets in endometrial biopsies, peripheral and menstrual blood in healthy controls
A feasibility study compared the profiling of immune cells in endometrial biopsies, peripheral blood and menstrual blood samples. In all samples, the number of live cells (7AAD–) was high: 99.76 ± 0.16% in peripheral blood, 93.77 ± 6.88% in menstrual blood and 79.74 ± 11.86% in endometrial biopsies. Menstrual blood samples were further split into the first 24 h (menstrual blood day 1) and the second 24 h (menstrual blood day 2) after start of menstruation. The predominant lymphocyte populations in endometrial biopsies and menstrual blood day 1 were CD3+ T cells and CD56+ NK cells (Figure 1). In comparison to peripheral blood, T cells were less prevalent in endometrial biopsies and menstrual blood (peripheral blood versus endometrial biopsies P = 0.039, peripheral blood versus menstrual blood day 1 P = 0.002, peripheral blood versus menstrual blood day 2 P = 0.001), while CD56+ NK cells were markedly higher in endometrial biopsies and menstrual blood day 1 (peripheral blood versus endometrial biopsies P = 0.006, peripheral blood versus menstrual blood day 1 P = 0.016, all Figure 1a). However, menstrual blood day 2 samples had a distinct lymphocyte composition, particularly in terms of CD56+ NK cells, which were closer to peripheral blood (peripheral blood versus menstrual blood day 2 P = 0.95, Figure 1a). In comparison to endometrial biopsies and menstrual blood day 1, peripheral blood displayed an inverse relationship (both P < 0.001) for NK cell subpopulations of CD56brightCD16dim and CD56dimCD16bright cells. Interestingly, CD56+ cells on menstrual blood day 1 were 86.8 ± 8.08% CD56brightCD16dim, indicating a more cytoregulatory phenotype, as were CD56+ cells in endometrial biopsies. On menstrual blood day 2, CD56+ cell counts converged towards the numbers in peripheral blood (P = 0.0001 when compared with endometrial biopsies). Whilst not significantly different to endometrial biopsies and menstrual blood day 1, the figure of around 60% CD56brightCD16dim cells indicates a more peripheral blood-like cytotoxic phenotype (all Figure 1c). Based on these findings, it was decided to limit further analysis to menstrual blood day 1 because it more accurately resembled the uterine immune milieu. No effect was found of the endometrial biopsies on the menstrual blood lymphocyte composition of the cycle following the endometrial biopsies (all P > 0.05, see Supplementary Figure 2).
Figure 1Immune cell populations in peripheral blood, endometrial biopsies and menstrual blood in control patients. Scatter plot showing lymphocyte populations determined by fluorescence activated cell sorting analysis (mean ± SD). (a) Main lymphocyte populations, subpopulations are then divided to (b) CD3+ T cells and (c) CD56+ NK cells. Data presented as % of CD45+ cells (a, b) and % of CD56+ cells (c). Statistical analysis by analysis of variance and Dunn–Bonferroni multiple comparison test.
Comparison of lymphocyte subpopulations in peripheral and menstrual blood of RPL and uINF patients and controls
Peripheral blood
Peripheral lymphocyte subpopulations of the study population are shown in Figures 2a, 3a and 3c as well as Supplementary Table 2. When compared with controls, RPL and uINF patients had significantly higher peripheral CD56+ NK cell counts (8.4 ± 3.5%; 11.42 ± 4.05%; 12.86 ± 4.29%; control versus RPL P = 0.021; control versus uINF P = 0.009). Within the CD56+ NK cell population, uINF patients showed a significantly higher percentage of CD56dimCD16brightNKp46+ cells than controls and RPL patients (79.52 ± 8.89, 59.94 ± 14.31, 58.91 ± 13.22, uINF versus control P = 0.0012, uINF versus RPL P < 0.001).
Figure 2Main lymphocyte subsets in controls, recurrent pregnancy loss (RPL) and unexplained infertility (uINF) patients. Violin plot showing mean, quartiles and data distribution of lymphocyte populations determined by fluorescence activated cell sorting analysis on (a) peripheral blood and (b) menstrual blood day 1. Data presented as % of CD45+ cells. Statistical analysis by Kruskal–Wallis test followed by Dunn test in case of significant result.
Figure 3T cell and NK cell subsets in controls, recurrent pregnancy loss (RPL) and unexplained infertility (uINF) patients. Violin plot showing mean, quartiles and data distribution of CD3+ T cell subpopulations (a, b) and CD56+ NK cell subpopulations (c, d) determined by fluorescence activated cell sorting analysis on peripheral blood (a, c) and menstrual blood day 1 (b, d). Data presented as % of CD45+ cells (a, b) and % of CD56+ cells (c, d). Statistical analysis by Kruskal–Wallis test followed by Dunn test in case of significant result.
Menstrual blood lymphocyte subpopulations of the study population are shown in Figures 2b, 3b and 3d as well as Supplementary Table 3. RPL patients displayed markedly higher CD56+ NK cell numbers in comparison to controls (31.13 ± 7.52% versus 36.73 ± 5.4%; P = 0.002). In addition, compared with controls, the fraction of CD56dimCD16bright NK cells within the entire CD56+ NK cell population was decreased in individuals with RPL and uINF (20.42 ± 11.53%, 16.34 ± 14.65%, 15.7 ± 5.91%, respectively; control versus RPL P = 0.011, control versus uINF P = 0.02). Further, compared with controls, uINF patients had lower CD3+ T cell counts (47.93 ± 11.11%, 38.81 ± 5.04%, P = 0.01), with decreased CD4+ (25.78 ± 7.91%, 15.66 ± 4.88%, P = 0.001) and CD8+ T cells (15.32 ± 4.14% versus 8.43 ± 2.85%, P < 0.001). Interestingly, uINF patients showed higher CD25highCD127dim/neg regulatory T cell counts than controls (1.84 ± 0.51%, 1.34 ± 0.49%, P = 0.007). Regarding the subpopulation of CD56brightCD16dim NK cells (Table 2), uINF patients had higher percentages of NKG2D+ NK cells (45.99 ± 13.83% versus 28.02 ± 13.57%, P = 0.010), while RPL and uINF patients showed higher proportions of NKp46+ NK cells (66.21 ± 15.36%, 68.12 ± 11.84%, respectively, versus 56.11 ± 11.45%, control versus RPL P = 0.009, control versus uINF P = 0.006). The number of NKG2D+ cells was also higher in the CD56dimCD16bright NK cell subset in uINF patients, but this was only significant when comparing to RPL patients (41.70 ± 11.42% versus 29.05 ± 15.65%, P = 0.045).
Table 2NK cell subsets in controls, RPL and uINF patients in menstrual blood
Embryo implantation remains one of the most enigmatic processes in human reproduction. Therefore, understanding the mechanisms governing implantation is of the utmost importance to help women suffering from uINF and RPL, as well as to improve success rates in treatments with assisted reproductive technologies. So far, if performed, uterine immune profiling has relied on a biopsy – an invasive and painful procedure. This study provides proof-of-concept of a non-invasive profiling of the uterine immune milieu with lymphocytes isolated from menstrual blood collected in menstrual cups as compared with endometrial biopsies. Furthermore, this approach was used to compare the uterine immune milieu in healthy controls, uINF and RPL patients. Peripheral blood, menstrual blood and endometrial biopsies were compared to evaluate whether endometrial biopsies and menstrual blood consist of comparable lymphocyte subsets. This was not performed in previous studies analysing menstrual blood with fertility disorders (
A combination of immune cell types identified through ensemble machine learning strategy detects altered profile in recurrent pregnancy loss: a pilot study.
Comparative analysis of NK cell subsets in menstrual and peripheral blood of patients with unexplained recurrent spontaneous abortion and fertile subjects.
). Within the current study, endometrial biopsies showed the clearest contrast to peripheral blood considering CD56+ NK cell subsets: while the cytotoxic subset of CD56dimCD16bright NK cells was more prevalent in peripheral blood, the cytoregulatory CD56brightCD16dim NK cells were more prevalent in endometrial biopsies and within the first 24 h of menstruation in menstrual blood. Menstrual blood of the second 24 h, however, mainly consisted of other subsets, more resembling those of peripheral blood, or at least of a combination of peripheral blood and menstrual blood – with a different CD56bright/dimCD16bright/dim combination. The initial endometrial shedding during the first 24 h and a larger concentration of peripheral blood in the menstrual effluent throughout the subsequent days of menstruation might explain this observation (
). This shift was not observed in menstrual blood, which was consistent with previous results in menstrual blood and might be related to CD8+ T cell-rich lymphoid aggregates seen mostly in the stratum basalis of the endometrium and not shed with the functionalis layer (
). A possible caveat of using menstrual blood instead of endometrial biopsies is that by the time menstrual blood lymphocytes are collected, the menstrual cycle has ended, and the endometrial structure is disintegrating. Furthermore, the period of collection does not represent the time of implantation. Nonetheless, based on this and earlier studies, it can be concluded that the menstrual blood lymphocytes are still viable cells, with an NK cell profile that is very similar to endometrial biopsies, and the NK cells can still produce cytokines (
Enhancement of peripheral blood CD56(dim) cell and NK cell cytotoxicity in women with recurrent spontaneous abortion or in vitro fertilization failure.
Peripheral natural killer cytotoxicity and CD56(pos)CD16(pos) cells increase during early pregnancy in women with a history of recurrent spontaneous abortion.
Pre-conceptional natural killer cell activity and percentage as predictors of biochemical pregnancy and spontaneous abortion with normal chromosome karyotype.
). In this study, only uINF patients had an elevated number of peripheral NKp46+CD56dimCD16bright NK cells, highlighting the complexity and possibly misleading approach of peripheral blood analysis in patients who may have an altered uterine immune milieu not reflected by peripheral blood.
Several studies have found increased uNK cells in RPL patients (
). In this study, CD56+ menstrual blood NK cells were significantly higher in RPL and uINF patients, indicating an altered uterine immune environment. A previous study on 15 RPL women compared with 15 healthy controls revealed no significant differences in menstrual blood NK cell numbers (
Comparative analysis of NK cell subsets in menstrual and peripheral blood of patients with unexplained recurrent spontaneous abortion and fertile subjects.
). Here, this analysis is extended to include the cytotoxicity of NK cell populations. In the current study, RPL and uINF patients had elevated expressions of NK2D and NKp46, especially in the CD56brightCD16dim subsets. The NKG2D and NKp46 receptors are regarded as one of the main activation receptors for NK cytotoxicity, and are, along with NKp30 and NKp44, considered as natural cytotoxicity receptors (
). A study on decidual NK cells in 21 patients with sporadic pregnancy losses showed a higher frequency of NKp44 and NKp46 in comparison to healthy pregnancies (
). It can be speculated that a higher expression of NKp46 and NKp44 receptors may sensitize NK cells to activation signals and lower the threshold to trigger (uterine) NK cells, because activation of NKp46 and NKG2D can directly activate NK lysis. The development of NK receptor expression throughout the first trimester of pregnancy might further have an influence on the release of cytokines and angiogenic factors, in accordance with the previously documented regulation of decidual NK secretion patterns (
. It has been demonstrated that increased NKG2D expression can protect the fetus against pathogens, because the NKG2D ligands MICA/B and ULBP1–5 are primarily triggered by cellular stressors such as cell damage or pathogen infection (
). An aberrant expression pattern of activating receptors, as seen in RPL and uINF patients in this study, could however lead to an altered NK activation homeostasis, leading to RPL or even infertility.
The current study set-up, with two consecutive menstrual blood collections separated by an endometrial biopsy in the luteal phase, enabled the study of endometrial scratching (intentionally disrupting the endometrial surface by performing a biopsy), the aim of which is to contribute to an increased endometrial receptivity and thereby higher implantation rates by inducing a local inflammatory response (
Endometrial scratch injury for women with one or more previous failed embryo transfers: a systematic review and meta-analysis of randomized controlled trials.
). This intervention did not result in a significant impact on menstrual blood lymphocyte counts here. Previous studies have reported an impact on cytokines such as leukaemia inhibitory factor or oncostatin M (
). So, rather than influencing lymphocyte populations, the technique could impact cytokine concentration and thereby influence implantation. A positive effect on implantation is, however, questionable, as large randomized controlled trials and a Cochrane meta-analysis have indicated that endometrial scratching had no effect on live birth rate (
The strengths of this study include well-phenotyped participants and that the immunological milieu of the endometrium is directly compared for endometrial biopsy and menstrual blood. However, the small sample size limits the ability to extrapolate the findings to other populations, as well as to perform further subgroup analyses in controls, RPL and uINF. In first-trimester decidua, single-cell RNA sequencing (scRNAseq) has revealed three subpopulations of uNK, which have been formerly assumed to comprise a single population (
). They were first described as decidual NK (dNK) 1, 2 and 3, but have now also been discovered in non-pregnant endometrium and can also be identified by flow cytometry using CD49a, CD9, CD39 and CD103 (
). The present study did not focus on these subsets but describes the pNK and menstrual blood NK cells based on their CD56dim/brightCD16dim/bright and cytotoxicity receptors. Future studies could try to also identify and describe these subsets in the menstrual blood, in larger cohorts, potentially in a multicentre setting.
It should also be noted that there were significant differences in age and BMI between controls, RPL and uINF patients in this study, which could affect the results. In previous research on RPL patients and healthy controls, pNK and uNK cell counts were not affected by clinical criteria such BMI, age or the time since the previous pregnancy loss (
). With a maximum age difference of 5 years and a mean BMI of 25.6 kg/m2 in RPL patients compared with 21.7 kg/m2 in controls, the modest variations seen in the current study sample are unlikely to influence the outcomes presented.
In conclusion, this study presents a reliable and painless method to analyse the uterine immune milieu. This biopsy-free method showed significant differences in the menstrual blood lymphocyte composition between uINF, RPL patients and controls. In future studies, the method's non-invasive nature could be used to identify and monitor patients who benefit from (immunomodulatory) treatment options.
Author roles
HSN, AMK and PE developed the study concept. Data acquisition was performed by KV, PE, IB-M, ADB, MLB, CSE and MRR; KV wrote the first draft of the manuscript. NlCF and HSN supervised the project and provided guidance. All authors participated in critical discussion of the results, critically revised the manuscript and approved the final version of the paper.
Data availability
Data will be made available on request.
Acknowledgements
We would like to thank Renate Van Der Molen for advice on establishing the isolation procedure for the menstrual blood, especially concerning the medium used.
This study was funded by the William Demant Fonden. KV was supported by the ESHRE Travelling Fellowship. The funding institutions were not involved in the study design, analysis, interpretation of data, writing the paper or the decision to submit the manuscript for publication.
A combination of immune cell types identified through ensemble machine learning strategy detects altered profile in recurrent pregnancy loss: a pilot study.
Peripheral natural killer cytotoxicity and CD56(pos)CD16(pos) cells increase during early pregnancy in women with a history of recurrent spontaneous abortion.
Comparative analysis of NK cell subsets in menstrual and peripheral blood of patients with unexplained recurrent spontaneous abortion and fertile subjects.
Enhancement of peripheral blood CD56(dim) cell and NK cell cytotoxicity in women with recurrent spontaneous abortion or in vitro fertilization failure.
No difference in natural killer or natural killer T-cell population, but aberrant T-helper cell population in the endometrium of women with repeated miscarriage.
Diagnosis and Therapy Before Assisted Reproductive Treatments. Guideline of the DGGG, OEGGG and SGGG (S2k Level, AWMF Register Number 015-085, February 2019) - Part 1, Basic Assessment of the Woman.
Diagnosis and Treatment Before Assisted Reproductive Treatments. Guideline of the DGGG, OEGGG and SGGG (S2k Level, AWMF Register Number 015-085, February 2019) - Part 2, Hemostaseology, Andrology, Genetics and History of Malignant Disease.
Natural conception rates in couples with unexplained or mild male subfertility scheduled for fertility treatment: a secondary analysis of a randomized controlled trial.
Endometrial scratch injury for women with one or more previous failed embryo transfers: a systematic review and meta-analysis of randomized controlled trials.
Pre-conceptional natural killer cell activity and percentage as predictors of biochemical pregnancy and spontaneous abortion with normal chromosome karyotype.
Kilian Vomstein is a gynaecologist at The Fertility Clinic, Department of Obstetrics and Gynecology, Copenhagen University Hospital Hvidovre and at the Recurrent Pregnancy Loss Unit Copenhagen, Denmark. His research focuses on the immunology of the feto–maternal interface – especially NK cells – and the interaction with the local microbiome in recurrent pregnancy loss and recurrent implantation failure.
Key message
Compared to controls, recurrent pregnancy loss and unexplained infertility patients have a different menstrual blood-NK-subtype profile, indicating an altered cytotoxicity in these patients. Menstrual blood can be used instead of endometrial biopsies in a clinical set-up to study immune alterations in a non-invasive way.
Article info
Publication history
Published online: March 28, 2023
Accepted:
March 22,
2023
Received in revised form:
March 10,
2023
Received:
January 16,
2023
HSN has received speaker's fees from Ferring Pharmaceuticals, Merck Denmark A/S, Gedeon Richter, Astra Zeneca, Cook Medical and Ibsa Nordic (outside the submitted work). KV received speaker's fees from Merck Denmark A/S and support for travel to conferences from Ferring Pharmaceuticals (outside the submitted work). NlCF has received grants from Gedeon Richter, Merck and Cryos (outside the submitted work). The other authors did not report any potential conflicts of interest. All authors declared no conflict of interest in relation to this work.
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