Adverse impact of CD138+ cells in proliferative-phase endometrium on pregnancy outcomes in fresh IVF/ICSI cycles.

In our study we analyzed the impact of CD138 + cells in proliferative-phase endometrium on infertile women. CD138 + cells seemed to be an adverse indicator for pregnancy outcomes in fresh IVF/ICSI cycles, with a certain value in predicting non-pregnancy. Furthermore, pregnancy outcome was poor for patients with ≥ 2 CD138 + cells/HPF in the endometrium, and it may worsen as the number of CD138 + cells increase.

Chronic endometritis (CE) is a chronic inflammatory condition of the endometrium and generally has no obvious symptoms or only causes light symptoms (pelvic discomfort, spotting, and leucorrhea). [ 1 ] CE can easily be overlooked by patients and ignored by gynecologists. However, research has increasingly suggested that CE is associated with infertility in women. [ 2 , 3 ] Indeed, the prevalence of CE is 15% in infertile women who undergo in vitro fertilization (IVF) cycles [ 4 ] and obviously elevated in women with recurrent implantation failure or recurrent pregnancy loss (RPL). [ 2 ] Therefore, the effective diagnosis and treatment of CE may benefit infertile patients seeking help getting pregnant through assisted reproductive techniques (ART). At present, there are no unified standards or guidelines for the diagnosis of CE. Currently, the literature indicates that plasma cell infiltration within endometrial stromal tissue is the most specific and sensitive manifestation of this disease. [ 5 , 6 ] Syndecan-1 (CD138) is a transmembrane-type heparan sulfate proteoglycan relatively specifically expressed on the membrane of plasma cells, and immunohistochemical (IHC) staining for CD138 revealed a significantly higher sensitivity for CE diagnosis. [ 5 , 7 , 8 ] Although CD138 + plasma cell detection by IHC staining has been extensively applied by assisted reproduction centers, the definition of clinically relevant CE has no consensus cutoff value. [ 9 ] Jonhston et al [ 10 ] recommended finding more than 1 CD138 + cell per high-power field (HPF) for CE diagnosis, while Chen et al [ 11 ] suggested 5 or more CD138 + cells per HPF as indicative of CE. The diagnostic criterion of CE was considered to be ≥ 5 CD138 + cells in 10 nonoverlapping HPFs in a study by Bouet et al [ 12 ] and ≥ 5 CD138 + cells in 20 nonoverlapping HPFs by Kitaya et al [ 5 ] Due to the widely varying diagnostic criteria for CE, patients could be either over- or undertreated by clinical interventions for CE. A recent study demonstrated that, among the patients who had experienced previous embryo transfer (ET) failure, the clinical pregnancy rate and embryo implantation rate in the next frozen embryo transfer decreased significantly when CD138 + cells were present in the secretory phase endometrium. [ 13 ] Another study indicated that detection of secretory-phase endometrial CD138 + cells could predict subsequent reproductive outcome in RPL women and more than 4 to 6 cells/HPF suggested a higher risk for adverse outcomes. [ 14 ] Li et al [ 9 ] found that more than 5 CD138 + cells/HPF was adverse for influencing pregnancy outcomes, and the endometrial tissue samples were similarly collected in secretory phase. Few studies have specifically focused on the impact of CD138 + cells in the proliferative-phase endometrium on pregnancy outcomes in fresh ET cycles. Therefore, in this study we aimed to explore the impact of proliferative-phase endometrial CD138 staining on pregnancy outcomes in fresh ET IVF or intracytoplasmic sperm injection (ICSI) cycles.

Conceptualization: Jie Li, Lijuan Yang. Data curation: Jie Li, Dujuan Xu, Ling Ma. Formal analysis: Jie Li. Investigation: Dujuan Xu, Ling Ma, Lin Li. Methodology: Jie Li, Lin Li. Writing – original draft: Jie Li. Writing – review & editing: Jie Li, Lijuan Yang.

We retrospectively collected information from 273 patients who underwent IVF/ICSI with fresh embryo transfer following an endometrial curettage in the proliferative-phase from January 2020 to May 2022 in the Reproductive Hospital of Jiangxi University of Traditional Chinese Medicine. These patients had no or only 1 previously failed cycle. Patients with uterine abnormalities, endometriosis, intrauterine adhesions (moderate-severe), adenomyosis, uterine mediastina and ovarian tumors, untreated hydrosalpinx, or abnormal uterine bleeding were excluded. All data of patients on demographics, baseline values, and pregnancy outcomes were available. We obtained ethical approval from the Medical Ethics Committee of Nanchang Reproductive Hospital (the Reproductive Hospital of Jiangxi University of Traditional Chinese Medicine) (approval number 2022.005). A gonadotropin-releasing-hormone antagonist or gonadotropin-releasing-hormone agonist long regimen was used for ovarian stimulation in all patients. When 60% of the follicles reached 16 mm, ovulation was triggered by subcutaneous injection of 250 μg recombinant human choriogonadotropins-ping alfa solution (Merck Serono S.P.A, BA061210). Oocytes were retrieved 34 to 36 hours later and were fertilized by IVF/ICSI. Embryos were in G1-plus/G2-plus media until day 3 or day 5/6 and then transferred into the patient’s uterine cavity. Clinical pregnancy was defined as the presence of a gestational sac with yolk sac, fetal heart, and fetal bud detected by color Doppler ultrasound on the 28th day after ET. Endometrial tissue was obtained by curettage within 3 to 5 days following menstruation in the previous cycle of IVF/ICSI and then used for the detection of plasma cells by IHC staining. Paraffin-embedded sections of 4% formalin-fixed endometrial samples were routinely dewaxed and hydrated. Anti-CD138 (Maixin Biotech, 1015201016) was used for IHC staining following a standardized protocol. The CD138 + cells were observed and counted under high magnification microscopy, and the CD138 + cells/HPF count was recorded. All statistical analyses were performed using the SPSS software (IBM SPSS Statistics 26, SPSS Inc., Chicago, IL), and P  < .05 was considered significant. Continuous data are presented as mean ± standard deviation, and categorical data are presented as percentage (count). All continuous data in this study were non-normal, and the Mann–Whitney U test was applied to assess intergroup differences. For comparing categorical data, the Chi-squared test, continuity-corrected Chi-squared test, or Fisher exact-probability test was performed, where applicable. We used receiver operating characteristic curve analysis to determine the relationship between the number of CD138 + cells per HPF and the clinical pregnancy outcome. The optimal cutoff value was identified by the highest combined sensitivity and specificity using Youden index.

Among the total of 273 patients recruited, 149 patients became pregnant (i.e., pregnant group) in the fresh transfer IVF/ICSI cycles and 124 did not become pregnant (i.e., nonpregnant group). Patient demographics and relevant baseline values of the pregnant and nonpregnant groups are compared in Table 1 . There were no statistically significant differences in cycle type, age, BMI, duration of infertility, infertility type, ovarian stimulation regimens, basal levels (i.e., follicle-stimulating hormone, luteinizing hormone, estradiol, and anti-Müllerian hormone), or endometrial thickness on HCG Day between the 2 groups. The number of embryos transferred and the rate of high-quality embryos transferred also revealed no significant differences. Of note, the CD138 + cells/HPF of the nonpregnant group was significantly higher than the pregnant group (2.36 ± 4.24 vs 1.31 ± 3.41, P  = .008). Demographics and baseline values of the pregnant and non-pregnant groups in the study. Variables are expressed as mean ± standard deviation unless otherwise stated. AMH = anti-Müllerian hormone, BMI = body mass index, CD138 = syndecan-1, E2 = estradiol, FSH = follicle-stimulating hormone, GnRH = gonadotropin-releasing-hormone, HCG = serum β human chorionic gonadotropin, HPF = high-power field, ICSI = intracytoplasmic sperm injection, IVF = in vitro fertilization, LH = luteinizing hormone, No. = number. We estimated the value of the CD138 + cell count for predicting the pregnancy outcome in fresh transfer IVF/ICSI cycles. The receiver operating characteristic curve of CD138 + cells/HPF was drawn (Fig. 1 ), and the area under the curve was 0.572 (95% CI [0.504, 0.641]; P  = .035). The optimal cutoff value was 2 CD138 + cells/HPF, with a sensitivity of 33.1% and specificity of 81.2%. ROC curve analysis of CD138 + cells/HPF in predicting non-pregnancy. CD138 = syndecan-1, HPF = high-power field, ROC = receiver operating characteristic. Next, we analyzed the pregnancy outcome after grouping based on a cut off value of CD138 + cells. Patients were divided into a negative group (CD138 + cells/HPF < 2, n   = 204) and a positive group (CD138 + cells/HPF ≥ 2, n   = 69) according to the cut off value of 2 CD138 + cells/HPF. Compared with the negative group, the positive group had a significantly lower clinical pregnancy rate (71.8% vs 40.6%, P  < .001). We further established additional subgroups: Group1 (CD138 + cells/HPF  8, n   = 20). The clinical pregnancy rates with respect to Groups 1, 2, and 3 were 71.8%, 44.9%, and 30.0%, respectively ( P  < .001), revealing a gradually decreasing trend (Fig. 2 ). Pregnancy outcomes in different subgroups as stratified by CD138 + cells/HPF. Group1 (CD138 + cells/HPF  8, n = 20). CD138 = syndecan-1, HPF = high-power field.

CE is generally an asymptomatic condition and, therefore, lacks attention in the field of gynecology, [ 15 ] although recent years have witnessed substantially increased research regarding CE in reproductive medicine. [ 16 ] Because of the high prevalence of CE in unexplained infertile women [ 17 ] and in patients experiencing recurrent implantation failure [ 12 , 18 , 19 ] or RPL, [ 12 , 20 ] it emerged as an important clinical challenge of reproductive success. Moreover, CE is difficult to diagnose and troublesome for clinicians. Despite the identification of plasma cells by IHC staining of CD138 being the gold standard diagnostic method, unified diagnostic criteria for CE have not yet been established. [ 2 , 21 ] Some studies suggested that antibiotic treatment could improve the pregnancy outcomes of infertile patients with high CD138 + cell counts, [ 17 , 22 , 23 ] whereas a recent meta-analysis reported that antibiotic treatment did not improve pregnancy outcomes in patients with CE. [ 24 ] This may be attributable to the lack of reliable criteria for CE making it difficult to evaluate the effectiveness of treatments for CE in improving pregnancy outcomes. Thus, it is crucial to find a suitable cut off value of CD138 + cell count for diagnosing CE and guiding therapy. Generally, B lymphocytes are postulated to be located in the basal layer of the endometrium [ 16 ] and to differentiate into plasma cells when stimulated by inflammation. [ 25 ] The functional layer of the proliferative-phase is thinner than that of the secretory phase, so biopsy performed in the proliferative-phase could obtain more basal layer tissue. In practical work, we have observed it is easier to detect CD138 + cells, and we usually detect more CD138 + cells when we biopsy the endometrium in the proliferative-phase. A wide variation in the prevalence of CE had been observed across studies, from 2.8% to 56.8%. [ 26 – 29 ] This may be attributed to the nonunified diagnostic criteria for CE and the variation of biopsy timing (proliferative or secretory phase, or not specified). In this study, we evaluated the impact of CD138 + cells in proliferative-phase (3–5 days following menstruation) endometrium on pregnancy outcomes in fresh IVF/ICSI cycles. Our results demonstrated that pregnancy failure was associated with higher endometrial CD138 + cell counts compared with pregnancy; and CD138 + cells had a certain predictive value for pregnancy outcomes, with the area under the curve being 0.572. Further analysis revealed that the clinical pregnancy rate was only 40.6% (a decrease of about 30.0%) when endometrial IHC detection revealed ≥ 2 CD138 + cells/HPF. For > 8 CD138 + cells/HPF, the clinical pregnancy rate further decreased to 30.0%. From our results, women with ≥ 2 CD138 + cells/HPF in the endometrium have a significantly decreased chance of pregnancy, which further reduces as the CD138 + cell count increases. Fresh embryo transfers can minimize therapy time and reduce costs compared with frozen embryo transfer. However, for infertile patients, embryos are hard-won and extremely precious. In the endometrium of a CE patient, the distributions and functions of epithelial cells, stromal cells, and immune cells are altered. [ 2 ] Aberrant expression of adhesion molecules and cytokines (e.g., E-selectin, CXCL1, and CXCL13), [ 28 ] change of proliferative phenotype, [ 25 ] and decreased CD56 bright CD16 − or CD56 + CD16 − NK cells [ 30 ] all reduce the endometrial receptivity of CE patients. CE greatly impairs implantation and needs to be effectively addressed before ET, which means the diagnosis of CE is crucial. From the data of this study, we suggest that cancelation of fresh ET could be a better alternative for patients with ≥ 2 CD138 + cells/HPF in the proliferative-phase endometrium. Patients can freeze embryos and receive further ART after amelioration of endometrial inflammation. Certainly, this study had the limitation of the intrinsically retrospective nature of the design. The results were from a single center, so multicenter clinical studies are needed to confirm the validity and clinical applicability of the results. Furthermore, the sample size was not very large, and we did not display the pregnancy data of patients following effective antibiotic therapy. In the future, we will employ a prospective study with a larger sample size to seek suitable cutoff values of CD138 + cells for defining CE in both proliferative-phase and secretory-phase endometrium. Meanwhile, for patients exceeding the cut off value of CD138 + cells, we will assess the impact of antibiotic therapy on pregnancy outcomes to guide the regimens of ART.