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Aim: We aimed to assess the amniotic fluid glucose concentration cut‐off as an indicator of fetal chromosomal abnormalities, such as trisomy 13, 18, and 21. Methods: This prospective observational study included pregnant females who underwent amniocentesis. Participants were divided into two groups on the border of 22 weeks of gestational age (<22 and ≥22‐week groups). Results: In total, 224 pregnant females were included in the analysis. In the <22 week group, 15 females had trisomies 13/18/21 and 174 females had no trisomies. In the ≥22 week group, 18 females had trisomies 13/18/21 and 17 had no trisomies. In each group, there was a difference in amniotic fluid glucose concentration between fetuses with trisomies 13, 18, and 21 and other fetuses with normal karyotype or minor chromosomal abnormalities. In both groups, the amniotic glucose concentration was noticeably lower in trisomies 13/18/21 (p = 0.002 in the <22 week group; p = 0.039 in the ≥22 week group). According to receiver operating characteristic curves, the optimal cut‐off point of glucose concentration was 46 mg/dL in the <22 week group (odds ratio 6.55; 95% confidence interval 1.78–24.1) and 24 mg/dL in the ≥22 week group (odds ratio 8.40; 95% confidence interval 1.83–38.6). Conclusions: Our study suggested that glucose concentration in amniotic fluid is an indicator of trisomy 13, 18, and 21. Amniotic fluid glucose concentration itself does not diagnose fetal trisomy, but this may be helpful in selecting treatment facilities. [ABSTRACT FROM AUTHOR]

Aims: This study aims to comprehensively examine the employment and practices of embryologists in Japan's assisted reproductive technology (ART) laboratories, focusing on the impact of various factors such as ART cycle numbers, add‐ons, and regional differences. Additionally, it seeks to assess the extent to which Japanese ART facilities meet international minimum standards set by the American Society for Reproductive Medicine (ASRM). Methods: A survey was conducted from December 2021 to February 2022 among 621 ART facilities in Japan. The study categorized facilities into five ART cycle groups and compared the number of embryologists across these groups. It also examined the correlation between the number of embryologists, ART cycles, add‐ons, and regional differences. Data were analyzed using linear regression and multiple linear regression analyses. Results: The study's findings revealed a significant correlation between the total number of embryologists at each facility and the ART cycles. Notably, there were significant differences in the number of embryologists across all ART cycle categories. Of the 435 facilities, only 44.6% met the ASRM minimum embryologist staffing requirement. The regression analysis further highlighted the significance of ART cycles and preimplantation genetic testing for aneuploidies as factors. Moreover, the number of embryologists stationed at urban facilities was significantly higher than at nonurban facilities, indicating a potential regional disparity. Conclusion: In Japan, it was first found that more than 50% of ART facilities do not have sufficient embryologists in place relative to the number of ART cycles. Furthermore, the add‐ons and regional differences affect the placement of embryologists. [ABSTRACT FROM AUTHOR]

Aim: To investigate variation in the diagnosis and treatment of chronic endometritis (CE) at the national level in Japan. Methods: We performed a nationwide survey targeting all assisted reproductive technology (ART) facilities across Japan between 2021 and 2022. Diagnostic methods, criteria, and first‐ and second‐line treatment protocols for CE were collected via a questionnaire. Results: Among 616 ART facilities, 437 responded to the survey (response rate: 70.9%) of which 339 (77.6%) implemented diagnosis and treatment of CE. In the diagnosis of CE, 214 (63.1%) facilities used CD138 immunohistochemical staining of endometrial tissue, while hysteroscopy was the most frequently used as an adjunct diagnostic method (241 facilities, 71.1%). The most frequent cutoff value of CD138‐positive cells for diagnosing CE was 3–5 cells/20 high‐power fields (50%), but 7.9% (17 facilities) and 5.1% (11 facilities) used cutoff values of 1 and 2 cells, respectively. The most common first‐ and second‐line treatment methods were doxycycline (210 facilities, 61.9%) and ciprofloxacin + metronidazole (164 facilities, 48.0%), respectively. Conclusions: There is considerable variation in the number of CD138‐positive cells used for diagnosing CE. Establishing unified diagnostic criteria and therapeutic methods for CE is essential to provide standardized medicine for CE at the national level. [ABSTRACT FROM AUTHOR]

Aims: In anticipation of the future development of assisted reproductive technology (ART) and to smoothly introduce new technology, it is necessary to understand the current staffing status of the medical system and the current state of treatment, as well as the status of in vitro fertilization add‐ons, where the need for insurance coverage is currently a matter of debate. Methods: ART facilities in Japan were surveyed (437 valid responses, response rate: 71%). Current staffing status of the medical system, implementation rates of ART, add‐on treatments, and medical supplies were investigated. Results: Despite the abundance of embryologists, nurses, and obstetricians and gynecologists in facilities, the majority of facilities lacked counselors, anesthesiologists, and other essential medical professionals. Conventional ovarian stimulation was widely adopted (median 120 [interquartile range 60–300] cycles), followed by mild ovarian simulation (60 [30–200]). Additionally, freeze–thaw embryo transfer cycles (300 [120–750]) were performed more frequently than fresh embryo transfer cycles (30 [30–60]). Among the add‐ons, assisted hatching (85.1%), chronic endometritis examination (77.2%) and treatment (76.9%), artificial oocyte activation (67.3%), endometrial receptivity analysis (64.2%), and endometrial microbiome analysis (58.9%) were relatively widely employed. Conclusions: The implementation of frozen–thawed embryo transfer cycles, freeze‐all strategies, and add‐on treatments have become popular and widely accepted despite the lack of robust evidence regarding their safety and efficacy. [ABSTRACT FROM AUTHOR]

Purpose: Dynamic morphological changes in the chromosome and cytoskeleton occur in mammals and humans during early embryonic development, and abnormalities such as embryonic chromosomal aneuploidy occur when development does not proceed normally. Visualization of the intracellular organelles and cytoskeleton allows elucidation of the development of early mammalian embryos. The behavior of the DNA and cytoskeleton in early mammalian embryos has conventionally been observed by injecting target molecule mRNAs, incorporating a fluorescent substance‐expressing gene, into embryos. In this study, we visualized the chronological behavior of male and female chromosome condensation in mouse embryos, beginning in the two‐pronuclear zygote, through the first division to the two‐cell stage, using fluorescent chemical probes to visualize the behavior of DNA, microtubules, and microfilaments. Method: Mouse two‐pronuclear stage embryo were immersed in medium containing fluorescent chemical probes to visualize DNA, microtubules, and microfilaments. Observation was performed with a confocal microscope. Results: This method allowed us to observe how chromosome segregation errors in first somatic cell divisions in mouse embryos and enabled dynamic analysis of a phenomenon called lagging chromosomes. Conclusions: By applying this method, we can observe any stage of embryonic development, which may provide new insights into embryonic development in other mammals. [ABSTRACT FROM AUTHOR]

Purpose: This study investigated the consistency between results of preimplantation genetic testing for aneuploidy performed on trophectoderm (TE) cells and remaining blastocyst cells. Methods: TE biopsy was performed on 29 surplus cryopreserved human blastocysts. Biopsy samples and remaining blastocysts were processed using the VeriSeq PGS kit, and chromosomal statuses were compared by next‐generation sequencing. Results: Discordance was observed in the chromosomal status of 11 out of 29 blastocysts between the biopsied TE and remaining blastocysts. Concordance was observed in 11 of 12 blastocysts classified as euploid by TE biopsy and in 7 of 17 blastocysts classified as aneuploid. There was 100% concordance (7/7) in cases diagnosed as aneuploid with no mosaicism by TE biopsy. However, discordance was observed in all 10 cases showing mosaicism or partial chromosomal abnormality. Conclusion: Chromosomal status analysis based on TE biopsy does not accurately reflect the chromosomal status of the whole blastocyst. The chromosomal status is usually the same between the TE and remaining blastocyst cells in cases diagnosed as euploid or aneuploid with no mosaicism. However, mosaic blastocysts and those with other types of structural rearrangements have a higher risk of inconsistency, warranting caution during embryo selection. [ABSTRACT FROM AUTHOR]

This prospective study evaluated the accuracy of non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) using cell-free DNA in spent culture medium, as well as that of preimplantation genetic testing for aneuploidy (PGT-A) using trophectoderm (TE) biopsy after culturing beyond implantation. Twenty frozen blastocysts donated by 12 patients who underwent IVF at our institution were investigated. Of these, 10 were frozen on day 5 and 10 on day 6. Spent culture medium and TE cells were collected from each blastocyst after thawing, and the embryos were cultured in vitro for up to 10 days. The outgrowths after culturing beyond implantation were sampled and subjected to chromosome analysis using next-generation sequencing. Chromosomal concordance rate, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), false-positive rate (FPR), and false-negative rate (FNR) of niPGT-A and PGT-A against each outgrowth were analyzed. The concordance rate between the niPGT-A and outgrowth samples was 9/16 (56.3%), and the concordance rate between the PGT-A and outgrowth samples was 7/16 (43.8%). NiPGT-A exhibited 100% sensitivity, 87.5% specificity, 88.9% PPV, 100% NPV, 12.5% FPR, and 0% FNR. PGT-A exhibited 87.5% sensitivity, 77.8% specificity, 87.5% PPV, 75% NPV, 14.3% FPR, and 22.2% FNR. NiPGT-A may be more accurate than PGT-A in terms of ploidy diagnostic accuracy in outgrowths. [ABSTRACT FROM AUTHOR]

Studies have shown that some electrolytes, including Na+ and K+, play important roles in embryonic development. However, these studies evaluated these electrolytes by using inhibitors or knockout mice, with no mention on the changes in the intracellular electrolyte concentrations during embryogenesis. In this study, we used the electrolyte indicators CoroNa Green AM and ION Potassium Green-2 AM to directly visualise intracellular concentrations of Na+ and K+, respectively, at each embryonic developmental stage in mouse embryos. We directly observed intracellular electrolyte concentrations at the morula, blastocyst, and hatching stages. Our results revealed dynamic changes in intracellular electrolyte concentrations; we found that the intracellular Na+ concentration decreased, while K+ concentration increased during blastocoel formation. The degree of change in intensity in response to ouabain, an inhibitor of Na+/K+ ATPase, was considered to correspond to the degree of Na+/K+ ATPase activity at each developmental stage. Additionally, after the blastocyst stage, trophectoderm cells in direct contact with the blastocoel showed higher K+ concentrations than in direct contact with inner cell mass, indicating that Na+/K+ ATPase activity differs depending on the location in the trophectoderm. This is the first study to use CoroNa Green AM and ION Potassium Green-2 AM in mouse embryos and visualise electrolytes during embryonic development. The changes in electrolyte concentration observed in this study were consistent with the activity of Na+/K+ ATPase reported previously, and it was possible to image more detailed electrolyte behaviour in embryo cells. This method can be used to improve the understanding of cell physiology and is useful for future embryonic development studies. [ABSTRACT FROM AUTHOR]

Purpose: Immature human oocytes from resected ovaries can be used for research and fertility preservation, though it is unknown whether it is feasible to transport oocytes for these purposes. This study examined in vitro maturation (IVM) outcomes after the transportation of human follicular fluid (HFF) containing oocytes. Methods: Fourteen patients with endometrial adenocarcinoma were enrolled. Oocytes obtained from the resected ovaries of seven patients were transported with HFF by railway (transportation group). Samples of HFF from the other seven patients were not transported, and IVM was performed promptly (non‐transportation group). The results of oocyte retrieval and IVM were compared. Results: The average ages in the transportation and non‐transportation groups were 40.1 ± 2.0 and 39.6 ± 1.8 years, respectively, and the average numbers of collected oocytes were 8.1 ± 8.4 and 5.1 ± 5.1, respectively. There was a significant negative correlation between the number of collected oocytes and age. The proportions of oocytes that reached meiosis II (maturation rate) after IVM were 38.6% and 69.2% in the transportation and non‐transportation groups, respectively (P = 0.013). Conclusion: In this preliminary study, the usefulness of the transportation of HFF was limited. Further studies on maintaining oocyte normality during transportation are necessary for becoming the effective method for research and clinical use. We transported human follicular fluid for about 260 minutes between two universities 250 km apart for research material. Even after transportation, about 38% of immature oocytes reached meiosis II via in vitro maturation (IVM). We compared the IVM results between the transportation group and the non‐transportation group. [ABSTRACT FROM AUTHOR]

Recent studies reported morphokinetic indices for optimal selection of embryos in assisted reproductive technology (ART). The morphokinetics in blastocyst stage include the collapse and re-expansion rates after thawing. However, evaluation methods using these morphokinetics have not been established, mainly because the underlying molecular mechanisms remain unclarified. In this study, we focused on the relationship between these morphokinetic observation of the blastocyst behaviour and the number of cells constituting the blastocyst. We evaluated 38 surplus human frozen-thawed blastocysts using time-lapse cinematography and recorded their expansion, contraction, and hatching. A total of 28 blastocysts expanded in culture (cross-sectional area ≥ 5,000 π μm2). In comparison to the ones that did not, the expanded group presented significantly more number of inner cell mass (ICM) and trophectoderm (TE) cells, which eventually develop into the fetus and placenta, respectively (ICM: Expanded 10.2 ± 6.3 vs. Non-Expanded 6.0 ± 12.3, p < 0.05; TE: Expanded 165.7 ± 74.8 vs. Non-Expanded 57.0 ± 29.4, p < 0.05). Moreover, a positive correlation was found between the expansion rate (up to 4 h) and the number of TE cells (r = 0.558, p = 0.0021). Additionally, blastocysts that hatched had a significantly higher number of TE cells than those that did not (hatching 225.2 ± 61.2 vs. no hatching 121.1 ± 48.6, p < 0.0001). The number of TE cells per unit of cross-sectional area correlated negatively with the contraction time (r = –0.601, p = 0.0007). No correlation between the number of ICM cells and these morphokinetics was detected. In conclusion, our study demonstrates that different morphokinetics of frozen-thawed blastocysts reflect the number of TE cells. The differentiation of blastocysts containing sufficient TE cells would be beneficial for implantation and prognosis of a subsequent pregnancy. Thus, evaluation of these morphokinetics can be an effective method to screen good embryos for ART. [ABSTRACT FROM AUTHOR]

Aim In recent years, the importance of fertility preservation ( FP) has increased. In vitro maturation ( IVM), an important technique in FP, has started to be used in the clinic, but controversies persist regarding this technique. Here, a survey of IVM for FP is provided. Methods Based on a literature review, the applications of FP, methods of FP, IVM of oocytes that had been collected in vivo and ex vivo, maturation of oocytes after IVM for FP, cryopreservation of oocytes for FP, explanation of the procedures to patients, and recent research on FP using IVM were investigated. Results Although IVM for FP remains controversial, the application of FP is expected to expand. Depending on the age and disease status of the patient, various methods of oocyte collection and ovarian stimulation, as well as various needle types and aspiration pressures, have been reported. The maturation rate of IVM in FP ranges widely and requires optimization in the future. In regard to cryopreservation for matured oocytes, the vitrification method is currently recommended. Conclusion Regarding FP for patients with cancer, the treatment of cancer is prioritized; thus, the time and use of medicines are often constrained. As several key points regarding IVM remain unclear, well-designed and specific counseling for patients is necessary. [ABSTRACT FROM AUTHOR]

The hatching of a blastocyst where the blastocyst portions on the inside and the outside of the zona pellucida feature a figure-of-eight shape is termed “8”-shaped hatching; this type of hatching has been reported to affect the proper presentation of the inner cell mass (ICM) in both human and mouse embryos. Here, our aim was to investigate the factors that affect ICM presentation during “8”-shaped hatching. We performed IVF by using B6D2F1 female mice and ICR male mice, and used the 104 captured blastocysts. Embryos were maintained in KSOM at 37°C in a 5% CO2, 5% O2, and 90% N2 environment, and their growth behavior was monitored individually and continuously using time-lapse cinematography. At 120 h after insemination, embryos were immunostained and examined under a confocal microscope. We used the hatching form to identify “8”-shaped hatching, and we classified the “8”-shaped-hatching blastocysts into two groups, one in which the hatching site was near the ICM center, and the other in which the hatching site was far from the ICM center. We measured each group for ICM size and the number of Oct3/4-positive cells. Of the 95 hatching or hatched embryos, 74 were “8”-shaped-hatching blastocysts, and in these embryos, the ICM was significantly wider when the hatching site was near the ICM than when the hatching site was far from the ICM (P = 0.0091). Moreover, in the “8”-shaped-hatching blastocysts in which the ICM was included in the blastocyst portion outside the zona pellucida―the portion defined as the “outside blastocyst”―after the collapse of this outside blastocyst, the ICM adhered to the trophectoderm of the outside blastocyst, opposite the hatching site. Our results indicate that in “8”-shaped-hatching blastocysts, the hatching site and the collapse of outside blastocyst affect ICM formation. Thus, the assessment of “8”-shaped hatching behaviors could yield indices for accurately evaluating embryo quality. [ABSTRACT FROM AUTHOR]

What are the effects of using a fertility education chatbot, i.e. automatic conversation programme, on knowledge, intentions to improve preconception behaviour and anxiety? A three-armed, randomized controlled trial was conducted using an online social research panel. Participants included 927 women aged 20–34 years who were randomly allocated to one of three groups: a fertility education chatbot (intervention group), a document about fertility and preconception health (control group 1) or a document about an irrelevant topic (control group 2). Participants' scores on the Cardiff Fertility Knowledge Scale and the State-Trait Anxiety Inventory, their intentions to optimize preconception behaviours, e.g. taking folic acid, and the free-text feedback provided by chatbot users were assessed. A repeated-measures analysis of variance showed significant fertility knowledge gains after the intervention in the intervention group (+9.1 points) and control group 1 (+14.9 points) but no significant change in control group 2 (+1.1 points). Post-test increases in the intentions to optimize behaviours were significantly higher in the intervention group than in control group 2, and were similar to those in control group 1. Post-test state anxiety scores were significantly lower in the intervention group than in control group 1 and control group 2. User feedbacks about the chatbot suggested technical limitations, e.g. low comprehension of users' words, and pros and cons of using the chatbot, e.g. convenient versus coldness. Providing fertility education using a chatbot improved fertility knowledge and intentions to optimize preconception behaviour without increasing anxiety, but the improvement in knowledge was small. Further technical development and exploration of personal affinity for technology is required. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the meaning of meiotic maturation kinetics and duration of pronucleus presence (DPP) for parthenogenetic activation outcome., Design: Retrospective study., Setting: University hospital., Patient(s): Eight patients with endometrioid adenocarcinoma and 65 patients who underwent in vitro fertilization (IVF) with intracytoplasmic sperm injection (ICSI)., Intervention(s): After collection of oocytes from nonstimulated ovaries of patients with endometrioid adenocarcinoma, in vitro maturation (IVM) and parthenogenetic activation performed with time-lapse imaging; after ICSI, embryos similarly incubated with time-lapse imaging., Main Outcome Measure(s): Timing of the release of the first polar body (fPB), DPP, and developmental stage with IVM and parthenogenetic activation; after ICSI, assessment of DPP and preimplantation developmental stage., Result(s): With IVM, 55.2% of oocytes matured; 53.1% of fPBs were released within 24 hours, and 46.9% of fPBs were released after 24 hours. Regarding developmental stage, oocytes that released fPB later during IVM tended to develop more than oocytes that released the fPB within 24 hours. For embryos from parthenogenetic activation the DPP was statistically significantly shorter than the DPP of embryos from ICSI. With ICSI, the DPP was statistically significantly shorter in embryos that developed to ≥8 cells than embryos whose final development included ≤7 cells. The development rate in parthenogenetic activation was statistically significantly lower than that in ICSI., Conclusion(s): Embryo development is negatively affected by DPP that is too short or too long. When the DPP was short with parthenogenetic activation, embryo development did not proceed, indicating that DPP is an important determinant of parthenogenetic activation outcomes as with the timing of fPB release., (Copyright © 2020 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

In addition to overeating, starvation also reduces fecundity in mammals. However, little is known about the molecular mechanisms linking food intake to fertility, especially in males. Gastric inhibitory polypeptide (GIP), which is released from intestinal K-cells after meal ingestion, stimulates insulin secretion from pancreatic β-cells through the action of incretin and has several extrapancreatic effects. Here, we identified GIP receptor (Gipr) expression in mouse spermatids. Microarray analysis revealed that pregnancy-specific glycoprotein 17 (Psg17), a potential CD9-binding partner, was significantly decreased in GIP receptor-knockout (Gipr-/-) testes. Glycosylphosphatidylinositol-anchored PSG17 was expressed on the surface of acrosome-reacted sperm, and Gipr-/- sperm led to a lower fertilization rate in vitro, compared with that of Gipr+/+ sperm, both in the absence and presence of the zona pellucida. Plasma GIP concentrations and Psg17 messenger RNA (mRNA) were immediately increased in the testis after a single meal, whereas ingestion of a chronic high-fat diet markedly decreased Gipr and Psg17 mRNA. These results suggest that reduced GIP signaling, by decreased GIP levels or the downregulation of Gipr, is associated with the reduction of fecundity due to starvation or overeating. Thus, proper regulation of GIP signaling in the testis could be a potential unique therapeutic target for male infertility in obese and diabetic individuals., (Copyright © 2017 Endocrine Society.)

Contraction during the blastocyst stage is observed during embryonic development of various mammals, including humans, but the physiological role of this process is not well understood. Using time-lapse monitoring (TLM), we studied the influence of vitrification and contractions on embryonic development in mice. Mouse embryos were cultured at the 2-cell stage. At the 8-cell stage, embryos were randomly divided into a fresh group (FG) and vitrified group (VG) and observed for up to 144 h. Strong contractions (i.e., contractions causing a decrease in volume of more than 20% and expansion of the perivitelline space) occurred significantly more often in unhatched embryos than hatching embryos in both groups. Regarding hatching embryos, contractions in the pre-hatching stage were significantly more frequent in the VG than the FG. Furthermore, mRNA expression levels of genes related to contractions were determined at three time points, the 8-cell stage, early blastocyst stage, and 20 h after blastocoel formation, with quantitative reverse transcription-polymerase chain reaction. There was no significant difference in Hspa1a expression between the FG and VG, but Hspa1a overexpression was observed just after thawing and tended to decrease gradually thereafter in some blastocysts. Furthermore, in the VG, Atp1a1 tended to show higher expression in the strong contraction group than in the weak contraction group. Overall, vitrification is an excellent method for cryopreservation but could increase contractions in the pre-hatching stage and may increase energy demands of the embryo. Observation of contraction by TLM may improve the evaluation of embryo quality.

Recently, a new technique was developed for non-catalytically mixing microdroplets. In this method, an alternating-current (AC) electric field is used to promote the antigen-antibody reaction within the microdroplet. Previously, this technique has only been applied to histological examinations of flat structures, such as surgical specimens. In this study, we applied this technique for the first time to immunofluorescence staining of three-dimensional structures, specifically, mammalian eggs. We diluted an antibody against microtubules from 1:1,000 to 1:16,000, and compared the chromatic degree and extent of fading across dilutions. In addition, we varied the frequency of AC electric-field mixing from 5 Hz to 46 Hz and evaluated the effect on microtubule staining. Microtubules were more strongly stained after AC electric-field mixing for only 5 minutes, even when the concentration of primary antibody was 10 times lower than in conventional methods. AC electric-field mixing also alleviated microtubule fading. At all frequencies tested, AC electric-field mixing resulted in stronger microtubule staining than in controls. There was no clear difference in a microtubule staining between frequencies. These results suggest that the novel method could reduce antibody consumption and shorten immunofluorescence staining time.

Endometrial cancer (EC) rates are rising in Japan. Lymph node (LN) metastasis is an important prognostic factor in EC, and its risk is increased with higher tumor grade, deep myometrial invasion, larger tumor size, and lymphovascular space invasion (LVSI). Current methodologies to assess these factors are unreliable. We previously showed the association between C-reactive protein (CRP) 1846C>T (rs1205) polymorphism and LN metastasis in esophageal, non-small cell lung, and breast cancers. The CRP gene is located on chromosome 1q21-q23, and the polymorphism in the noncoding region (1846C>T) of this gene decreases serum CRP levels. We investigated the relationship between CRP 1846C>T genetic polymorphism and LN metastasis or LVSI in 130 EC patients using polymerase chain reaction-restriction fragment length polymorphism. The CRP 1846C/T genotype was C/C in 11 patients, C/T in 58 patients and T/T in 61 patients. The patients were divided into two groups based on their CRP 1846 genotypes: "C/C" and "C/T + T/T". Nine (7%) and 18 (13%) patients, all with the polymorphism, had LN metastasis and moderate or prominent lymphatic invasion, respectively. LN metastasis and/or severe lymphatic invasion were observed in the C/T + T/T group, while patients with the C/C genotype had no LN metastases or severe lymphatic invasion. Univariate and multivariate logistic regression models revealed that the C/T + T/T patients had a significant likelihood of developing LN metastasis and/or severe lymphatic invasion. Our results suggest that CRP genetic polymorphism is a novel risk predictor of LN metastasis and/or lymphatic invasion in EC.