Window of implantation (WOI) genes have been comprehensively identified at the single cell level. DNA methylation changes in cervical secretions are associated with in vitro fertilization embryo transfer (IVF-ET) outcomes. Using a machine learning (ML) approach, we aimed to determine which methylation changes in WOI genes from cervical secretions best predict ongoing pregnancy during embryo transfer. A total of 2708 promoter probes were extracted from mid-secretory phase cervical secretion methylomic profiles for 158 WOI genes, and 152 differentially methylated probes (DMPs) were selected. Fifteen DMPs in 14 genes (BMP2, CTSA, DEFB1, GRN, MTF1, SERPINE1, SERPINE2, SFRP1, STAT3, TAGLN2, TCF4, THBS1, ZBTB20, ZNF292) were identified as the most relevant to ongoing pregnancy status. These 15 DMPs yielded accuracy rates of 83.53%, 85.26%, 85.78%, and 76.44%, and areas under the receiver operating characteristic curves (AUCs) of 0.90, 0.91, 0.89, and 0.86 for prediction by random forest (RF), naïve Bayes (NB), support vector machine (SVM), and k-nearest neighbors (KNN), respectively. SERPINE1, SERPINE2, and TAGLN2 maintained their methylation difference trends in an independent set of cervical secretion samples, resulting in accuracy rates of 71.46%, 80.06%, 80.72%, and 80.68%, and AUCs of 0.79, 0.84, 0.83, and 0.82 for prediction by RF, NB, SVM, and KNN, respectively. Our findings demonstrate that methylation changes in WOI genes detected noninvasively from cervical secretions are potential markers for predicting IVF-ET outcomes. Further studies of cervical secretion of DNA methylation markers may provide a novel approach for precision embryo transfer.

The causes of implantation failure remain a black box in reproductive medicine. The exact mechanism behind the regulation of endometrial receptivity is still unknown. Epigenetic modifications influence gene expression patterns and may alter the receptivity of human endometrium. Cervical secretions contain endometrial genetic material, which can be used as an indicator of the endometrial condition. This study evaluates the association between the cervical secretion gene methylation profile and pregnancy outcome in a frozen-thawed embryonic transfer (FET) cycle. Cervical secretions were collected from women who entered the FET cycle with a blastocyst transfer (36 pregnant and 36 non-pregnant women). The DNA methylation profiles of six candidate genes selected from the literature review were measured by quantitative methylation-specific PCR (qMSP). Bioinformatic analysis of six selected candidate genes showed significant differences in DNA methylation between receptive and pre-receptive endometrium. All candidate genes showed different degrees of correlation with the pregnancy outcomes in the logistic regression model. A machine learning approach showed that the combination of candidate genes’ DNA methylation profiles could differentiate pregnant from non-pregnant samples with an accuracy as high as 86.67% and an AUC of 0.81. This study demonstrated the association between cervical secretion methylation profiles and pregnancy outcomes in an FET cycle and provides a basis for potential clinical application as a non-invasive method for implantation prediction.

Preimplantation genetic testing has been used widely in recent years as a part of assisted reproductive technology (ART) owing to the breakthrough development of deoxyribonucleic acid (DNA) sequencing. With the advancement of technology and increased resolution of next generation sequencing (NGS), extensive comprehensive chromosome screening along with small clinically significant deletions and duplications can possibly be performed simultaneously. Here, we present a case of rare chromosomal aberrations: 46,XY,dup(15)(q11.2q13),t(16;18)(q23;p11.2), which resulted in a normally developed adult but abnormal gametes leading to recurrent pregnancy loss (RPL). To our best knowledge, this is the first report of t(16;18) translocation with such a small exchanged segment detected by NGS platform of MiSeq system in simultaneous 24-chromosome aneuploidy screening.

STUDY QUESTION: Are the clinical pregnancy rates and cell lineage-related markers of equal-grade Day 5 (D5) and Day 6 (D6) embryos the same? SUMMARY ANSWER: High-grade D5 (HG D5) blastocysts have a higher live birth rate than high-grade D6 (HG D6) blastocysts, which could be associated with differences in intrinsic OCT4 expression. WHAT IS KNOWN ALREADY: In IVF cycles, D5 blastocysts are generally considered to be of higher quality and are preferred for embryo transfer over D6 blastocysts. However, the differences between delayed D6 embryos and D5 embryos remain poorly understood. This study aimed to perform an immunofluorescence (IF)-based analysis to evaluate cell content-related differences between same-grade D5 and D6 blastocysts and to assess how these differences may be associated with the clinical pregnancy outcomes following frozen-thawed embryo transfer cycles. STUDY DESIGN, SIZE, DURATION: This retrospective study included 774 frozen-thawed single blastocyst transfer (SBT) cycles at the Taipei Fertility Center from March 2020 to February 2023. All D5 (D5-SBT) and D6 (D6-SBT) frozen-thawed cycles (n = 774) were classified as high grade (≥BB, n = 694) or low grade (<BB, n = 80). PARTICIPANTS/MATERIALS, SETTING, METHODS: For the cell content analyses, IF staining with the pluripotent marker OCT4 was used to determine the cell numbers in the inner cell mass (ICM), and the trophectoderm (TE) marker GATA3 was used to evaluate the TE region. Same-grade frozen-thawed D5 blastocysts (n = 20), D6 blastocysts (n = 20), and D5 blastocysts cultured to D6 (ExD6, n = 17) were used to assess differences in the cell number in the TE and ICM by evaluating OCT4 and GATA3 expression. The total number of cells was visualized using DAPI staining. OCT4 and GATA3 transcripts were analysed with quantitative reverse transcription polymerase chain reaction (qRT-PCR). MAIN RESULTS AND THE ROLE OF CHANCE: For the clinical results, in the single euploid embryo transfer cycle, the overall biochemical pregnancy rate (HCG+, 71.8% vs 52.3%, P < 0.0001), clinical pregnancy rate (SAC+, +66.7% vs 47.7%, P = 0.0001) and live birth rate (59.5% vs 37.9%, P < 0.0001) were significantly higher in the D5-SBT group than in the D6-SBT group. Among HG embryos, the D5-SBT group consistently demonstrated better clinical outcomes. A generalized linear mixed model revealed that the odds ratios were significantly elevated only for D5 embryos but not for other confounders, highlighting the importance of developmental timing. On the other hand, the staining results showed no significant differences in the numbers of TE, ICM, or total cells between the same-grade D5 and D6 blastocysts. However, a notable decrease in OCT4-positive cells was observed in D6 embryos, suggesting a reduction in embryo quality (D5 vs D6: 64 ± 4% vs 49 ± 5% per embryo). ExD6 blastocysts had a significantly increased number of TE cells compared to D6 blastocysts, reflecting greater expansion of the TE population. Gene expression analysis results aligned with these findings, showing a significant decrease in the OCT4/GATA3 ratio in TE cells from D6 embryos compared to D5 embryos LIMITATIONS, REASONS FOR CAUTION: This study exclusively analysed euploid embryos in clinical assessments, omitting cases of mosaic embryo transfer. Due to the limited sample size for embryo staining, assessment of additional pluripotent markers was not feasible. WIDER IMPLICATION OF THE FINDINGS: Our research demonstrated a significant decrease in the live birth rate with D6 blastocysts compared to D5 blastocysts. The similar proportion of TE cells between D5 and D6 blastocysts suggests that reduced numbers of OCT4-positive cells in D6 blastocysts, rather than TE proliferation, may influence the embryo developmental potential. STUDY FUNDING/COMPETING INTEREST(S): No specific funding was used for this study. W.F.C. is a co-investigator at Taipei Fertility Center and an employee of Taipei Medical Technology Co., Ltd All other authors declare no conflict of interest related to the content of this study. TRIAL REGISTRATION NUMBER: N/A.