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Aim: The objective of this study was to assess whether PGT conducted with previously untested vitrified embryos affect the clinical outcomes., Methods: A total of 49 patients who underwent biopsy on vitrification-warming embryos for PGT were enrolled from January 2016 to January 2019. The cleavage-stage embryos were thawed and cultured into the blastocyst stage for biopsy. During this period, 195 patients underwent routine PGT and FET, whose embryos were biopsied before frozen were used as the control group. The clinical outcomes were further compared between the two groups after a 1:2 PSM., Results: There were 47 transferable blastocysts in 30 patients, while 19 patients without transferable embryos, who performed biopsy on vitrification-warming embryos for PGT. During this study period, 27 patients have already underwent FET with the clinical pregnancy rate was 66.7% (18/27). After 1:2 PSM, 24 patients in the biopsy on vitrification-warming embryo group and 48 patients in the control group were compared, the clinical pregnancy rate (68.8% vs. 70.8%, p = 0.86), miscarriage rate (18.2% vs. 11.8%, p = 0.86), or live birth rate (52.1% vs. 62.5%, p = 0.40) had no significant difference. And the transferable blastocyst rate or the clinical pregnancy rate in the vitrification-warming cleavage-stage embryo group was not significantly different from those in the vitrification-warming blastocyst group. In addition, the PGT clinical outcomes of biopsy on vitrification-warming embryos had no significant difference between IVF-fertilized embryos and ICSI-fertilized embryos., Conclusion: Biopsy on the vitrification-warming embryos with a dual vitrified cryopreservation does not affect the embryo quality or the PGT clinical outcomes., (© 2022 Japan Society of Obstetrics and Gynecology.)

Mitochondria play a critical role in cell function and embryo development. Recently, increasing studies have investigated whether mitochondrial DNA (mtDNA) can be used as a predictive biomarker of embryo implantation. However, the results of its effect on implantation are still controversial. To further understand the clinical application value of mtDNA content for reproductive potential, we analyzed the influence of relative mtDNA quantity on embryo quality and transfer outcomes based on the results of second-generation sequencing of preimplantation genetic testing patients in our center. Biopsied trophectoderm (TE) from aneuploid blastocysts contained much larger amounts of mtDNA than those from euploid blastocysts ( p  < 0.000). In an analysis of only euploid blastocysts ( n  = 769), female age had no effect on mtDNA content ( p  = 0.216). TE cells biopsied on day 5 ( n  = 355) contained significantly higher amounts of mtDNA compared to those biopsied on day 6 ( n  = 388) or day 7 ( n  = 26) ( p  < 0.000). Higher quality trophoblast was associated with lower mtDNA content ( p  = 0.026), but quality of inner cell mass was not correlated with quantity of mtDNA ( p  = 0.112). For transferred embryos, the biopsied date and mtDNA content were significantly associated with embryo implantation and live birth outcomes. Day-5 euploid blastocysts with lower quantities of mtDNA exhibited higher implantation rate and live birth rate. However, our data indicated that mtDNA content may not be considered an independent predictive marker, it may be a useful reference for the selection of day-5 transferred euploid blastocysts.

Human chorionic gonadotropin (hCG) can attract regulatory T cells (Tregs) into the fetal-maternal interface regulating maternal immune tolerance in pregnancy. The objective of this study was to investigate whether hCG recruits the Tregs into endometrium by inducing chemokines. The number of Tregs in the endometrium was analyzed by immunohistochemistry. The expression of CCL2 was analyzed in vivo and in vitro with hCG stimulation. CD4 + CD25 + Tregs were isolated from peripheral blood for Tregs migration assay with hCG, CCL2 siRNA and CCR2 antagonist stimulation. Our results showed that the number of endometrial Tregs in RIF patients was significantly decreased (9.4 ± 5.3 vs. 23.1 ± 3.1, P < 0.01), while intrauterine infusion of 2000 IU hCG increased the endometrial Tregs (18.6 ± 9.8 vs. 9.4 ± 5.3, P < 0.05) and CCL2 expression (0.21 ± 0.01 vs. 0.17 ± 0.01, P < 0.01). Horn injecting with 10 IU hCG also increased the endometrial Tregs in pseudopregnant mice (46 ± 16.8 vs. 7 ± 4.3, P < 0.01). Furthermore, the CCL2 protein and mRNA levels were significantly increased in human endometrial stromal cells (hESCs) with the stimulation of hCG. Migration assays showed that hESCs with hCG stimulation promoted Tregs migration (2597 ± 833.2 vs. 1115 ± 670.7, P < 0.05), while the number of migrated Tregs significantly decreased with CCL2 siRNA (1061 ± 105.4 vs. 2598 ± 294.7, P < 0.05) or CCR2 antagonist (356.7 ± 138.8 vs. 2597 ± 833.2, P < 0.05) treatment. In conclusion, intrauterine perfusion of hCG might promote the recruitment of Tregs into endometrium by inducing chemokine CCL2., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: It is still uncertain whether surgical evacuation adversely affects subsequent embryo transfer. The present study aims to assess the influence of surgical evacuation on the pregnancy outcomes of subsequent embryo transfer cycle following first trimester miscarriage in an initial in vitro fertilization and embryo transfer (IVF-ET) cycle., Methods: A total of 645 patients who underwent their first trimester miscarriage in an initial IVF cycle between January 2013 and May 2016 in Nanjing Drum Tower Hospital were enrolled. Surgical evacuation was performed when the products of conception were retained more than 8 h after medical evacuation. Characteristics and pregnancy outcomes were compared between surgical evacuation patients and no surgical evacuation patients. The pregnancy outcomes following surgical evacuation were further compared between patients with ≥ 8 mm or < 8 mm endometrial thickness (EMT), and with the different EMT changes., Results: The EMT in the subsequent embryo transfer cycle of surgical evacuation group was much thinner when compared with that in the no surgical evacuation group (9.0 ± 1.6 mm vs. 9.4 ± 1.9 mm, P = 0.01). There was no significant difference in implantation rate, clinical pregnancy rate, live birth rate or miscarriage rate between surgical evacuation group and no surgical evacuation group (P > 0.05). The live birth rate was higher in EMT ≥ 8 mm group when compared to < 8 mm group in surgical evacuation patients (43.0% vs. 17.4%, P < 0.05)., Conclusions: There was no significant difference in the pregnancy outcomes of subsequent embryo transfer cycle between surgical evacuation patients and no surgical evacuation patients. Surgical evacuation led to the decrease of EMT, especially when the EMT < 8 mm was association with a lower live birth rate.

Research Question: Can next-generation sequencing (NGS) based on copy number variation sequencing (CNV-Seq) identify normal/balanced embryos in balanced reciprocal translocation carriers and what are their reproductive outcomes?, Design: One hundred couples with balanced reciprocal translocation who underwent a total of 134 preimplantation genetic testing (PGT) cycles between January 2015 and October 2017 were evaluated. Trophectoderm cells of blastocysts were biopsied for CNV-Seq-based NGS. All the balanced/normal blastocysts were vitrified and cryopreserved. Single balanced/normal blastocysts were warmed and transferred in the subsequent frozen embryo transfer (FET) cycle., Results: During the study period, 400 blastocysts were analysed by NGS-PGT, of which 109 (27.25%) were balanced and euploid. A total of 52 blastocysts were transferred in the FET cycle. Clinical pregnancy was confirmed in 34 women (65.38%), with a miscarriage rate of 2.94%; 26 healthy term babies were born, including 24 singletons and one set of twins, while eight couples had ongoing pregnancies. Amniocentesis revealed a fetal chromosome status that was consistent with the NGS-PGT results. Female carriers had a significantly higher blastocyst rate than did the male carriers (37.01% versus 31.27%, P = 0.04). The transferable blastocyst rate was higher in couples treated with gonadotrophin-releasing hormone (GnRH) antagonist than in those treated with GnRH agonist (38.20% versus 24.37%, P = 0.01). However, neither carrier sex nor ovarian stimulation protocol influenced the clinical pregnancy rate., Conclusions: CNV-Seq-based NGS is an efficient and reliable PGT method for balanced reciprocal translocation., (Copyright © 2019 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Objective: The aim of this retrospective cohort study was to assess the effect of subchorionic hematoma (SCH) on pregnancy outcomes in IVF/ICSI patients., Methods: We retrospectively analyzed 1097 pregnancies achieved by in vitro fertilization and embryo transfer (IVF-ET) or frozen-thawed embryo transfers (FETs) between January 2013 and June 2013 at the IVF center of Nanjing Drum Tower Hospital. The prevalence of SCH was 12.1% in this group (133/1097). We compared the pregnancy outcomes between the SCH group and non-SCH group, while the risk factors for SCH were also evaluated., Results: There was no significant difference between SCH group and non-SCH group with regard to patients' age, spouse's age, endometrial thickness, miscarriage rate (5.6% versus 6.2%, p > 0.05), second trimester fetus loss rate (5.6% versus 7.7%, p > 0.05) or live birth rate (89.5% versus 86.1%, p > 0.05). While the birth weight in singleton pregnancy in SCH group was significant lower (3207.8 ± 595.7 g versus 3349.2 ± 59.7 g, p = 0.03). SCH was more common in fresh embryo transfer patients than that in FET patients (16.6% versus 5.1%, p < 0.01), fresh embryo transfer was a high risk for SCH with OR 3.67, 95% CI: 2.28-5.90., Conclusion: We concluded that SCH was associated with lower birth weight in singleton pregnancy, but SCH did not increase pregnancy loss rate in IVF/ICSI patients, and fresh embryo transfer may contribute to SCH onset.

Purpose: To investigate the usefulness of preimplantation genetic diagnosis (PGD) for the patient affected by congenital contractural arachnodactyly (CCA) and spinal and bulbar muscular atrophy (SBMA)., Methods: Multiple displacement amplification (MDA) was performed for whole genome amplification (WGA) of biopsied trophectoderm (TE) cells. Direct mutation detection by sequencing and next-generation sequencing (NGS)-based single nucleotide polymorphism (SNP) haplotyping were used for CCA diagnosis. Direct sequencing of the PCR products and sex determination by amplification of sex-determining region Y (SRY) gene were used for SBMA diagnosis. After PGD, the unaffected blastocyst (B4) was transferred in the following frozen embryo transfer (FET)., Results: In this PGD cycle, sixteen MII oocytes were inseminated by ICSI with testicular spermatozoa. Four blastocysts (B4, B5, B10, B13) were utilized for TE cell biopsy on day 5 after ICSI. After PGD, B4 was unaffected by CCA and SBMA. B5 was affected by CCA and carried SBMA. B10 was unaffected by CCA and carried SBMA. B13 was affected by CCA and unaffected by SBMA. B4 was the only unaffected blastocyst and transferred into the uterus for the subsequent FET cycle. The accuracy of PGD was confirmed by amniocentesis at 21 weeks of gestation. A healthy boy weighing 2850 g was born by cesarean section at the 38th week of gestation., Conclusions: PGD is a valid screening tool for patienst affected of CCA and SBMA to prevent transmission of these genetic diseases from parents to children., Competing Interests: Compliance with ethical standards The couple signed informed consent forms for ICSI treatment, PGD, and follow-up. This project was approved by the Ethics Committee of the Drum Tower Hospital, which is affiliated to Nanjing University Medical College. Conflict of interest The authors declare that they have no conflict of interest.

Background: Although repeated cryopreservation is an occasional occurrence, the effect on perinatal outcomes is unclear. Therefore, the aim of this study was to evaluate the perinatal outcomes of singletons after embryo re-cryopreservation. Methods: In this retrospective study, a total of 647 singleton live births after blastocyst freeze-thaw embryo transfer cycles were investigated. They were divided into two groups: vitrified-warmed blastocysts (once-vitrified group) and vitrified-warmed blastocysts derived from thawed cleaved embryos (re-vitrified group). Propensity score matching (PSM) was used to control for potential confounding factors. Results: A total of 592 infants were included in the once-vitrified group, and 55 infants were included in the re-vitrified group. After PSM, 108 cases were generated for comparison. The median gestational age was 38 weeks for both groups, and the birthweights were comparable (3390.6 ± 601.5 g vs. 3412.8 ± 672.6 g, P > 0.05). The incidence of preterm birth (PTB) (20.4% vs. 16.7%), low birthweight (LBW) (3.7% vs. 7.4%), macrosomia (11.1% vs. 16.7%) and large for gestational age (LGA) (29.6% vs. 22.2%) were not significantly different between the two groups. Logistic regression analysis indicated that double vitrification-warming procedures did not affect the occurrence of PTB (OR, 2.58 [95% CI, 0.77, 8.63]), LBW (OR, 0.83 [95% CI, 0.08, 8.29]), macrosomia (OR, 0.60 [95% CI, 0.13, 2.69]), or LGA (OR, 1.51 [95% CI, 0.53, 4.27]) (P > 0.05, for all). Conclusion: Our findings demonstrate that double vitrification-warming procedures do not increase the risk of adverse neonatal outcomes compared with those of once-vitrified embryos. [ABSTRACT FROM AUTHOR]

Purpose: To evaluate the clinical utility of serum luteinizing hormone (LH) level in predicting frozen embryo transfer (FET) outcomes among the patients with an ovulation induction (OI) cycle. Methods: A total of 250 patients who underwent OI cycle frozen–thawed embryo transfer from January 2018 to June 2020 in Drum Tower Hospital affiliated with Nanjing University Medical School were retrospectively analyzed. The primary outcomes were clinical pregnancy rate and the live birth rate. Results: The results showed that a significant difference in Serum LH level on the day of human chorionic gonadotropin (hCG) administration was observed between the clinical pregnancy group and no clinical pregnancy group (p = 0.002), while there was no significant difference between the live birth group and no live birth group (p = 0.06). Multiple logistic regression analysis of factors related to clinical pregnancy showed serum LH level on the day of hCG administration was related to improved clinical pregnancy rate (OR was 1.02, 95% CI: 1.0–1.03, p = 0.02), while serum LH level had no significant effect on live birth rate. The ROC curves revealed the serum LH level was significantly correlated with clinical pregnancy rate and live birth rate; the cut-off point of serum LH level on the day of hCG administration was 8.46 miu/mL for clinical pregnancy rate (AUC 0.609, p = 0.003). Conclusion: In patients with OI FET, serum LH level on the day of hCG administration might be a biomarker for the prediction of clinical pregnancy. The prediction that patients who underwent OI cycle frozen–thawed embryo transfer with serum LH levels below 8.46 mIU /mL might be pregnant appeared to be meaningful. [ABSTRACT FROM AUTHOR]

Objective: To minimize twin birth rate by establishing an elective single cleavage embryo transfer strategy based on a twin live birth prediction model from fresh double cleavage embryos transfer (cleavage DET) patients.Methods: A total of 2478 patients underwent fresh cleavage DET in Nanjing Tower Hospital were enrolled to establish the twin live birth prediction model by logistic regression analysis and the cutoff value was calculated by ROC curve. Another 300 fresh cleavage DET patients and 550 cleavage single-embryo transfer (SET) patients were selected to testify the sensitivity, specificity and usefulness of this model.Results: The twin live birth probability (TLBP) = eX/(eX + 1), e is a natural logarithm, X = -1.763 - (0.319 × female age) + (0.329 × endometrial thickness) + (0.282 × the number of transferred top embryos) - (0.314 × previous transfer times), and the cutoff value of TLBP was 24.2%. The sensitivity of this model for predicting twin live birth was 75.6%, while the specificity was 52.5% in the external validation of 300 DET patients. Furthermore, the validation of 550 SET patients showed that the live birth rate of TLBP value positive patients was significantly higher than that in negative patients (54.3% vs. 35.5%, p < .001). When adopted an elective single cleavage embryo transfer strategy, the patients with a positive TLBP value choose SET, while still undergo DET who with a negative TLBP value, the live birth rate would maintain as 56.7%; however, the twin birth rate would significantly decline to 7.4%.Conclusion: Female age, endometrial thickness, the number of transferred top embryos and previous embryo transfer times were critical variables for the twin live birth prediction model. An elective single cleavage embryo transfer strategy according to this model can maintain the relatively high live birth rate, meanwhile get the acceptable low twin birth rate. [ABSTRACT FROM AUTHOR]

Purpose: To study the association between the numbers of oocytes retrieved and the cumulative live birth rates (LBR) in women aged 35-40 years undergoing long GnRH agonist IVF/ICSI cycles.Methods: A total of 931 women aged 35-40 years who underwent their first cycle of IVF/ICSI treatment between January 2010 and December 2013 at Nanjing Drum Tower Hospital were identified and reviewed. The main endpoint of this study was the cumulative LBR after one complete oocyte retrieval, which included fresh and all subsequent frozen-thaw embryo transfer cycles. Odds ratios (OR) and 95% confidence interval (CI) for live birth were estimated by multivariate logistic regression analysis. Furthermore, all the women were divided into four groups based on the number of oocytes retrieved: 0-4, 5-9, 10-14 or ≥15 oocytes group. Variables were then compared among groups.Results: We found that 634 out of the 931 patients (68.1%) achieved at least one live birth. The number of oocytes retrieved was an independent predictive factor for live birth, with OR 1.20 (95% CI 1.15-1.26) when adjusted for age (years), duration of infertility and Gn (gonadotrophin) doses. The cumulative LBR in the four different oocyte groups was 35.6, 68.8, 83.4 and 89.2%, respectively. When the 1-4 oocytes group was issued as a reference, the ORs for cumulative LBR gradually increased to 3.66, 6.74 and 11.77 in other three oocytes groups, respectively. The moderate-severe ovarian hyperstimulation syndrome (OHSS) rate was dramatically increased in the ≥15 oocytes group (6.9%) when compared to that in the 10-14 oocytes group (0.8%), while the cumulative LBR only increased 5.8% (from 83.4 to 89.2%).Conclusions: The ideal number of oocytes retrieved in women aged 35-40 years is 10-14 oocytes, which achieves a high cumulative LBR while maintaining an acceptable low OHSS rate. [ABSTRACT FROM AUTHOR]

This study investigated the usefulness of next-generation sequencing (NGS)-based single nucleotide polymorphism (SNP) haplotyping for preimplantation genetic diagnosis (PGD) of hemoglobin H (Hb H) disease. Multiple displacement amplification (MDA) was used for whole genome amplification (WGA) of biopsied trophectoderm (TE) cells. Gap-PCR and NGS-based SNP haplotyping was used to distinguish the two genotypes of -α3.7/αα and –SEA/αα for PGD of Hb H disease. One out of the ten blastocysts (B11) was successfully diagnosed as genotype -α3.7/αα by Gap-PCR, whereas the others revealed allele dropout (ADO) (B1, B2, B4, B5, B7, B8, B12, and B15) or amplification failure (B10). However, NGS-based SNP haplotyping successfully diagnosed the -α3.7/αα and –SEA/αα genotypes from the MDA products of the biopsied TE cells. The haplotyping result showed that B4, B7, B8, B10, B11, B12, and B15 were carriers of the -α3.7deletion (-α3.7/αα), whereas B1, B2, and B5 were carriers of the –SEAdeletion (–SEA/αα). A blastocyst (B11) was transferred into the uterus in a subsequent frozen embryo transfer (FET) cycle after PGD. A healthy infant with a -α3.7/αα genotype weighing 2,800 g was born by cesarean section at the 38thweek of gestation. This result indicates that NGS-based SNP haplotyping is a valid screening tool for the PGD of Hb H disease. [ABSTRACT FROM PUBLISHER]

The aim of this study was to determine if an association existed between serum human chorionic gonadotrophin (HCG) level at 12 h after trigger and IVF and intracytoplasmic sperm (ICSI) treatment outcomes. Women undergoing initial IVF–ICSI and embryo transfer treatment using the long luteal phase gonadotrophin-releasing hormone agonist protocol between April 2012 and March 2013 for tubal factor were included ( n = 699). In the clinical pregnancy group, HCG after trigger was significantly elevated (276.0 ± 5.1 versus 198.5 ± 6.1 mIU/mL; P < 0.001). The optimal cut-off value proposed by the receiver operating characteristic analysis (area under curve = 0.730) for HCG was 201.2 mIU/ml. Compared with the lower HCG group, the clinical pregnancy rate in the higher HCG group was increased in obese and non-obese patients (77.8% versus 57.3%, P < 0.05; 85.6% versus 53.0%, P < 0.01, respectively). Adjusted for age and body mass index, an increase of HCG was associated with a better IVF–ICSI treatment outcome (OR 4.39, 95% CI 2.99 to 6.45). Clinical pregnancy rate was significantly higher across increasing quartiles of HCG. An elevated level of serum HCG at 12 h after trigger was associated with a better IVF–ICSI outcome. [ABSTRACT FROM AUTHOR]

Is there a simple and effective method for male patients with genetic disorders in families with no identified haplotype and with Robertsonian translocations to avoid the transfer of embryos carrying translocated chromosomes? Single spermatozoa were separated to identify by next-generation sequencing (NGS) those that were genetically abnormal, to establish a sperm-based single-nucleotide polymorphism (SNP) haplotype. Blastocysts that developed to day 5 or 6 were then biopsied for whole genome amplification and screening for chromosomal aneuploidy. Normal embryos were selected by comparison with a single-sperm-based SNP haplotype and were transferred. The results were verified by second trimester amniocentesis. Two blastocysts obtained from patients with neurofibroma type 1 (NF1) were found to be normal after NGS according to single-sperm-based SNP haplotype analysis (13 SNP sites). Three and one blastocysts, respectively, were obtained from the patients with Robertsonian translocation. Blastocysts B9 and B7 were found to be normal after NGS according to the single-sperm-based SNP haplotype analysis (12 and 13 SNP sites selected on chromosomes 14 and 22 for the first patient; 12 and 9 SNP sites selected on chromosomes 13 and 14 for the second patient). Successful pregnancies after blastocyst transfer occurred in all three patients. The identification of embryos was verified by mid-trimester amniocentesis. All three patient couples successfully delivered healthy babies. This study preliminarily summarized the process of single-sperm-based SNP haplotyping, which could be applied as preimplantation genetic testing for male patients without identified disease-causing haplotypes and with Robertsonian translocations. [ABSTRACT FROM AUTHOR]