PURPOSE:Recurrent pregnancy loss (RPL) is frequently associated with embryonic aneuploidy, yet paternal genetic contributors remain poorly understood. In this study, we investigated the genetic cause of a couple with oligoasthenoteratozoospermia (OAT) and a history of RPL involving recurrent sex chromosome aneuploidies. METHODS:Evaluation included preimplantation genetic testing for aneuploidy (PGT-A) with parental origin analysis, semen analysis, and sperm fluorescence in situ hybridization (FISH). Whole-exome sequencing (WES) was performed to identify pathogenic variants, which were subsequently validated by Sanger sequencing. RESULTS:WES revealed a homozygous C12orf40 frameshift variant (c.232_233insTT) in the proband and his affected brother. Both brothers exhibited markedly elevated sperm sex chromosome error rates (proband nullisomy:18.8%; brother disomy:10.3%). Across three PGT-A cycles, only 41.7% (5/12) of the blastocysts were euploid, and 85.7% (6/7) of the aneuploid embryos involved sex chromosome abnormalities. Parental origin analysis indicated 72.7% of abnormalities were paternal in origin. CONCLUSION:This study identified a previously unreported phenotype of the C12orf40 c.232_233insTT variant, expanding its spectrum from complete meiotic arrest to incomplete arrest with severe meiotic errors and residual spermatogenesis. It provides the first evidence linking this variant to severe OAT and RPL, highlighting a crucial paternal genetic contributor to reproductive failure.

Background Early diagnosis and precise non-invasive detection of colorectal cancer (CRC) is slated to greatly improve patient prognosis and promote the development of personalized treatment. Circulating tumor DNA (ctDNA), characterized by cancer-specific genetic and epigenetic features, has become an important biomarker with the development of liquid biopsy technologies. Methods In this study, extensive methylation and transcriptome sequencing data from The Cancer Genome Atlas (TCGA) were analyzed to identify five genes- ADHFE1, C9orf50, IKZF1, SDC2, and SEPT9- previously reported in colorectal cancer (CRC) studies. A range of statistical methods was employed to develop a novel CRC-specific diagnostic model. The model's performance was initially evaluated using tissue sample datasets from public sources and subsequently validated in plasma samples collected from 61 CRC patients and 26 normal controls. The association between model scores and tumor burden was further examined using plasma data. Public datasets were obtained from TCGA-COAD, TCGA-READ, and GEO accession numbers GSE75546, GSE77954, GSE77965, GSE42752, GSE48684, GSE68060, GSE77718, GSE101764, and GSE40279. Results The tissue-derived model achieved high diagnostic specificity and sensitivity in public tissue datasets and accurately differentiated CRC patients from normal controls in plasma samples (AUC=0.907). Model scores were significantly associated with tumor volume and treatment response, demonstrating the potential to monitor tumor burden non-invasively. Conclusion The five-gene ctDNA methylation model demonstrated high diagnostic accuracy and correlated with tumor burden in this exploratory study, highlighting its potential as a minimally invasive liquid-biopsy tool for CRC assessment. However, the findings are limited by the modest plasma cohort size, the lack of matched tissue-plasma methylation data, and the exploratory study design; therefore, larger prospective cohorts are required for further clinical validation.

Comprehensive chromosomal status of blastocyst from women with polycystic ovary syndrome (PCOS) was limited. This study aimed to identify possible differences in the preimplantation blastocyst chromosome aberrations between PCOS women and controls receiving preimplantation genetic testing (PGT). This was a multi-center retrospective cohort study including a total of 707 blastocysts from 147 PCOS women and 3006 blastocysts from 821 control women receiving PGT between 2015 and 2021. Embryonic chromosomal aberration spectrums were compared between PCOS and controls. Mixed effects generalized linear model was conducted to explore possible influence of PCOS-related endocrinological disorders on embryonic chromosomal abnormalities. Blastocysts from PCOS demonstrated significantly lower aneuploidy rate (15.2

STUDY QUESTION:What is the effect of pretreatment with oral contraceptive pills (OCPs) on oocyte and embryo quality and pregnancy rates in women with polycystic ovary syndrome (PCOS) scheduled for IVF/ICSI cycles? SUMMARY ANSWER:In women with PCOS who underwent a first or second IVF/ICSI cycle with a GnRH antagonist protocol and were randomized to start ovarian stimulation immediately, the quality of cleavage-stage embryos was non-inferior to pretreatment with OCP. WHAT IS KNOWN ALREADY:PCOS in Asian populations is characterized by high levels of circulating LH in the early follicular phase. Previous studies indicated that inappropriately high LH levels might affect oocyte maturation and fertilization rates, and impaired embryo quality, consequently resulting in higher rates of impaired pregnancy and miscarriage in women with PCOS. OCPs are frequently used as pretreatment to lower LH levels in PCOS patients. STUDY DESIGN SIZE DURATION:We performed a randomized controlled trial. After informed consent, women diagnosed with PCOS scheduled for their first or second IVF/ICSI cycle with a GnRH antagonist protocol were randomized to receive OCPs (OCP group) or start ovarian stimulation immediately, regardless of the day of the menstrual cycle (non-OCP group). Using a non-inferiority hypothesis, the sample size was calculated at 242 women. The study lasted from 7 February 2018 to 31 August 2021. PARTICIPANTS/MATERIALS SETTING METHODS:A total of 242 infertility patients with PCOS undergoing the first or second cycle of IVF or ICSI were enrolled and randomized into two groups. In the OCP group, recombinant FSH was started on Day 7 of the washout period after pretreatment with OCP. In the non-OCP group, recombinant FSH was started immediately regardless of the day of the menstrual cycle. All participants received standardized GnRH antagonist ovarian stimulation. The freeze-all strategy was applied to all participants. The primary outcome was the number of good-quality embryos on Day 3 after insemination. Secondary outcomes included the rates of blastocyst formation, implantation, clinical pregnancy, and live birth from the first frozen/warmed embryo transfer cycles and cumulative live birth rates. MAIN RESULTS AND THE ROLE OF CHANCE:We randomized 242 women to receive OCP (n = 121) or start immediately with ovarian stimulation (n = 121). The number of good-quality embryos on Day 3 in the OCP group was non-inferior to the non-OCP group (OCP group versus non-OCP group, 6.58 ± 4.93 versus 7.18 ± 4.39, AD -0.61, 95% CI: -1.86 to 0.65, P = 0.34). The rates of blastocyst formation (55.4% versus 52.9%, relative risk (RR) 1.11, 95% CI: 0.96 to 1.28, P = 0.17), implantation (63.0% versus 65.5%, RR 0.90, 95% CI: 0.53 to 1.53, P = 0.79), clinical pregnancy (67.9% versus 68.8%, RR 0.96, 95% CI: 0.54 to 1.71, P = 1.0), and live birth rate (52.8% versus 55.1%, RR 0.92, 95% CI: 0.53 to 1.56, P = 0.79) of the first frozen/warmed embryo transfer cycles were all comparable between the OCP and non-OCP group, respectively. Cumulative live birth rates were also similar in the OCP and non-OCP groups (78.3% versus 83.5%, respectively RR 0.71, 95% CI: 0.36 to 1.42, P = 0.39). LIMITATIONS REASONS FOR CAUTION:Only patients with PCOS in Southern China were recruited. Therefore, caution is necessary when generalizing our results to all such patients with PCOS. Also, since a freeze-only strategy was used, the results of this study are only applicable when infertile women with PCOS undergo the freeze-only method. The obvious treatment difference between the two groups meant that the study was designed as an open-label study for women and doctors. The study had a randomized controlled design that minimized bias. WIDER IMPLICATIONS OF THE FINDINGS:Pretreatment with OCPs to lower LH levels in patients with PCOS before ovarian stimulation in IVF or ICSI cycles may not improve the quality of cleavage-stage embryos. STUDY FUNDING/COMPETING INTERESTS:This study was funded by the National Key Research and Development Program of China (No. 2023YFC2705503). This study was supported in part by the Investigator-Initiated Studies Program (grant from MSD and Organon). BWM reports consultancy, travel support, and research funding from Merck. He reports consultancy from Organon and Norgine, and also reports holding stock from ObsEva. No conflicts of interest are declared for the other authors. TRIAL REGISTRATION NUMBER:Chinese Clinical Trial Registry (No. chiCTR1800014822). URL: https://www.chictr.org.cn/showproj.html?proj=25280. TRIAL REGISTRATION DATE:7 February 2018. DATE OF FIRST PATIENT’S ENROLLMENT:22 February 2018.

Objective: To investigate whether common genetic polymorphisms are associated with gonadotropin levels after down-regulation with daily gonadotropin-releasing hormone agonist and whether the polymorphisms of candidate variants influence the ovarian response to exogenous gonadotropins. Design: Genetic association study. Setting: University-affiliated in vitro fertilization center. Patients: Subjects enrolled in an exploratory exome-wide association study (n= 862), a replication exome-wide association study (n= 86), and a classifier validation study (n= 148) were recruited from September 2016 to October 2018, September 2019 to September 2020, and January 2021 to December 2021, respectively. The included patients were aged %40 years and had a basal follicle-stimulating hormone (FSH) %12 IU/L. Interventions: All participants received a luteal phase down-regulation long protocol. Genome DNA was extracted from the peripheral blood leukocytes. For the exploratory and replication cohorts, exome sequencing was conducted on a HiSeq 2500 sequencing platform. The multiplex polymerase chain reaction amplification technique and next-generation sequencing also were performed in the exploratory and replication cohorts. For the samples of the validation cohort, Sanger sequencing was performed. Main Outcome Measures: The primary endpoint was the gonadotropin levels after down-regulation, and the secondary endpoints were hormone levels and follicle diameters during stimulation, the total dose of FSH, duration of FSH stimulation, number of oocytes retrieved, and clinical pregnancy rate. Results: In the exploratory cohort, we identified that FSHB rs6169 (P=2.71 x 10(-24)) and its single-nucleotide polymorphisms in high linkage disequilibrium were associated with the down-regulated FSH level. The same locus was confirmed in the replication cohort. Women carrying the C allele of FSHB rs6169 exhibited higher average estradiol level during stimulation (P=6.82 x 10(-5)), shorter duration of stimulation, and less amount of exogenous FSH (P-duration=0.0002; P-dose=0.0024). In the independent validation set, adding rs6169 genotypes into the prediction model for FSH level after down-regulation enhanced the area under the curve from 0.560 to 0.712 in a logistic regression model, and increased prediction accuracy by 41.05% when a support vector machine classifier was applied. Conclusion: The C allele of FSHB rs6169 is a susceptibility site for the relatively high level of FSH after down-regulation, which may be associated with increased ovarian FSH sensitivity. (Fertil Steril (R) 2023;120:671-81. (c) 2023 by American Society for Reproductive Medicine.)

The need to identify a missing person (MP) through kinship analysis of DNA samples found at a crime scene has become increasingly prevalent. DNA samples from MPs can be severely degraded, contain little DNA and mixed with other contributors, which often makes it difficult to apply conventional methods in practice. This study developed a massively parallel sequencing-based panel that contains 1661 single-nucleotide polymorphisms (SNPs) with low minor allele frequencies (MAFs) (averaged at 0.0613) in the Chinese Han population, and the strategy for relationship inference from DNA mixtures comprising different numbers of contributors (NOCs) and of varying allele dropout probabilities. Based on the simulated dataset and genotyping results of 42 artificial DNA mixtures (NOC = 2-4), it was observed that the present SNP panel was sufficient for balanced mixtures when referenced to the closest relatives (parents/offspring and full siblings). When the mixture profiles suffered from dropout, incorrect assignments were markedly associated with relatedness, NOC and the dropout level. We, therefore, indicate that SNPs with low MAFs could be reliably interpreted for MP identification through the kinship analysis of complex DNA mixtures. Further studies should be extended to more possible scenarios to test the feasibility of this present approach.

OBJECTIVE:To compare live-birth rates between letrozole application and artificial cycle for endometrium preparation during frozen embryo transfer (FET) cycle among women with polycystic ovarian syndrome (PCOS). METHODS:A randomized controlled trial was conducted. Women with PCOS were randomized to letrozole application for ovulation induction compared with artificial cycle for endometrial preparation during FET. The primary outcome was live-birth rate per embryo transfer. Secondary outcomes included pregnancy-related outcomes, perinatal outcomes, and maternal complication rates. Assuming α=0.05 and 80% power, 186 patients per group were required to demonstrate a difference of 15% in live-birth rate: 205 patients (at least) per group were randomized to allow for a 10% dropout rate. RESULTS:Four hundred twenty patients were enrolled from 2018 to 2021. Two hundred ten patients were assigned to the letrozole application group, and 210 were assigned to the artificial cycle group. There was no difference in the live-birth rate (42.4% vs 42.9%, P =>.99). There was no difference in secondary outcomes, including clinical pregnancy rate (51.4% vs 56.2%, P =.378), implantation rate (51.8% vs 55.8%, P =.401), and miscarriage rate (8.6% vs 11.0%, P =.511). For perinatal outcomes, singleton birth weight was significantly higher in the artificial cycle group (3,108±56 g vs 3,301±58, P =.018), and the incidence of gestational diabetes mellitus (GDM) was significantly higher in letrozole application group (14.6% vs 5.6%, P =.050). The other outcome was no difference in maternal complications. CONCLUSION:There was no difference in pregnancy outcomes between letrozole application compared with artificial cycle for endometrial preparation in women with PCOS who underwent FET. The risk of GDM was higher in the letrozole application group, and the singleton birth weight was lower in the artificial cycle group. CLINICAL TRIAL REGISTRATION:Chinese Clinical Trial Registry, ChiCTR1800014746.

In forensic applications, there is an increasing demand for the analysis of DNA profiles arising from missing person identification (MPI) cases. A specific DNA profile may originate from a single source or more than one contributor (i.e., a DNA mixture). When direct references are not available, indirect relative references can be used to identify missing persons by kinship analysis. As a novel kind of multiallelic marker, microhaplotypes have proven promising for relatedness determination and mixture deconvolution. Herein, we developed a large panel of 185 microhaplotype markers and demonstrated its application in different scenarios of relationship inference through a simulation study and real pedigree analysis, combined with probabilistic genotyping models for data interpretation. Based on single-source profiles, it was shown that the present microhaplotype panel was sufficient for pairwise close relative testing (parent/child, full-sibling and 2nd-degree relative). For more distant relatives (3rd-degree relatives), there was a clear improvement when data from one well-chosen extra relative were available. We further sought to evaluate the theoretical systematic effectiveness and actual performance of microhaplotype markers in identifying the contribution of a missing pedigree member to a two-person mixture (as a minor donor). It was observed that 100% correct assignments were made in the balanced mixtures (with no dropout) when referenced to close relatives. When the mixture profiles suffered from dropout, incorrect assignments of minor donors were markedly associated with relatedness and the dropout level. Meanwhile, the studied scenarios generally exhibited zero or very low false-positive rates, indicating a low probability of incorrectly assigning an unrelated contributor as a close relative of the reference. Our results indicate that microhaplotype data can be reliably interpreted for identifying missing persons through kinship analysis based on DNA profiles of single-source samples or two-person mixtures. Furthermore, this study could be extended to more complex scenarios, such as determining the relatedness of contributors in (or among) mixed DNA profiles, if combined with different statistical frameworks.

To determine whether single endometrial polyp (EP) or multiple EP (polyp number ≥ 6) are associated with chronic endometritis (CE).

In steroidogenic cells, steroids are synthesized de novo from cholesterol stored in lipid droplets (LDs). The size of LDs regulated by adipose differentiation-related protein (ADRP) is closely related to cholesterol ester hydrolysis. Many studies reported that cadmium (Cd) had dual effects on steroidogenesis in granulosa cells (GCs). However, the role of LD and its regulation in abnormal steroidogenesis caused by Cd exposure remain unknown. In current study, female rats were exposed to CdCl2 during gestation and lactation, and influence of such exposure was investigated in ovarian GCs of female offspring. The size of LDs was found much smaller than normal in GCs; ADRP was down-regulated and hormone-sensitive lipase (HSL) phosphorylation was increased, followed by up-regulation of steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage enzyme (CYP11A1); the expression of 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase beta-2 (PLCβ2) and protein kinase C alpha type (PKCα) were both decreased accompanying the ADRP down-regulation. This series of events resulted in a high level of progesterone in serum. Similar results were demonstrated in GCs treated with 20 μM CdCl2 for 24 h in vitro. The protein level of ADRP was decreased after gene silencing of PLCβ2/PKCα, and the knockdown of PLCβ2/PKCα/ADRP led to micro-sized LD formation. We found that Cd exposure down-regulated ADRP by inhibiting the PLCβ2-PKCα signaling pathway, reduced the size of LDs, and promoted HSL phosphorylation. StAR and CYP11A1 were both up-regulated following the hydrolysis of cholesterol ester, which led to a high production of progesterone. LD thereby is a target subcellular organelle for Cd to affect steroid hormone synthesis in ovarian GCs. These findings might help to uncover the mechanism of ovarian dysfunction and precocious puberty caused by Cd pollution.

Introduction:Although pre-treatment with a GnRH agonist can reduce the size of adenomyosis lesions, the supra-physiological hormone level induced by controlled ovarian hyperstimulation (COH) may negate the usefulness of the GnRH agonist in patients with adenomyosis lesions, leading to continued poor outcomes in fresh embryo transfer cycles duringin vitrofertilization (IVF). It is unclear whether GnRH agonist pre-treatment before starting the long GnRH agonist protocol for IVF/ICSI (intracytoplasmic sperm injection) can improve cumulative live birth rate (CLBR) of infertile women with adenomyosis. Method:In this retrospective cohort study, a total of 374 patients diagnosed as adenomyosis (477 cycles) underwent IVF/ICSI with long GnRH agonist protocol with or without GnRH agonist pre-treatment between January 2009 and June 2018. Logistic regression was used to assess the association between GnRH agonist pre-treatment and pregnancy outcome after adjusting for confounding factors. Results:The live birth rate in fresh embryo transfer cycles was higher in the non-pre-treatment group than in the GnRH agonist pre-treatment group (37.7 vs. 21.2%,P= 0.028); the adjusted odds ratio (OR) for the long agonist protocol without pre-treatment was 1.966 (95% CI: 0.9-4.296,P= 0.09). The CLBR was higher in the non-pre-treatment group than in the GnRH agonist pre-treatment group (40.50 vs. 27.90%,P= 0.019); the adjusted OR for the long agonist protocol without pre-treatment was 1.361 (95% CI: 0.802-2.309,P= 0.254). Conclusion:Our results indicated that GnRH agonist pre-treatment before starting the long GnRH agonist protocol does not improve the live birth rate in fresh embryo transfer cycles or CLBR in infertile women with adenomyosis after IVF/ICSI treatment when compared to that in non-pre-treated patients. A subsequent prospective randomized controlled study is needed to confirm these results.

AIM:To investigate whether treated hyperprolactinemia has an impact on pregnancy outcomes in in vitro fertilization/intracytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET). METHODS:A retrospective cohort study was conducted on 535 women who underwent IVF/ICSI-ET between January 2012 and December 2016, of which 123 had treated hyperprolactinemia (case group), 369 were matched controls. Besides, 43 remained hyperprolactinemic after treatment consisted of abnormal group. Cumulative live birth rate (CLBR) after one oocyte retrieval cycle was taken as the primary outcome. A time-to-event analysis using Fine and Gray's test was used to compare CLBR between case and control groups. RESULTS:The median prolactin level was 80.00 ng/mL before dopamine agonist treatment in case group, and it reduced to 14.80 ng/mL after the treatment, similar to the level of control group (15.17 ng/mL, P = 0.316). No significant differences in baseline characteristics were found between case and control groups. The CLBR after one oocyte retrieval cycle were 69.1% (85/123) and 66.4% (245/369) in the case group and control group, respectively (P = 0.580). No significant differences were found between case and control groups in perinatal outcomes. Pregnancy and perinatal outcomes of abnormal group were similar to those of case and control groups. CONCLUSION:Impact of treated hyperprolactinemia on CLBR and perinatal outcomes in IVF-ET was not evident.

Objective: To analyze the influence of the start point of luteal support on clinical pregnancy rate, implantation rate, and live birth rate of in vitro fertilization and embryo transfer (IVF-ET) cycles. Design: Single-center prospective randomized controlled trial. Setting: University-affiliated IVF unit. Patient(s): Women% 35 years of age with day 3 FSH levels < 15 mIU/mL, who were undergoing their first IVF-ET cycles and received ovarian stimulation with the use of a GnRH agonist long protocol. Intervention(s): The patients were randomized on the day of hCG trigger to receive luteal phase support either 1 day after oocyte retrieval (group A) or on the day of oocyte retrieval (group B). Main Outcome Measure(s): Clinical pregnancy rate, implantation rate, miscarriage rate in the first trimester of pregnancy, and live birth rate per embryo transfer cycle. Result(s): Two hundred thirty-three patients were enrolled in this study: 117 were assigned to group A and 116 to group B. The clinical pregnancy rate (group A vs. group B: 55.3% vs. 51.5%), implantation rate (38.4% vs. 38.0%), and miscarriage rate (7.7% vs. 7.5%) were similar between the two groups. The live birth rate also did not significantly differ between the two groups (45.7% vs. 46.6%). Conclusion(s): Our study indicated that the initiation of progesterone supplementation 1 day after oocyte retrieval did not decrease the clinical pregnancy rate, implantation rate, or live birth rate in women undergoing IVF-ET cycles with the use of the GnRH agonist long protocol. (C) 2017 by American Society for Reproductive Medicine.

Researchers have sought to develop an effective protocol for paternity analysis using cell-free DNA (cfDNA) in maternal plasma. The use of massively parallel sequencing (MPS) technology for SNP testing is attractive because of its high-throughput capacity and resolution to single-base precision. In this study, we designed a customized SNP panel for cfDNA sequencing that includes 720 short amplicons (< 140 bp) targeting SNPs on the autosome and Y chromosome. The systemic performance was evaluated using the Ion Torrent PGM, indicating balanced coverage among most of the included loci, except for 78 poorly performing SNPs that were observed to have an inconsistent allele balance, lower coverage reads or high background signals. Then, the custom panel was used to perform cfDNA genotyping in maternal plasma from 20 pregnancies in the first and second trimesters (9 to 21 weeks). By establishing an allele fraction cutoff of 2.0%, 53 to 128 autosomal SNP loci were considered informative for paternal origin. Validation results in foetal samples showed that 49.43% to 100% of the real paternal alleles were accurately identified, with incorrect alleles encountered in 3 cases. The concentration of foetal cfDNA ranged from 4.28% to 10.70%. Our results show that this amplicon-based sequencing strategy could be utilized in analysing paternally inherited alleles in maternal plasma. However, further studies and optimization are required for a more detailed and accurate interpretation of the cfDNA sequencing results based on MPS technology.

The aim of this study was to determine whether an interchromosomal effect (ICE) occurred in embryos obtained from reciprocal translocation (rcp) and Robertsonian translocation (RT) carriers who were following a preimplantation genetic diagnosis (PGD) with whole chromosome screening with an aCGH and SNP microarray. We also analyzed the chromosomal numerical abnormalities in embryos with aneuploidy in parental chromosomes that were not involved with a translocation and balanced in involved parental translocation chromosomes.

BACKGROUND Recent advances in massively parallel sequencing (MPS) technology have provided efficient methods for noninvasive prenatal paternity testing (NIPAT). However, a well‐accepted protocol has not been established. The present study developed an MPS‐based approach for NIPAT and compared the performance of two recently reported methods for MPS data interpretation. STUDY DESIGN AND METHODS We selected 1795 unlinked polymorphic single‐nucleotide polymorphisms (SNPs) and performed paternity analysis in 34 real parentage test cases with maternal plasma samples using the Illumina HiSeq platform. Sequencing data were interpreted by the straightforward counting method for the identification of paternal alleles and mathematical algorithms for paternity index (PI) calculation, respectively. RESULTS Based on the sequencing data from each family case, both of the two statistical approaches produced a significant separation between the biological father and 90 unrelated males (p < 0.0001) when sufficient effective loci were attained. Nevertheless, up to 30.82% of real paternal alleles were filtered by a predefined cutoff and resulted in insufficient effective loci, especially in plasma samples with low fetal fraction (approx. 90.60% were filtered). In contrast, the PI calculation model utilized all maternal homozygous SNPs as effective loci (approx. 40% of total SNPs) and successfully identified the correct biological father, with the log‐transformed combined PI (Lg(CPI)) value varying from 68.23 to 158.01 in each family case. CONCLUSION Our study illustrates that the Bayesian approach represents the better choice in NIPAT data interpretation. Further, the adoption of more informative markers (e.g., tri‐allelic SNPs, tetra‐allelic SNPs, and micro‐haplotypes) or deeper sequencing is recommended for the improvement of the testing efficiency.

BACKGROUND:Researchers have sought to develop a noninvasive protocol for paternity analysis that uses fetal cell-free DNA (cfDNA) in maternal plasma. Massively parallel sequencing (MPS) is expected to overcome this challenge because it enables the analysis of millions of DNA molecules at a single-base resolution. STUDY DESIGN AND METHODS:Seven women were involved in prenatal paternity testing cases. Before conventional invasive procedures, cfDNA was isolated from maternal plasma. Fetal tissues were then collected, as were blood samples from the alleged fathers. A custom array was designed that targeted 1497 regions containing single-nucleotide polymorphisms. These regions were massively parallel sequenced. RESULTS:In these seven cases, the mean nonmaternal allele fractions in maternal plasma ranged from 3.22% to 6.17%. Setting the allele fraction cutoff of 2.5%, 300 to 491 loci were considered informative for paternal origin and no genetic incompatibilities with the alleged fathers were found. These results were concordant with those of conventional short tandem repeat genotyping. Validation results performed using fetal samples showed that sequencing noise was completely filtered out, and 78.35% to 99.19% of the paternal alleles were accurately genotyped. The fetal cfDNA concentrations ranged from 7.12% to 13.81%, and the overall sequencing error rates ranged from 0.40% to 0.93%. CONCLUSION:In our study, we evaluate a straightforward method that can be used to identify paternal alleles based on analyses of paternal alleles and sequencing errors in maternal plasma. Our results support the notion that an MPS-based method could be utilized in noninvasive fetal genotyping and prenatal paternity analyses.

Abstract Objective: It has been shown in animal models that circadian clock exists in corpora luteum which is essential for maintaining pregnancy. However, it is unknown whether circadian clock exists in corpora luteum and its relation with steroidogenesis in human ovary. Study design: Human luteinized granulosa cells from patients who underwent in vitro fertilization treatment were purified and cultured in vitro. Accumulation patterns of circadian gene and steroidogenesis-related gene mRNAs in human luteinized granulosa cells were observed during the 48 hours after treatment with human chorionic gonadotropin (hCG) by quantitative PCR. Results: We found that the circadian genes CLOCK, PER2, and BMAL1 were expressed in cultured human luteinized granulosa cells. Among these genes, only expression of PER2 displayed oscillating patterns with a 16-h period in these cells after stimulation by hCG. Expression of CLOCK and BMAL1 did not show significant oscillating patterns. Expression of the steroidal acute regulatory protein (STAR) gene showed an oscillating pattern that was similar to that of PER2. Expression of CYP11A1, HSD3B2, and CYP19A1 increased significantly after hCG stimulation; however, none of these genes displayed significant oscillating patterns. Conclusions: Molecular circadian clock exists in human luteinized granulosa cells and may be related with steroidogenesis in human ovary.

To observe the effect of gene expression and tumorigenicity in hybrid cells of human embryonic stem cells (hESCs) and ovarian cancer cells in vitro and in vivo using a mouse model, and to determine its feasibility in reprogramming tumour cells growth and apoptosis, for a potential exploration of the role of hESCs and tumour cells fusion in the management of ovarian cancer. Stable transgenic hESCs (H1) and ovarian cancer cell line OVCAR-3 were established before fusion, and cell fusion system was established to analyse the related indicators. PTEN expression in HO-H1 cells was higher than those in the parental stem cells and lower than those in parental tumour cells; the growth of OV-H1 (RFP + GFP) hybrid cells with double fluorescence expressions were obviously slower than that of human embryonic stem cells and OVCAR-3 ovarian cancer cells. The apoptosis signal of the OV-H1 hybrid cells was significantly higher than that of the hESCs and OVCAR-3 ovarian cancer cells. In vivo results showed that compared with 7 days, 28 days and 35 days after inoculation of OV-H1 hybrid cells; also, apoptotic cell detection indicated that much stronger apoptotic signal was found in OV-H1 hybrid cells inoculated mouse. The hESCs can inhibit the growth of OVCAR-3 cells in vitro by suppressing p53 and PTEN expression to suppress the growth of tumour that may be achieved by inducing apoptosis of OVCAR-3 cells. The change of epigenetics after fusion of ovarian cancer cells and hESCs may become a novel direction for treatment of ovarian cancer.

Objective This study examined microRNA-92 (miR-92) expression level in relation to the mRNA level of its potential target gene, estrogen receptor β1 (ERβ1), in female patients diagnosed with pelvic organ prolapse (POP). Study design Between July 2012 and September 2014, a total of 104 patients were recruited at the First Affiliated Hospital of Sun Yat-sen University, which included 56 POP patients and 48 non-POP control subjects. Based on POP-Q score, the POP patients were further categorized into POP II and POP III groups. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to quantify miR-92 expression level. ERβ1 tissue expression was measured by western blot and immunohistochemistry (IHC) methods. SPSS 19.0 software was used for statistical analysis. Results No remarkable differences were observed between the POP group and non-POP group, and between the POP II and POP III groups, with respect to age, body mass index (BMI), parity, menopause status, and family history of POP. The expression level of miR-92 in the POP group was dramatically higher than the non-POP group (P < 0.05). Consistent with the disease status, miR-92 expression level in POP III group was markedly higher than the POP II group (P < 0.05). Western blot analysis revealed significantly reduced levels of ERβ1 in the POP group compared to the non-POP group, with similar results obtained between the POP III and POP II groups (all P < 0.05). IHC results showed ERβ1 staining mainly in the nucleus and semi-quantitative measurements, expressed as positive expression rate, revealed that ERβ1 level in the POP group was clearly lower than non-POP group. Finally, statistical analysis of IHC results from uterosacral ligament tissue showed inverse correlation between miR-92 and ERβ1 expression levels in POP patients (P < 0.05). Conclusions Our results revealed increased miR-92 expression and decreased ERβ1 level in uterosacral ligaments of women diagnosed with POP, compared to non-POP subjects POP III patients exhibited more severe changes than POP II patients. Further, ERβ1expression is inversely correlated to miR-92 expression. Taken together, our results suggest that miR-92 and ERβ1 expression levels may be used as reliable diagnostic markers for assessing the severity of POP.