BACKGROUND:Menstrual blood-derived mesenchymal stem cels are proved to be a new type of adult stem cels. To date, little is reported on the methods of establishing this kind of cel lines systematicaly.
OBJECTIVE:To isolate, culture, identify and finaly establish menstrual blood-derived mesenchymal stem cel lines.
METHODS:Menstrual blood-derived mesenchymal stem cels were isolated from the menstrual blood of healthy female donors and cultured accordingly using density gradient centrifugation method. Cel morphology and proliferation characteristics were observed, and proliferative ability was detected by cel counting kit-8 assay. Cels were colected periodicaly for karyotype analysis. Expressions of cel surface antigens such as CD34, CD38, CD44, CD45, CD73, CD90, CD105 and Oct-4 were analyzed using flow cytometry. Cel pluripotency were identified by inducing adipogenesis and osteogenesis in vitro.
RESULTS AND CONCLUSION:We successfuly established four menstrual blood-derived mesenchymal stem cel lines from the menstrual blood of six healthy volunteers. The cultured cel lines had a typical spindle shape and kept normal 46, XX karyotype. The cels grew rapidly and entered into the logarithmic growth phase at 48 hours after passaging. The results of flow cytometry showed that menstrual blood-derived mesenchymal stem cels were positive for CD44, CD73, CD90, CD105 and Oct-4, while negative for CD38, CD34 and CD45. Menstrual blood-derived mesenchymal stem cels were positive for oil red O staining at 22 days after adipogenic induction and also positive for alizarin red staining at 2 weeks after osteogenetic induction. In conclusion, our study suggests that menstrual blood-derived mesenchymal stem cels with stable karyotype and pluripotency can proliferate quickly, and we can provide a new resource for stem cel therapy and regenerative medicine through establishing the menstrual blood-derived mesenchymal stem cel lines.

BACKGROUND: Ethanol chemical damage method can be used to establish thin endometrium rat model. Expression of proliferation-related proteins is decreased in thin endometrium animal model, which contributes to the study of thin endometrium-related mechanism and provides guidance for clinical treatment. OBJECTIVE:To explore the changes in vimentin and vascular endothelial growth factor expression in rat modelsof high concentrations of ethanol-induced thin endometrium. METHODS: 0.3 mL 95% ethanol was slowly injected in the right uterus of female Sprague-Dawley rats to establish rat models of thin endometrium. An equal volume of physiological saline was injected into the left uterus as control sides. RESULTS AND CONCLUSION:The thickness of endometrium in the injured uterus was significantly thinner than the control ones. Number of glands reduced, structure became disorder, and vimentin and vascular endothelial growth factor expression markedly diminished. These results suggest that 95% ethanol injury can effectively establish rat models of thin endometrium. Proliferation-related protein expression altered in the thin endometrium.

Objective: To explorer factors related to spontaneous reduction in twin pregnancy following assisted reproductive technology. Methods: 2 848 twin pregnant women with treatment of vitro fertilization-embryo transfer (IVF-ET) or intra cytoplasmic sperm injection (ICSI) cycles were enrolled at Assisted Reproductive Centre of the First Affiliated Hospital of Nanjing Medical University, Nanjing Maternity Hospital and Shengjing Hospital of China Medical University from January 2013 to December 2016 respectively. Basic features of subjects, relevant clinical indicators, factors of assisted reproductive therapy and pregnancy outcome were collected from clinical assisted reproductive technology management system. According to the pregnancy outcome, the subjects with spontaneous reduction were classified as case group (n=686), and those with normal twin birth were classified as control group (n=2 162). The features of subjects in the two groups were compared. Non-conditional logistics regression model was used to analyze the related factors of the occurrence of spontaneous reduction. Results: The age of case group and control group were (30.6±4.3) and (30.2±4.0) years old respectively. After the adjustment of male sterile factor, compared to the subjects with luteinizing hormone level on the day of human chorionic gonadotropin administration (HCG) <1.43 IU/L, OR (95%CI) of the subjects with value at 2.59-5.10 IU/L was1.62 (1.08-2.42).Compared to the subjects with number of transferred embryo as 1, OR (95%CI) of the subjects with value as 3 was 0.23 (0.07-0.74). Compared to the subjects with stage of transferred embryo as cleavage stage, OR (95%CI) of the subjects with blastula stage was 0.42 (0.27-0.67). Conclusion Luteinizing hormone level on day of HCG, number and stage of embryo transfer are related factors to spontaneous reduction in twin pregnancy following assisted reproductive technology.

Purpose: Male overweight and obesity could affect sperm quality and reproductive health. However, the impact of body mass index (BMI) on assisted reproductive technology (ART) outcomes in oligospermia and/or asthenospermia patients is yet lacking. This study aims to assess the impact of paternal BMI on ART and neonatal outcomes among oligozoospermia and/or asthenospermia patients undergoing in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI). Materials and Methods: In this study, 2,075 couples undergoing their first fresh embryo transfer between January 2015 and June 2022 were recruited. Following the World Health Organization’s (WHO’s) categories, couples were stratified into three cohorts based on paternal BMI: normal weight (18.5–24.9 kg/m2), overweight (25.0–29.9 kg/m2), and obese (≥30.0 kg/m2). Modified Poisson regression models were used to assess the associations of paternal BMI with fertilization, in vitro embryonic development, and pregnancy outcomes. Logistic regression models were performed to investigate the associations of paternal BMI with pregnancy loss and neonatal outcomes. Furthermore, stratified analyses were performed based on fertilization methods, male infertility cause, and maternal BMI. Results: Higher paternal BMI is associated with a lower likelihood of achieving normal fertilized (p-trend=0.002), Day 3 transferable (p-trend=0.007), and high-quality embryos (p-trend=0.046) in IVF cycles, rather than in ICSI cycles. Paternal BMI of oligospermia or asthenospermia was negatively correlated with day 3 transferable (p-trend=0.013 and 0.030) and high-quality embryos (p-trend=0.024 and 0.027). Moreover, for neonatal outcomes, paternal BMI was positively associated with macrosomia (p-trend=0.019), large for gestational age (LGA) (p-trend=0.031), and very LGA (p-trend=0.045). Conclusions Our data suggested that higher paternal BMI was associated with fetal overgrowth, reduced fertilization, and embryonic development potential. Among males with oligospermia and/or asthenospermia, the impact of overweight and obesity on the choice of fertilization method and the long-term effects on their offspring need to be further investigated.

OBJECTIVE: To assess the value of single sperm sequencing in preimplantation genetic diagnosis. METHODS: A male patient with achondroplasia due to a de novo FGFR3 variant was subjected to single sperm isolation and sequencing. Twenty single sperm samples were isolated by mechanical immobilization, and their whole genome was amplified. PCR primers were designed for the variant site and 25 flanking single nucleotide polymorphism (SNP) loci, and the PCR products were sequenced to determine the chromosomal haplotype which did not harbor the pathogenic variant. Biopsy samples of 12 embryonic trophoblasts were taken. Following whole genome amplification, high-throughput sequencing was carried out to detect the carrier status of the embryos. Wild type blastocysts were selected for transplantation. Amniotic fluid samples were taken at 19 weeks of gestation to confirm the status of the fetus. RESULTS: Eight SNP were selected by single sperm sequencing, with which the haplotypes were successfully constructed. Preimplantation genetic testing indicated that 5 embryos have carried the pathogenic variant and 7 did not. Testing of amniotic fluid sample during the second trimester of pregnancy confirmed that the fetus did not carry the FGFR3 gene c.1138G>A variant. CONCLUSION For male patients carrying de novo pathogenic variants, SNP sites can be selected through single sperm sequencing, and haplotypes can be constructed by linkage analysis for preimplantation genetic diagnosis.