Nanopore-based metagenomics analysis reveals microbial presence in amniotic fluid: A prospective study.

Background: Current research on amniotic fluid (AF) microbiota yields contradictory data, necessitating an accurate, comprehensive, and scientifically rigorous evaluation. Objective: This study aimed to characterise the microbial features of AF and explore the correlation between microbial information and clinical parameters. Methods: 76 AF samples were collected in this prospective cohort study. Fourteen samples were utilised to establish the nanopore metagenomic sequencing methodology, whereas the remaining 62 samples underwent a final statistical analysis along with clinical information. Negative controls included the operating room environment (OE), surgical instruments (SI), and laboratory experimental processes (EP) to elucidate the background contamination at each step. Simultaneously, levels of five cytokines (IL-1ß, IL-6, IL-8, TNF-α, MMP-8) in AF were assessed. Results: Among the 62 AF samples, microbial analysis identified seven without microbes and 55 with low microbial diversity and abundance. No significant clinical differences were observed between AF samples with and without microbes. The correlation between microbes and clinical parameters in AF with normal chromosomal structure revealed noteworthy findings. In particular, the third trimester exhibited richer microbial diversity. Pseudomonas demonstrated higher detection rates and relative abundance in the second trimester and Preterm Birth (PTB) groups. S. yanoikuyae in the PTB group exhibited elevated detection frequencies and relative abundance. Notably, Pseudomonas negatively correlated with activated partial thromboplastin time (APTT) (r = -0.329, P = 0.016), while Staphylococcus showed positive correlations with APTT (r = 0.395, P = 0.003). Furthermore, Staphylococcus negatively correlated with birth weight (r = -0.297, P = 0.034). Conclusion: Most AF samples exhibited low microbial diversity and abundance. Certain microbes in AF may correlate with clinical parameters such as gestational age and PTB. However, these associations require further investigation. It is essential to expand the sample size and undertake more comprehensive research to elucidate the clinical implications of microbial presence in AF.

[Clinical phenotype and genetic analysis of a fetus with recombinant chromosome 8 syndrome].

OBJECTIVE: To explore the clinical characteristics and molecular genetic mechanism of a fetus with recombinant chromosome 8 (Rec8) syndrome. METHODS: A fetus who was diagnosed with Rec8 syndrome at the Provincial Hospital Affiliated to Shandong First Medical University on July 20, 2021 due to high risk for sex chromosomal aneuploidy indicated by non-invasive prenatal testing (NIPT) (at 21st gestational week) was selected as the study subject. Clinical data of the fetus was collected. G-banded karyotyping and chromosomal microarray analysis (CMA) were carried out on the amniotic fluid sample. Peripheral blood samples of the couple were also subjected to G banded karyotyping analysis. RESULTS: Prenatal ultrasonography at 23rd gestational week revealed hypertelorism, thick lips, renal pelvis separation, intrahepatic echogenic foci, and ventricular septal defect. The karyotype of amniotic fluid was 46,XX,rec(8)(qter→q22.3::p23.1→qter), and CMA was arr[GRCh37]8p23.3p23.1(158049_6793322)×1, 8q22.3q24.3(101712402_146295771)×3. The karyotype of the pregnant woman was 46,XX,inv(8)(p23.1q22.3), whilst that of her husband was normal. CONCLUSION: The Rec8 syndrome in the fetus may be attributed to the pericentric inversion of chromosome 8 in its mother. Molecular testing revealed that the breakpoints of this Rec8 have differed from previously reported ones.

[Genetic features of a case with mosaic ring chromosome 4 and a review of the literature].

OBJECTIVE: To explore the genetic basis, clinical phenotype and pathogenesis for a child with mosaicism ring chromosome 4. METHODS: Clinical data of the child was collected. Peripheral blood chromosomal karyotype G banding analysis, chromosomal microarray analysis (CMA), fluorescence in situ hybridization (FISH) were carried out for the child, in addition with a review of the literature. RESULTS: The child was born full-term with low birth weight, facial dysmorphism, patent ductus arteriosus and ventricular septal defect. His karyotype was determined as mos46,XY,r(4)(p16.3q35.2)[259]/45,XY,-4[25]/47,XY,r(4)(p16.3q35.2), +r(4)(p16.3q35.2)[8]/46,XY,der(4)del(4)(p16.3)inv(4)(p16.3q31.1)[6]/46,XY,dic?r(4;4)(p16.3q35.2;p16.3q35.2)[4]/48,XY,r(4)(p16.3q35.2),+r(4)(p16.3q35.2)×2[3]/46,XY,r(4)(p1?q2?)[2]; CMA result was arr[GRCH37]4p16.3(68 345-2 981 614)×1; FISH result was 45,XY,-4[12]/45,XY,-4×2,+mar1.ish r1(4)(WHS-,D4Z1+)[1]/ 46,XY,-4,+mar1.ishr1(4)(WHS-,D4Z1+)[73]/46,XY,-4,+mar2.ishr2(4)(WHS-,D4Z1++)[1]/47,XY,-4,+mar1×2.ishr1(4) (WHS-, D4Z1+)×2[4]/46,XY,del(4)(p16.3).ish del(4)(p16.3)(WHS-,D4Z1+)[9]. CONCLUSION: In this case, the ring chromosome 4 as a de novo variant has produced a number of cell lines during embryonic development and given rise to mosaicism. The clinical phenotype of ring chromosome 4 is variable. The instability of the ring chromosome itself, presence of mosaicism, chromosome breakpoint and range of deletion and/or duplication may all affect the ultimate phenotype.

Biological investigations on therapeutic effect of chitosan encapsulated nano resveratrol against gestational diabetes mellitus rats induced by streptozotocin.

The chitosan encapsulation with bioactive compounds (resveratrol) is a significant method that can be used to raise the stability and effectiveness of substances in gestational diabetes management. In this study, the resveratrol-zinc oxide complex is encapsulated with chitosan (CS-ZnO-RS). The synthesized CS-ZnO-RS could be used to deliver the resveratrol with minimized side effects and also improved bioavailability. CS-ZnO-RS were characterized by various techniques such as particle size analyzer, DSC, FT-IR, TEM, SEM, and AFM. The electron microscopic and particle analyzer confirmed that the synthesized CS-ZnO-RS were monodispersed, spherical and its average size was 38 nm. The drug-releasing profile showed that 95% of RS is released from CS-ZnO-RS within 24 h. In vitro studies confirmed that α-glucosidase and α-amylase inhibitory activities were closely related to the concentration of CS-ZnO-RS. The highest inhibition of α-glucosidase (77.32%) and α-amylase (78.4%) was observed at 500 µg/mL. Furthermore, the treatment of CS-ZnO-RS significantly decreased the blood glucose levels in gestational diabetes mellitus induced rats and maintained the lipid content toward the normal rats. In addition, the CS-ZnO-RS reduced the level of inflammation factors (IL-6 and MCP-1) and endoplasmic reticulum stress (GRP78, p-IRE1α, p-eIF2α, and p-PERK).

[Result of prenatal diagnosis for 151 high-risk women by noninvasive prenatal screening based on high-throughput sequencing].

OBJECTIVE: To assess the value of combined fetal karyotyping and chromosomal microarray analysis (CMA) for the verification of high-risk pregnancy signaled by noninvasive prenatal screening (NIPS) based on high-throughput sequencing. METHODS: One hundred and fifty-one pregnant women with high risks for aneuploidies of chromosomes 13, 18, 21, X and Y or pathological copy number variations (CNVs) by NIPS were subjected to amniocytic karyotyping and CMA analysis. RESULTS: One hundred and forty-two women were found to have a high risk for fetal chromosomal aneuploidies, which included 83 cases of trisomy 21, 17 cases of trisomy 18, 2 cases of trisomy 13, and 40 cases of sex chromosome aneuploidies. Amniocytic karyotyping and CMA analysis has confirmed 81 cases of trisomy 21, 15 cases of trisomy 18, 10 cases of 47,XXY, 4 cases of 47,XXX, 2 cases of 47,XYY and 1 case of 46,X,del(X)(q26.1). Two trisomy 21, two trisomy 18, 2 trisomy 13, and 23 cases of sex chromosomal aneuploidies were verified as false positives. For 9 women with pathological fetal CNVs detected by NIPS, combined fetal karyotyping and CMA has confirmed 1 case of chromosome 13 microdeletion, 1 case of chromosome 18 microduplication, and 1 case of chromosome 18 deletion. For a case with 30 Mb duplication of chromosome 2 and 25 Mb duplication of chromosome 8, CMA analysis had no positive finding, while fetal umbilical cord blood karyotyping has yielded a 46,XX,dup(2)(p23.1p25.3)[13]/46,XX[87] karyotype. The remaining 5 cases were confirmed as false positive results. CONCLUSION: Combined fetal karyotyping and CMA has provided a powerful tool for verifying high-risk fetuses signaled by NIPS.

Severe mitral stenosis in term pregnancy: management by simultaneous cesarean section and mitral valve replacement. Case report.

A 38-year-old woman in the 37th week of pregnancy was admitted for acute presentation of severe cough and aggravated dyspnea. She was previously diagnosed with rheumatic heart disease (RHD) at the age of 31. During the present pregnancy, she reported at 37 weeks and had RHD, severe mitral stenosis, secondary pulmonary hypertension, and heart failure. As a consequence, the patient underwent an emergency cesarean section followed by reoperative mitral valve replacement, which was successfully under a cardiopulmonary bypass (CPB). In this report, the authors present the successful experience of management of severe mitral stenosis in pregnancy.

Genetic effects of a 13q31.1 microdeletion detected by noninvasive prenatal testing (NIPT).

Microdeletions of chromosome 13q31.1 are relatively rare. These types of deletions may cause different genetic effects on genotypes and/or phenotypes. There are several ways to detect microdeletions; noninvasive prenatal testing (NIPT) is the newest detection method. In this study, we aimed to investigate the genetic effects of a 13q31.1 microdeletion detected by NIPT and to reconfirm the feasibility of this procedure in predicting sub-chromosomal copy number variations (CNVs). The 13q31.1 microdeletion, which has previously been described as a disease-associated fragment, was detected by NIPT in a pregnant woman. To validate the finding and to explain the origin of this sub-chromosomal CNV, we collected fetal amniotic fluid and parental blood samples and tested the samples using array-based comparative genomic hybridization (aCGH). Karyotype analysis was performed on all of the samples to rule out balanced or mosaic anomalies. The aCGH results confirmed the NIPT findings. We detected the same type of microdeletion in the fetus and the mother via aCGH. The mother had a normal phenotype; therefore, in a post-test genetic counseling session, we predicted a normal phenotype for the fetus. After delivery, the normal phenotype of the newborn confirmed our prediction. Based on the present study, this 13q31.1 microdeletion may be considered as a chromosomal polymorphism. This study also reconfirmed the feasibility of obtaining a molecular karyotype of a fetus via NIPT.

Effect of two kinds of different combined oral contraceptives use on bone mineral density in adolescent women.

BACKGROUND: Steroid hormonal contraceptives are highly effective and widely used. Most studies have shown a negative effect of combined oral contraceptives (COCs) on the bone mineral density (BMD) of adolescents. The study was conducted to compare BMD among users of ethinylestradiol/desogestrel, users of ethinylestradiol/cyproterone acetate and nonhormonal control subjects in women aged 16-18 years. STUDY DESIGN: The study included 450 women 16-18 years of age. One hundred fifty women were using ethinylestradiol/desogestrel, 150 women were using ethinylestradiol/cyproterone acetate, and 150 women were using nonhormonal contraception as control subjects. BMD ofthe lumbar spine and femoral neck was obtained using dual-energy X-ray absorptiometry, and mean BMD changes in COCs users and nonusers were compared. RESULTS: At 24 months of treatment, lumbar spine and femoral neck mean BMD values in women (n=127) who used ethinylestradiol/desogestrel were slightly lower compared with baseline, but these effects did not reach statistical significance (p=.837 and p=.630, respectively). The mean lumbar spine and femoral neck BMD values in women (n=134) who used ethinylestradiol/cyproterone acetate were slightly higher compared with baseline, but there was no statistical significance (p=.789 and p=.756, respectively). The increases in mean percent change in lumbar spine and femoral neck BMD in the ethinylestradiol/cyproterone acetate group were less than those in the control group (1.88% vs. 0.30% and 0.98% vs. 0.49%, respectively). There were no significant differences in mean BMD of the lumbar spine and femoral neck between the users of ethinylestradiol/desogestrel or ethinylestradiol/cyproterone acetate and nonusers (p>.05). CONCLUSION: Our study indicates that 2 years of COCs therapy had no significant effect on bone density in adolescents, but it remains unknown whether therapy longer than 2 years has a significant adverse effect on the attainment of peak bone mass.

Effects of the crude extract of Polygala tenuifolia Willd on human sperm in vitro.

The aim of the present study is to analyze sperm membrane changes and the spermicidal effect in treatment with the crude extract from Polygala tenuifolia Willd (PTW) in vitro. The root of PTW was extracted in distilled water. Normal human spermatozoa were used to assess the spermicidal activity (Sander-Cramer assay) of the extract from the PTW root. The hypo-osmotic swelling (HOS) test and the eosin Y (EY) staining were used to detect the integrity of sperm membrane and vitality. The sperm chromatin dispersion (SCD) test was performed to determine sperm DNA integrity. N-9 was used as a reference standard and semen added to physiological saline was used as the control. Semen samples were donated by 42 healthy fertile men. The crude extract from the root of PTW could immobilize and kill 100% spermatozoa within 20 s in vitro at the concentrations of 20.0 and 10.0 mg/ml; at the concentration of 5.0 mg/ml, spermatozoa were immobilized in (39.5±3.2) s. In the groups of the crude extract from the root of PTW and N-9 solution, the rate of the normal HOS (tails swollen) and the white head (unstained) was 0%, and the rate of the abnormal HOS (tails unswollen) and red head (stained) was 100%. Sperm DNA fragmentation showed no change in exposure to the crude extract from the root of PTW and N-9 solution. The sperm revival test did not show any spermatozoa that recovered their motilities. The rapid spermicidal activity of the crude extract from the root of PTW in vitro may occur by the disruption of the sperm membrane integrity.

[Mechanism for sperm immobilization activity of extract from Platycodon grandiflorum in vitro].

OBJECTIVE: To explore the mechanism of spermicidal effect of crude extract and platycodin-D from Platycodon grandiflorum (PG) root in vitro. METHODS: Between February 2006 and December 2009, 38 fertile and healthy adult males were selected as donors. PG root was extracted and platycodin-D purified. Grouping was as follows: crude extract from PG root, platycodin-D, nonoxynol-9 (N-9, as a reference standard) and semen-added physiological saline (as control). Spermicidal experiments were carried out in vitro (Sander-Cramer test). The hypo-osmotic swelling (HOS) test and modified Eosin-Giemsa (EG) staining were used to detect the integrity of sperm membrane. Four types of sperm morphology were divided through HOS-EG test: Type A: spermatozoa with swelling in tails and head white staining HOS(+)-EG(-) (membrane intact); Type B: spermatozoa with no swelling in tails (membrane-damaged) and head white staining HOS(-)-EG(-); Type C: spermatozoa with tail swelling and head red HOS(+)-EG(+); Type D: spermatozoa with no swelling in tails and head red HOS(-)-EG(+). Sperm chromatin dispersion (SCD) test was performed to determine the integrity of sperm DNA. RESULTS: The crude extract from PG root could immobilize and kill 100% spermatozoa within 20 s in vitro at the concentrations of 50.0 g/L and 20.0 g/L (v:v = 1:1 in semen). When the semen sample was exposed to the concentrations of 2.0 g/L and 1.0 g/L of platycodin-D, all spermatozoa were immobilized within 20 s. In the control group, the mean percentage of Types A, B, C and D was (69.0 ± 8.3)%, (3.4 ± 0.5)%, (10.2 ± 1.7)% and (17.4 ± 2.1)% respectively. In the groups of platycodin-D and N-9 solution, the rate of Types A and B was 0. The rate of Types C [(65.3 ± 3.8)%] and D [(34.7 ± 7.1)%] significantly increased versus control in the platycodin-D group (P < 0.01). Sperm DNA fragmentation had no change upon an exposure to the extract from PG root, platycodin-D and N-9 solution. And the sperm revival test showed none of the spermatozoa recovered their motility. CONCLUSION: The extract and platycodin-D from PG root have a quick sperm-killing effect in a short time in vitro by disrupting the integrity of sperm membrane (main head).

[Measurement of sperm DNA integrity by sperm chromatin dispersion test and TdT-mediated dUTP nick end labeling assay].

OBJECTIVE: To compare the effects of sperm chromatin dispersion (SCD) test and TdT-mediated dUTP nick end labeling (TUNEL) assay in assessing the DNA fragmentation in human sperm. METHODS: Motile sperms were isolated from the semen samples obtained from 20 healthy fertile men and 32 clinically infertile patients by swim-up technique, and underwent SCD and TUNEL to analyze the DNA fragmentation. RESULTS: The rate of sperm with DNA damage of the infertile patients was 12.8% +/- 5.8% tested by SCD, significantly higher than that of the healthy fertile men (7.6% +/- 3.3%, t = 3.576, P = 0.001), and the rate of sperm with DNA damage of the infertile patients was 11.1% +/- 5.1% tested by TUNEL assay, significantly higher than that of the healthy fertile men (6.8% +/- 2.8%, t = 3.467, P = 0.001). The proportion of sperm cell with abnormal DNA integrity measured by SCD test was correlated strongly with that determined by TUNEL for the infertile men (r = 0.841, P = 0.000) and for the fertile men too (r = 0.823, P = 0.000). The rate of sperm with DNA damage measured by SCD were not significantly different from those of TUNEL-positive sperm in fertile men (t = 1.996, P = 0.060). The rate of sperm with DNA damage measured by SCD was significantly higher than that measured by TUNEL among infertile patients (t = 3.023, P = 0.005). CONCLUSION: The presence of sperm DNA damage may lead to male infertility. SCD is simpler, cheaper and more reliable than TUNEL in testing the sperm DNA damage.

[Azoospermia factor microdeletions in idiopathic azoospermia and severe oligozoospermia].

OBJECTIVE: To observe the relationship between microdeletions of AZF( azoospermia factor) on Y chromosome in male with idiopathic azoospermia and severe oligozoospermia. METHODS: Only patients with an apparently normal 46,XY karyotype and normal FSH, LH and T were included in this study. Multiplex PCR was used to detect the sequence-tagged sites( STS) as follows :sY84, sY86, sY127, sY134, sY152, sY153, sY254, sY255, and ZFX/Y was used as internal control gene. RESULTS: No microdeletion was detected in the control whereas 8 microdeletion cases existed in 67 idiopathic azoospermia and severe oligozoospermia, including 4 in AZFc, 2 in AZFa + AZFc, 1 in AZFc + AZFb, and 1 in AZFb. The prevalence rate of microdeletion was 11.94%, which was statistically different from the control. CONCLUSION: Microdeletions in the AZF regions on the long arm of the Y-chromosome are associated with idiopathic azoospermic and severely oligozoospermic men. Multiplex PCR was a rapid and reliable method for screening microdeletions of AZF.

Successful pregnancy and birth after intrauterine insemination using caput epididymal sperm by percutaneous aspiration.

AIM: To manage male infertility with obstructive azoospermia by means of percutaneous epididymal sperm aspiration (PESA) and intrauterine insemination (IUI). METHODS: Ninety azoospermic patients with congenital bilateral absence of the vas deferens (BAVD, n=58) or bilateral caudal epididymal obstruction (BCEO, n=32) requesting for fine needle aspiration (FNA), PESA and IUI were recruited. The obstruction was diagnosed by vasography and determination of the fructose, carnitine and alpha-glucosidase levels in the seminal fluid. RESULTS: The mean sperm motility, density, abnormal sperm and total sperm count of the caput epdidymis were 16 %+/-22 %, (12+/-31) x 10(6)/mL, 55 %+/-36 % and (16+/-14) x 10(6), respectively. In the 90 couples, a total of 74 PESA procedures and 66 cycles of IUI were performed. Three pregnancies resulted, including one twin pregnancy giving birth to two healthy boys, one single pregnancy with a healthy girl and another single pregnancy aborted at week 6 of conception. The pregnancy rate per IUI cycle was 4.5 %. CONCLUSION: The birth of normal, healthy infants by IUI using PESA indicates that the caput epididymal sperm possess fertilization capacity. The PESA-IUI programme is a practical and economical procedure for the management of patients with obstructive azoospermia.