L. J. Li

Assisted reproductive technologies impair the expression and methylation of insulin-induced gene 1 and sterol regulatory element-binding factor 1 in the fetus and placenta.

OBJECTIVE To evaluate the cholesterol metabolism linked to assisted reproductive technology (ART) by analyzing the expression levels and DNA methylation patterns of the insulin-induced gene (INSIG), sterol regulatory element-binding protein (SREBP), and SREBP cleavage-activating protein in the fetus and placenta. DESIGN Experimental research study. SETTING An IVF center, university-affiliated teaching hospital. PATIENT(S) Four patients groups were recruited: pregnancies after IVF/intracytoplasmic sperm injection (ICSI) (n = 55), natural pregnancies (n = 40), multifetal reduction after IVF/ICSI (n = 56), and multifetal reduction after controlled ovarian hyperstimulation (COH) (n = 42). INTERVENTION(S) Expression and DNA methylation of INSIG-SREBP- SREBP cleavage-activating protein in the fetus and placenta samples were determined. MAIN OUTCOME MEASURE(S) The expression and DNA methylation patterns were tested by real-time quantitative polymerase chain reaction (PCR) and pyrosequencing. RESULT(S) In the ICSI treatment group, significantly higher levels of triglycerides and apolipoprotein-B were observed in cord blood compared with controls. Meanwhile, in ICSI-conceived fetuses, the expression of INSIG1 was significantly higher, and methylation rates were lower, than in the IVF and control groups. Furthermore, in the placenta, the INSIG1 and SREBF1 transcripts were also significantly higher with lower methylation rates in the ICSI group than in the IVF and control groups. CONCLUSION(S) Our results indicated that the dysregulation of INSIG1 and SREBF1 caused by ART were observed not only in the fetus but also in the placenta, primarily in the ICSI group. However, the long-term sequelae of this dysregulation should be closely followed.

Influences of sub-Alfvenic shear flows on nonlinear evolution of magnetic reconnection in compressible plasmas

Influences of sub-Alfvenic shear flows on the nonlinear evolution of the magnetic reconnection are studied in the framework of compressible resistive MHD and compressible Hall MHD. It is found for the first time that the sub-Alfvenic shear flow can either stabilize or destabilize magnetic reconnection, which is mainly determined by the plasma beta and the half thickness of the shear flow ( λ v ). The shear flow exhibits a suppressing effect on magnetic reconnection and the boosting effect nearly disappears for the beta plasma β ≲ 1 , which is associated with the presence of a pair of discontinuities in the upper and lower inflow region. The shear flow has the boosting effect on magnetic reconnection when the half thickness of the shear flow exceeds a critical value in the high beta plasma which is in a good agreement with the results from incompressible simulation. With the inclusion of the Hall effect,shear flow can still either stabilize or destabilize magnetic reconnection, but the boosting effect becomes weaker as the ion inertial length d i increases.

Genome-wide DNA methylation patterns in IVF-conceived mice and their progeny: A putative model for ART-conceived humans ☆

The aim of this study was to use a mouse model to gain an understanding of the safety of reproduction between humans conceived through assisted reproductive technology (ART). Mice derived from in vitro fertilization and embryo transfer (IVF-ET) were crossed. Their behavior, morphology, histology and genome-wide DNA methylation status in the brain were examined by the Morris water maze, H&E staining and methylated DNA immunoprecipitation coupled with DNA methylation microarrays. Although no significant differences in behavior or morphology were observed, we did find small clusters of CpG islands and promoters that were aberrantly methylated. Hypermethylation was more common than hypomethylation in each of the two generations. Some of the aberrant methylated promoters were validated by bisulfite sequencing. Our results show that IVF may slightly modify the somatic methylation pattern and that some of this aberrant methylation might be inherited by the following generation.

Small-volume vitrification for human spermatozoa in the absence of cryoprotectants by using Cryotop

Cryotop is a carrier that has been used successfully in the cryopreservation of human spermatozoa. Here, we explored a novel method to vitrify human spermatozoa without cryoprotective agents (CPAs) using Cryotop. Spermatozoa from 21 Normozoospermic patients were collected and vitrified without CPAs or with sucrose in small volume using Cryotop. The sperm recovery rate, motility, viability, chromatin damage and DNA fragmentation were assessed. No significant difference was observed in the sperm recovery rate and motility rate between the spermatozoa cryopreserved without CPAs and with sucrose. The post‐thawed spermatozoa cryopreserved without CPAs had a higher viability and lower damage to sperm chromatin and DNA than those cryopreserved with sucrose. These results suggest that small numbers of human spermatozoa can be successfully vitrified without CPAs using Cryotop.

Human Sperm Devoid of Germinal Angiotensin-Converting Enzyme Is Responsible for Total Fertilization Failure and Lower Fertilization Rates by Conventional In Vitro Fertilization

ABSTRACT In conventional in vitro fertilization (IVF), complete failure of fertilization occurs in 5% to 15% of treatments. Although the causes may be unclear, sperm defects appear to be the major contributor. However, a convincing test is not yet available that can predict the risk of fertilization failure. In this study, we found that germinal angiotensin-converting enzyme (gACE) (also called testicular ACE) was undetectable in sperm from patients who had total fertilization failure (TFF) and lower fertilization rates (LFRs) by IVF based on Western blot and indirect immunofluorescence analyses. Additionally, almost all of the patients without gACE on sperm (23 of 25) manifested a TT genotype of the rs4316 single-nucleotide polymorphism of ACE. Overall, our results indicate that the absence of gACE expression is responsible for TFF and LFRs by IVF. The rs4316 polymorphism of ACE might be associated with infertility in those patients. We conclude that sperm lacking gACE may be recognized before commencing IVF and that the patients may be directed instead to consider intracytoplasmic sperm injection.

Depression of HspA2 in human testis is associated with spermatogenic impairment and fertilization rate in ICSI treatment for azoospermic individuals

PurposeHeat shock protein A2 (HspA2) expression was quantitatively measured in human testis and its relationship with the spermatogenetic status and laboratory outcomes of intracytoplasmic sperm injection (ICSI) was investigated.MethodsTesticular tissues of azoospermia men were divided into four groups according to histopahtology: normal spermatogenesiss, hypospermatogenesis, maturation arrest and Sertoli cell-only syndrome (SCOS). HspA2 immunostaining was measured by Image Pro-Plus (IPP) and laboratory outcomes were calculated. The regression analysis between HspA2 expression and Johnsen score of as well as fertilization, cleavage and high quality embryo rate was performed.ResultsHspA2 was strongly present in the cytoplasm of spermatocytes and spermatides in normal testis. However, hypospermatogenesis and maturation arrest testicular tissues demonstrated light staining and no staining for SCOS. Quantitative image analysis showed that there were significant differences among groups (P = 0.000 & P = 0.001). HspA2 exspression was founded significantly correlated spermatogenetic status (R2 = 0.726, P = 0.000) as well as fertilization rate in ICSI (R2 = 0.569, P = 0.000).ConclusionsThe fertilization rate with ICSI is associated with HspA2 expression in the testis from which sperm retrieved and the alteration of HspA2 expression has been involved in spermatogenic impairment.

Persistence and intergenerational transmission of differentially expressed genes in the testes of intracytoplasmic sperm injection conceived mice.

Intracytoplasmic sperm injection (ICSI) is commonly used to solve male infertility problems. Previous studies showed that early environmental exposure of an embryo may influence postnatal development. To detect whether ICSI operations affect the reproductive health of a male or his offspring, we established assisted reproductive technologies (ART) conceived mouse models, and analyzed gene expression profiles in the testes of both ICSI and naturally conceived (NC) newborn F1 mice using micro-array analysis. Among the differentially expressed genes, we focused on the expression of eight male reproduction-related genes. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) was used to analyze the expression of these genes in the testes of both adult and old F1 generation mice and adult F2 generation mice. Our results showed that down-regulated and somatic cell-expressed genes in newborn mice retained their differential expression patterns in adult and old F1 generation individuals, implying the persistence and fetal origin of the alteration in the expression of these genes. The intergenerational transmission of differential gene expression was observed, but most changes tended to be reduced in adult F2 generations. Controlled ovarian hyperstimulation (COH) and in vitro fertilization (IVF) mice models were added to explore the precise factors contributing to the differences in ICSI offspring. The data demonstrated that superovulation, in vitro culture, and mechanical stimulation involved in ICSI had a cumulative effect on the differential expression of these male reproductive genes.

Decreased expression of SAM68 in human testes with spermatogenic defects

OBJECTIVE To assess the expression patterns of SAM68 in the testes of azoospermic patients with normal and abnormal spermatogenesis. DESIGN Retrospective study and in vitro study. SETTING University hospital. PATIENT(S) Testicular biopsies of azoospermic men with normal spermatogenesis (OAZ; n=20), with maturation arrest at the spermatocyte stage (MA; n=20), and with Sertoli cell-only syndrome (SCOS; n=10). INTERVENTION(S) No interventions with patients. Knockdown of Sam68 was performed in the GC-2spd(ts) cell line. MAIN OUTCOME MEASURE(S) SAM68 expression was analyzed using quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, and immunohistochemistry analysis in tissues. Moreover, Sam68 was knocked down in GC-2spd(ts) cells. Cell viability was measured using the MTT assay, and the apoptosis rate was detected using flow cytometry with the Annexin V-FITC kit. RESULT(S) Using qRT-PCR, the expression level of testicular SAM68 mRNA in MA and SCOS patients was statistically reduced compared with in OAZ patients. In addition, using qRT-PCR, Western blot, and immunohistochemistry analyses, mRNA and protein expressions of SAM68 were absent or barely detectable in testicular tissues in 45% (9 of 20) of patients with MA and in all patients with SCOS. Furthermore, decreased expression of Sam68 suppressed germ cell proliferation and induced apoptosis in transfected GC-2spd(ts) cells. CONCLUSION(S) Deficient SAM68 expression was observed in the human testis with MA at the spermatocyte stage and SCOS. These results may offer new perspectives on the molecular basis of abnormal spermatogenesis.

Alterations in the frequency of trinucleotide repeat dynamic mutations in offspring conceived through assisted reproductive technology

STUDY QUESTION How does the frequency of trinucleotide repeat dynamic mutations in offspring conceived through assisted reproductive technology (ART) compare with the frequency of these mutations in control offspring conceived from spontaneous pregnancies? SUMMARY ANSWER There is a slight increase in dynamic mutation instability in offspring conceived through ART compared with the naturally conceived offspring. WHAT IS KNOWN ALREADY There is evidence to suggest that ART can increase the risk of birth defects and karyotypic abnormalities. However, the accumulating evidence of an association between ART and de novo genetic aberrations is controversial. STUDY DESIGN, SIZE, DURATION A prospective clinical observational study was performed on 246 families recruited from an in vitro fertilisation (IVF) centre at a tertiary-care, university-affiliated teaching hospital from 2008 to 2012. The study included 147 ART families [75 IVF and 72 intracytoplasmic sperm injection (ICSI)] in the study group and 99 natural-conception families in the control group. PARTICIPANTS, SETTING, METHODS Parental, umbilical cord and infant peripheral blood samples were collected, and the trinucleotide repeats of the ATN1, AR, ATXN1, ATXN3, Huntington, DMPK and FMR-1 genes were investigated between the generations; these genes were chosen due to their ability to undergo dynamic mutation. The frequencies and sizes of the mutational repeats, as well as the intergenerational instability, were measured. MAIN RESULTS AND THE ROLE OF CHANCE In 2466 transmissions identified in the ART offspring, 2.11% (n = 52/2466) of the alleles were unstable upon transmission, while in the control group offspring, the frequency of dynamic mutation was 0.77% (n = 10/1300); this difference was statistically significant (P < 0.01). The unstable transmission alleles were detected in 32 (2.48%) of the 1288 alleles from the IVF offspring and in 20 (1.70%) of the 1178 alleles from the ICSI offspring; both of these frequencies were significantly different from that of naturally conceived offspring (0.77%) (P < 0.01 and P < 0.05, respectively). However, there were no significant differences in the sizes of the mutational repeats or in the rates of expansion or contraction among the three groups (P > 0.05). The repeat copy numbers of the examined genes were found to be within the normal ranges in all parents and infants. LIMITATIONS, REASONS FOR CAUTION One strength of our study is the relatively large sample size; we were able to detect mutations in seven common dynamic genes, and this large sample size allowed us to detect unstable alleles. Although we observed a clear alteration in the frequency of dynamic mutation in the ART offspring compared with controls, further studies are urgently needed to confirm this observation and determine the cause of this phenomenon. WIDER IMPLICATIONS OF THE FINDINGS DNA microsatellite analysis provides an important tool to assess genomic instability. In this study, we report an association between ART and the frequency of dynamic mutation. The instability could be a reflection of the core infertility problem, the controlled ovarian hyperstimulation and/or the in vitro culture conditions.

Bursty magnetic reconnection under slow shock‐generated whistler waves

Nonlinear dynamics of magnetic reconnection with an external sub-Alfvenic parallel shear flow is investigated by using two-dimensional compressible Hall MHD simulation. Two pairs of slow shocks in the inflow region are generated by the sub-Alfvenic shear flow in the MHD simulation. With inclusion of Hall effects, it is found that whistler waves are generated in the downstream region of slow shocks. The whistler waves propagating toward the reconnection region drive a large bursty enhancement in magnetic reconnection during its decaying phase.