BACKGROUND

DDX3Y (DBY), located within AZoospermia Factor a (AZFa) region of the human Y chromosome (Yq11), encodes a conserved DEAD-box RNA helicase expressed only in germ cells and with a putative function at G1–S phase of the cell cycle. Deletion of AZFa results most often in germ cell aplasia, i.e. Sertoli-cell-only syndrome. To investigate the function of DDX3Y during human spermatogenesis, we examined its expression during development and maturation of the testis and in several types of testicular germ cell tumours (TGCTs), including the pre-invasive carcinoma in situ (CIS) precursor cells which are believed to originate from fetal gonocytes.

METHODS

DDX3Y protein expression was analysed during development in different tissues by western blotting. The localization of DDX3Y in normal fetal and prepubertal testis tissue of different ages as well as in a series of distinct TGCT tissue samples (CIS, classical seminoma, spermatocytic seminoma, teratoma and embryonal carcinoma) was performed by immunohistochemistry.

RESULTS

Germ cell-specific expression of DDX3Y protein was revealed in fetal prospermatogonia but not in gonocytes and not before the 17th gestational week. After birth, DDX3Y was expressed at first only in the nuclei of Ap spermatogonia, then also in the cytoplasm similarly to that seen after puberty. In CIS cells, DDX3Y was highly expressed and located predominantly in the nuclei. In invasive TGCT, significant DDX3Y expression was found in seminomas of the classical and spermatocytic type, but not in somatically differentiated non-seminomas, consistent with its germ-cell specific function.

CONCLUSIONS

The fetal germ cell DDX3Y expression suggests a role in early spermatogonial proliferation and implies that, in men with AZFa deletion, germ cell depletion may begin prenatally. The strong expression of DDX3Y in CIS cells, but not in gonocytes, indicates phenotypic plasticity of CIS cells and suggests partial maturation to spermatogonia, likely due to their postpubertal microenvironment.

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BACKGROUND

Spermatogenesis is maintained by a dynamic balance between germ cell proliferation and apoptosis. Previous study has demonstrated that CD147 knockout mice are infertile with arrested germ cells. However, the question of whether and how CD147 may be involved in the apoptotic process during spermatogenesis remains elusive. The aim of this study was to evaluate the role of CD147 in the regulation of germ cell apoptosis in mice.

METHODS

CD147 function was blocked by anti-CD147 antibody in GC-1 (immortalized spermatogonia) and GC-2 (immortalized spermatocytes) cell lines and in testicular germ cells in vivo. Testes size and weight were examined after injection of anti-CD147 antibody into the seminiferous tubules of severe combined immunodeficiency mice. Germ cell apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and levels of p53 and two effectors, caspase 3 and poly ADP-ribose polymerase (PARP), using western blots.

RESULTS

The size and weight of the CD147-immunodepleted testes were decreased compared with that in control testes (P < 0.001). The TUNEL assay showed an increase in the number of apoptotic spermatocytes (P < 0.001 versus control) but not spermatogonia in Stages XI–XII of CD147-immunodepleted testes. In addition, in vitro experiments demonstrated that CD147 immunodepletion induced an increase in apoptosis in GC-2 cells (P < 0.001 versus control) but had no effect on GC-1 cells. Moreover, deprivation of CD147 induced apoptosis in spermatocytes through a p53-independent mechanism, which led to caspase 3 and PARP activation.

CONCLUSIONS

We have demonstrated that immunodepletion of CD147 induces p53-independent apoptosis in mouse spermatocytes but not spermatogonia.

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BACKGROUND

Animal models of endocrine dysfunction have associated male genital defects with reduced anogenital distance (AGD). Human studies have correlated shorter AGD with exposure to putative endocrine disruptors in the environment but have not examined AGD in hypospadiac boys. We measured AGD in boys with hypospadias and those with normal genitals.

METHODS

Data were collected prospectively on boys undergoing urologic procedures at the University of California San Francisco and the Children's Hospital of Oakland, CA, USA. Data included age, race, height, weight, BMI, urologic diagnoses and AGD. To minimize any potential effects of race on observed AGD, we examined only Caucasian boys. Differences between boys with hypospadias and those with normal genitals were examined through two-tailed Student's t-tests.

RESULTS

One hundred and nineteen Caucasian boys ranging in age from 4 to 86 months underwent AGD measurement, of which 42 and 77 were boys with normal genitals and hypospadias, respectively. The mean (±SD) AGD of boys with hypospadias was 67 ± 1.2 versus 73 ± 1 mm for boys with normal genitals (P = 0.002). In these age-unmatched patient groups, there were also differences in age, height and weight (P = 0.0001, 0.0002 and 0.0004, respectively). After age matching (all <2 years of age), boys with hypospadias (n= 26) still featured a shorter AGD than boys with normal genitals (n= 26; 62 ± 2 versus 68 ± 2 mm respectively, P = 0.033) but the differences in age, height and weight were no longer significant.

CONCLUSIONS

In humans, hypospadias may indeed be associated with reduced AGD. Additional studies are needed to corroborate these preliminary findings and to determine their etiology.

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BACKGROUND

Human embryonic stem cells (hESCs) are most commonly derived from the inner cell mass (ICM) of blastocyst stage embryos. While the majority of hESC lines originate from good-quality embryos donated after cryogenic storage, poor-quality embryos (PQEs) not suitable for clinical use have also been shown to generate hESC. This provides a newfound function for embryos that would otherwise be discarded following IVF or ICSI. Owing to their lack of clinical importance, however, data on the poorest embryos in a cohort go largely unreported in the literature. It is therefore of interest to better understand the availability of PQEs from IVF/ICSI cycles and to determine their ability to develop into blastocysts with good-quality ICMs for use in hESC derivation. In this study, we investigate the influence of patient parameters and embryo cohort on PQE incidence, blastocyst development, ICM quality and successful hESC derivation from donated PQEs.

METHODS

PQEs from 736 patient cycles that did not meet our clinical criteria for transfer or cryopreservation were cultured until Day 6 of development and assessed for blastocyst formation and ICM quality. A subset of blastocysts with good-quality ICMs were then used for hESC derivation attempts. Anonymous patient data such as maternal age, embryo history and cohort parameters were then retrospectively compiled and analysed.

RESULTS

PQEs made up 46.8% of two pronucleate embryos created from IVF/ICSI. Including embryos with abnormal fertilization, a mean of 3.6 ± 2.8 embryos were donated per cycle with 32.6% developing to the blastocyst stage. Good-quality ICM were produced in 13.9% of PQEs cultured. Of good-quality ICM, 15.4%  of those used in hESC derivation attempts resulted in a novel line. The PQEs that originated from older patients (>37 year) or from cycles that did not result in pregnancy had significantly diminished blastocyst development and ICM quality. Maternal age was also shown to further influence the ability of good-quality ICMs to generate hESC.

CONCLUSIONS

PQEs are an abundant source of embryos capable of developing to blastocysts with good-quality ICMs and subsequently generating novel hESC. We have shown that prognostic variables used to predict IVF/ICSI outcome can also help predict which PQEs have the best hESC developmental potential. Owing to the diversity of PQE origin, experiments designed to compare hESC derivation techniques or efficiency using PQEs should consider clinical IVF/ICSI parameters to establish groups with equal developmental competence. Additional investigation is needed to determine if these results are applicable to hESC derivation using good-quality embryos.

Source:
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BACKGROUND

Growth factors and cytokines are present in small quantities in the oviduct and uterus and some are synthesized by the growing embryo. Granulocytes–macrophage colony-stimulating factor (GM-CSF) is known as an important regulator, which enhances cell proliferation and reduces apoptosis in developing blastocysts, during normal fetal and placental development. The purpose of this study is to investigate whether adding GM-CSF to the culture media affects blastulation or the chromosomal status of mouse embryos.

METHODS

Murine embryos were cultured in vitro from the 2-cell stage until the blastocyst stage in the presence of different concentrations of GM-CSF of 0 ng/ml (control), 1, 2, 5 and 10 ng/ml. The development of each embryo was noted and the embryos were then spread for fluorescence in situ hybridization (FISH) using locus-specific probes (LSI) for chromosomes 2, 11 and 16 in all embryos.

RESULTS

No difference in the blastulation potential was noted with the addition of 1 and 2 ng/ml of GM-CSF compared with the controls, but there was a significant decrease (P < 0.001) in the blastulation rate in the 5 and 10 ng/ml concentrations. The rate of mosaicism/aneuploidy noted in all GM-CSF groups (1, 2, 5 and 10 ng/ml) was slightly higher than in the control group (0 ng/ml GM-CSF) but the differences were not significant. In the mosaic embryos from the GM-CSF cultured groups, the percentage of aneuploid cells was statistically higher than in the control group.

CONCLUSIONS

GM-CSF exerted a negative impact on blastocyst development at higher concentrations. GM-CSF did not affect the rates of mosaicism/aneuploidy, but did increase the percentage of aneuploid cells within the mosaic embryos. Adding GM-CSF to the culture media for clinical use requires further studies either on human or animal models to evaluate its long-term effects.

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BACKGROUND

The use of ovarian stimulation, to stimulate a multi-follicular response for assisted reproduction treatments, may force the production of oocytes from follicles that do not reach optimal maturation, possibly yielding oocytes that are not fully competent. The present study aimed to define the follicular environment and oocyte competence of unstimulated pre-ovulatory follicles, to compare it with that of similar-sized stimulated follicles. For this purpose, we analyzed the follicular hormonal milieu, the oocyte meiotic spindle, the embryo development and the cumulus cells gene expression (GE) profiles.

METHODS AND RESULTS

The study population was divided in two groups: (i) 42 oocyte donors undergoing unstimulated cycles and (ii) 18 oocyte donors undergoing controlled ovarian stimulation cycles (COS). Follicular fluid was analyzed to quantify the concentrations of estradiol (E2), progesterone (P), FSH, LH, testosterone (T) and androstendione (4). T was higher in the COS group, while 4, E2 and LH were significantly higher in unstimulated cycles. The cumulus oophorus cells (CC) surrounding the oocyte were removed and their GE profiles were analyzed with microarrays. There were 18 differentially expressed genes in CC: 7 were up-regulated and 11 were down-regulated in the COS cycles. The microarray was validated by qRT–PCR. The analysis of spindle structure revealed no significant differences between the groups, except for the parameter of length which presented differences. The fertilization ability and embryo morphology on Days 2, 3 and 4 did not show any significant differences between groups.

CONCLUSIONS

The use of ovarian stimulation induces changes in the follicular fluid and in CC GE that may affect immune processes, meiosis and ovulation pathways. Although these differences do not seem to relate to early-stage embryo morphology, the implications of some of the molecules, especially ALDH1A2, CTSL and ZNF33B at the CC level, deserve to be addressed in future studies.

Source:
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BACKGROUND

An efficient method for cryopreservation of human oocytes may offer solutions to legal and ethical problems in routine infertility programs and may also be used for fertility preservation for medical and social reasons.

METHODS

We conducted an observational longitudinal cohort multicentric study to investigate the efficacy and reproducibility of oocyte cryopreservation outcomes in IVF/ICSI cycles. Moreover, the effects of patient and cycle characteristics on the delivery rate (DR) were analyzed.

RESULTS

In 486 cycles performed in 450 couples, 2721 oocytes were warmed and 2304 of them survived cryopreservation (84.7%). Of the 2182 oocytes subjected to ICSI, the rates of fertilization and development to top-quality embryos were 75.2 and 48.1%, respectively. A total of 128 deliveries were obtained (26.3% per cycle and 29.4% per transfer) for 450 patients (28.4%) and 147 babies were live born from 929 embryos transferred (15.8%). The forward logistic regression analysis on a per patient basis showed that female age [odds ratio (OR): 0.93, 95% confidence interval (CI): 0.88–0.98], number of vitrified oocytes (OR: 1.08, 95% CI: 1.01–1.17) and the day of transfer (OR: 1.97, 95% CI: 1.14–3.42) influenced DR. By recursive partitioning analysis, it can be estimated that more than eight oocytes vitrified are required to improve the outcome (22.6 versus 46.4% DR, respectively). When fewer oocytes are available in women aged >38 years, results are dramatically reduced (12.6 versus 27.5% DR, respectively). Conversely, when >8 oocytes are available, blastocyst culture represents the most efficient policy (62.1% DR; data from one center only).

CONCLUSIONS

Oocyte vitrification is an efficient and reliable approach, with consistent results between centers and predictable DRs. It should be applied routinely for various indications. A predictive model is proposed to help patient counselling and selection.

Source:
http://humrep.oxfordjournals.org/rss/current.xml

BACKGROUND

Assessment of male fertility is traditionally based on microscopic evaluation of semen. However, the classical semen parameters do not adequately reflect sperm function, and their clinical value in predicting fertility is limited. We hypothesize that the sperm expression profile could reflect the fertilizing quality of spermatozoa and could be more informative for predicting the in vivo reproductive fitness of men with normal semen parameters.

METHODS

Sperm gene expression patterns of 68 normozoospermic donors (43 Phase I and 25 Phase II), used for therapeutic IUI, were analysed via TaqMan Arrays.

RESULTS

Significant differences in the expression of individual genes were observed between groups of donors with the lowest and highest pregnancy rates (PRs) after IUI. Additionally, we have developed a molecular means to classify the fertility status of semen donors for IUI based on the expression signature of four genes. In the Phase I study, this model had 90% sensitivity and 97% specificity for discriminating donors resulting in low PRs (cut-off value: <13.6%), far better than that obtained from the combination of sperm parameters. The translation of the model was validated in Phase II donors resulting in a sensitivity of 71.5% and a specificity of 78%.

CONCLUSIONS

Our findings contribute to the search for the most valuable genetic markers which are potentially useful as tools for predicting pregnancy. Our expression model could complement classical semen analysis in order to identify sperm donors with a less favourable IUI reproductive outcome despite having normal semen parameters. It may also be useful for the study of sperm function in couples with unexplained infertility.

Source:
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BACKGROUND

Animal experiments have suggested that a high intraperitoneal pressure (IPP) might adversely affect the surgical peritoneal environment. The present experimental study investigates the impact of IPP of a CO₂ pneumoperitoneum on human peritoneum.

METHODS

Patients undergoing laparoscopic surgery were subjected to either low (8 mmHg) or standard (12 mmHg) IPP. Normal peritoneum was collected from the parietal wall at the beginning of surgery and every 60 min thereafter. Expression levels of 168 genes that encode extracellular matrix proteins, adhesion molecules or inflammatory cytokine signaling molecules were measured in peritoneal tissues using real-time polymerase chain reaction (PCR)-based assay panels. Human peritoneal mesothelial cells (HPMCs) and human peritoneal fibroblasts (HPFBs) were incubated in a CO₂ insufflation chamber for 1 h at 12 or 8 mmHg. Hyaluronan (HA) synthesis and mRNA expression levels of hyaluronic acid synthases (HAS) and hyaluronidases (Hyal) in HPMCs and HPFBs were measured at 0, 4, 8, 12, 24 and 48 h after CO₂ gas exposure by ELISA and real-time PCR, respectively.

RESULTS

Expression levels of connective tissue growth factor (CTGF), matrix metalloproteinase-9, E-selectin, chemokine (C-X-C motif) ligand 2 (CXCL-2), Hyal-1 and Hyal-2 were significantly higher and those of HAS-1, HAS-3, thrombospondin-2 (TSP-2) and interleukin-10 were significantly lower in the 12 mmHg group compared with the 8 mmHg group. HA synthesis was significantly lower in the 12 mmHg group compared with the 8 mmHg group in HPMCs and HPFBs throughout the time course.

CONCLUSIONS

A low IPP (8 mmHg) may be better than the standard IPP (12 mmHg) to minimize the adverse impact on the surgical peritoneal environment during a CO₂ pneumoperitoneum.

Source:
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BACKGROUND

The aim of this study was to investigate the appropriate measures for diagnosing and treating perineal endometriosis (PEM) with anal sphincter involvement.

METHODS

Between January 1992 and April 2011, the clinical features, diagnosis and management of 31 patients who were diagnosed with PEM with anal sphincter involvement at the Peking Union Medical College Hospital were retrospectively analyzed using their clinical records. A range of 6–78 months of outpatient follow-up after surgery were conducted for these 31 patients but was extended by telephone interviews with 29 patients conducted in December 2011.

RESULTS

All 31 patients had a history of vaginal delivery. The level of serum CA₁₂₅ was elevated in only 2 (6.5%) cases. All cases received surgical treatment, which included narrow excision (NE, close to the edge of the endometrioma) with primary sphincteroplasty (PSp) for 30 cases and incomplete excision (IE) for 1 case. Of the 30 cases in the NE group, 20 (66.7%) received hormone therapy preoperatively. Up until December 2011, there was one recurrence (3.6%) of PEM in the NE group. PEM relapse occurred in the IE patient 6 years after the initial IE surgery. Perineal abscesses were found in one patient post-operatively. No complaint of dyspareunia and no fecal incontinence episodes were observed during follow-up.

CONCLUSIONS

Based on our own experience, NE and PSp may be indicated for the treatment of PEM with anal sphincter involvement.

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