Xinjie Zhuang

Effect of embryo culture media on percentage of males at birth

STUDY QUESTION Does embryo culture medium influence the percentage of males at birth? SUMMARY ANSWER The percentage of males delivered after ICSI cycles using G5™ medium was statistically significantly higher than after cycles where Global, G5™ PLUS, and Quinns Advantage Media were used. WHAT IS KNOWN ALREADY Male and female embryos have different physiologies during preimplantation development. Manipulating the energy substrate and adding growth factors have a differential impact on the development of male and female embryos. STUDY DESIGN, SIZE AND DURATION This was a retrospective analysis of the percentage of males at birth, and included 4411 singletons born from fresh embryo transfer cycles between January 2011 and August 2013 at the Center for Reproductive Medicine of Third Hospital Peking University. PARTICIPANTS/MATERIALS, SETTING, AND METHODS Only singleton gestations were included. Participants were excluded if preimplantation genetic diagnosis, donor oocytes and donor sperm were used. The database between January 2011 and August 2013 was searched with unique medical record number, all patients were present in the database with only one cycle. Demographics, cycle characteristics and the percentage of male babies in the four culture media groups were compared with analysis of variance or χ(2) tests. Multivariable logistic regression was done to determine the association between the sex at birth and culture media after adjusting for other confounding factors, including parental age, parental BMI, type of infertility, parity, number of embryos transferred, number of early gestational sacs, cycles with testicular sperm aspiration (TESA)/percutaneous epididymal sperm aspiration (PESA)/testicular sperm extraction (TESE), number of oocytes retrieved, cycles with blastocyst transfers, and gestational age within ICSI group. MAIN RESULTS AND THE ROLE OF CHANCE Within the IVF group, the percentage of males at birth for G5™, Global, Quinns and G5™ PLUS media were comparable (P > 0.05); however, within the ICSI group, the percentage of male babies in cycles using G5™(56.1%) was statistically significantly higher than in cycles that used Global (47.2%; P = 0.003), G5™ PLUS (47.7%; P = 0.005) or Quinns media (45.0%; P = 0.009). There were no statistically significant differences in the percentage of males at birth between cycles that used Global, G5™ PLUS and Quinns media (P > 0.05). Multivariable logistic regression indicated that culture media (G5™ versus Global, G5™ PLUS, and Quinns) were significantly associated with the sex at birth (P = 0.008) after adjusting for parental age, parental BMI, type of infertility, parity, number of embryos transferred, number of early gestational sacs, cycles with TESA/PESA/TESE, number of oocytes retrieved, cycles with blastocyst transfers, and gestational age. LIMITATIONS AND REASONS FOR CAUTION This study was not a randomized controlled trial and allocation of treatment cycles over the four media was not completely at random. Cigarette smoking was not included in the current study because this confounding factor was not registered in our database. Moreover, intra-variability of sperm selection between the five embryologists may directly affect the percentage of males. WIDER IMPLICATIONS OF THESE FINDINGS Our study suggests that human embryogenesis responds differently to G5™, Global, G5™ PLUS and Quinns Advantage Medium. This finding can be generalized to other commercial culture media. STUDY FUNDING/COMPETING INTERESTS National Natural Science Foundation of China for Young Scholars (81300483 and 81200466). The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER Not applicable.

Up-regulation of heme oxygenase-1 expression modulates reactive oxygen species level during the cryopreservation of human seminiferous tubules

OBJECTIVE To study the effect of freezing techniques and to optimize a method for trace amounts of testicular spermatozoa from biopsed seminiferous tubules. The level of reactive oxygen species (ROS) and the gene expression of heme oxygenase-1 was evaluated. DESIGN Prospective experimental study. SETTING University-based laboratory. PATIENT(S) Eighteen adults with male fator infertility underwent testicular biopsy surgery. INTERVENTION(S) Seminiferous tubular fragments from each man were evenly allocated to three groups: fresh control, slow freezing, and vitrifiaction groups. The morphology and ROS levels before and after freezing were evaluated for seminiferous tubular fragments. The expression of heme oxygenase-1 (HO-1) at both the transcriptional and protein levels was determined. MAIN OUTCOME MEASURE(S) The morphology was analyzed by light microscopy. The ROS levels were measured with ELISA. The proliferation and differentiation were evaluated by immunohistochemistry, and the expression of HO-1 was evaluated using a real-time polymerase chain reaction (PCR) and Western blotting. RESULT(S) Decreased ROS levels and increased HO-1 expression at the transcriptional and protein levels were observed after thawing the human seminiferous tubules. The ROS level was negatively correlated with HO-1 expression. Slow freezing was more effective than vitrification in terms of HO-1 up-regulation and ROS alteration. CONCLUSION(S) Based on our study, the slow freezing technique was more effective compared with the vitrification method.

Identification and Characterization of Xlr5c as a Novel Nuclear Localization Protein in Mouse Germ Cells

Background Spermatogenesis is the complex process by which diploid stem cells generate haploid germ cells in gamete production. Members of the Xlr (X-chromosome linked, lymphocyte regulated) superfamily play essential roles in spermatogenesis. The expression, localization and role in spermatogenesis of one such member, Xlr5c, has not been reported previously. Methodology/Principal Findings Xlr5c mRNA and protein levels in murine testes and other tissues were investigated using RT-PCR and Western blotting. Xlr5c was abundantly transcribed in mouse testes, particularly during the early stages of spermatogenesis and throughout prophase I in the nuclei of spermatocytes. Xlr5c was specifically localized at synaptonemal complexes(SCs) region in preleptotene and pachytene spermatocytes, as was the homologous Xlr protein Sycp3. Conclusions/Significance These results suggest that Xlr5c was abundantly transcribed in germ cells, localized at SCs region, where it may play a potential role during the early stages of spermatogenesis. Identification and characterization of this novel testis protein may offer a new perspective for understanding of the molecular mechanisms involved in germ cell differentiation.

Effect of male body mass index on live-birth sex ratio of singletons after assisted reproduction technology

OBJECTIVE To determine the effect of male body mass index (BMI) on the probability of achieving a live birth and the sex ratio of singletons at birth after IVF and intracytoplasmic sperm injection (ICSI) treatment. DESIGN A retrospective cohort study. SETTING University-affiliated infertility center. PATIENT(S) Patients seeking infertility treatment who received IVF or ICSI treatment with autologous oocytes from January 2009 to December 2013. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Live-birth sex ratio of singletons at birth stratified by male BMI and adjusted by parental age, parental BMI, type of infertility, parity, embryo culture media, and cause of infertility. RESULT(S) A total of 8,490 couples undergoing IVF or ICSI treatment resulted in 39.12% live births and gave birth to 2,377 live birth singletons and 943 twins. There was no significant difference in the live birth rate between groups stratified by BMI. The probability of live births for overweight and obese groups were not decreased compared with the normal-weight group; similar null findings existed in the IVF and ICSI subgroups. Of note, the sex ratio of offspring in the overweight and obese male groups was significantly higher than in the normal-weight group (1.27 vs. 1.07). Male BMI was significantly associated with sex ratio of singletons after adjusting for confounders. In twins, incidences of twins with male-male infants in the overweight/obese group were not different from the normal-weight group. CONCLUSION(S) Increased male BMI has no effect on live birth success, but has an increased probability of giving birth to male singletons.

Trim27 interacts with Slx2, is associated with meiotic processes during spermatogenesis.

ABSTARCT Formation of the XY body is believed to prevent recombination between X and Y chromosomes during meiosis. We recently demonstrated that SYCP3-like X-linked 2 (Slx2) could be involved in synaptonemal complex formation as well as XY body maintenance during meiosis. In order to further investigate the role and composition of XY body protein complexes in meiotic processes and spermatogenesis, a yeast 2-hybrid screening was performed, and the tripartite motif protein 27(Trim27) was found to interact with Slx2 and co-localized in the XY body. Trim27 has a tripartite motif (TRIM) consisting of a RING finger, B-box and coiled-coil domains, and is a transcriptional regulator that is expressed in various tumor cell lines. In this study, we showed that Slx2 and Trim27 were highly expressed in meiosis of mouse testis. And the Slx2/Trim27 interaction was confirmed in vivo by co-immunoprecipitation and mammalian 2-hybrid interaction assays. Moreover, cytoimmuno localization experiments revealed that Slx2/Trim27 was co-localized to the XY body of spermatocytes during meiosis, and immunohistochemical results revealed co-localization of Trim27 and γ-H2AX in the XY body of primary spermatocytes in the mouse testis. Trim27 may therefore be a transcriptional regulation protein connecting Slx2 and γ-H2AX, thereby promoting the formation of a more potent XY body protein complex in meiotic processes and spermatogenesis. In conclusion, Trim27 connecting Slx2 may regulate meiotic processes in multiple ways by influencing XY body formation and germ cell proliferation during spermatogenesis.

A clinical trial on the consistency of bilateral testicular tissue histopathology and Johnsen score: single side or bilateral side biopsy?

To evaluate and compare left and right testicular tissue histopathology and Johnsen score, and to investigate the necessity for bilateral testicular biopsy. We recruited180 patients with non-obstructiveazoospermia (NOA) on testicular biopsy who had undergonetesticular sperm aspiration (TESA). Pathological sections of testicular tissue were diagnosed by specially-assigned doctors, who evaluated pathological findings, determined the Johnsen score and confirmed for the presence or absence of sperm. Sperm positive rates for left and right testicular histopathology were 55.0% and 51.7% respectively, and the proportion of Johnsen scores≥8 for left and right testes were 53.3% and 50.0%, respectively. Cohen kappa values revealed that the identification of sperm in bilateral testicular samples was not consistent and was related to random effects; Optimized cut-off value for bilateral testicular volume was 11ml (Johnsen score ≥8), and optimized cut-off values of E2 on left and right testes were 144.5pmol/L and 133.5 pmol/L (Johnsen score≤7). However, age, serum prolactin (PRL), follicle stimulating hormone (FSH), luteinizing hormone (LH) and total testosterone (TT) levels were not accurate predictors for the existence of testicular sperm. There was nostatistical significance between left and right testicular histopathology in terms of sperm positive rates or Johnsen score; the Johnsen score were caused entirely by random effects and a score from one side could not represent the other side. Therefore, we recommend that both testes need to undergo surgery when NOA patients undergo testicular biopsy or sperm retrieval.

Effect of repeated cryopreservation on human embryo developmental potential

Repeated cryopreservation of surplus embryos from frozen-thawed cycles should occasionally be considered. The purpose of this retrospective cohort study was to evaluate the pregnancy and perinatal outcome of repeated cryopreservation by vitrification of human blastocysts derived from slowly frozen-thawed day 3 embryos. In total, 571 vitrified-warmed blastocyst transfer cycles were investigated. The vitrified-warmed blastocysts were derived from slowly frozen-thawed cleavage embryos (twice-cryopreserved group) or fresh embryos (control group) cultured to the blastocyst stage. Age, body mass index, endometrial thickness, blastocyst developmental rate and number of embryos transferred were not significantly different between twice-cryopreserved and control groups. Clinical pregnancy and implantation rates were also similar. Compared with controls, the miscarriage rate was significantly higher in the twice-cryopreserved group (33.93% versus 19.07%, P = 0.017). This resulted in a significantly lower live birth rate in the twice-cryopreserved group than in controls (29.13% versus 39.18, P = 0.038). No differences were observed in mean gestational age, birthweight and sex ratio of newborns between groups. In conclusion, acceptable clinical pregnancy outcomes may be expected from transfer of twice-cryopreserved human embryos. While the neonatal outcome is not affected, the correlation between the risk of higher pregnancy loss and repeated cryopreservation needs further investigation.