Wenyan Song

Clinical outcome of emergency egg vitrification for women when sperm extraction from the testicular tissues of the male partner is not successful

The development of an effective oocyte cryopreservation system will have a significant impact on the clinical practice of reproductive medicine. However, the important option of emergency oocyte cryopreservation has yet to be well documented. In this report, we review the cases of 15 women with male partners who were diagnosed with nonobstructive azoospermia and for whom testicular sperm extraction on the day of oocyte retrieval failed. Emergency oocyte vitrification was performed and after two months, the vitrified oocytes were warmed and the surviving oocytes inseminated with frozen-thawed donor sperm by intracytoplasmic sperm injection (ICSI). A total of 117 mature oocytes from the 15 women were vitrified and warmed. The post-warming survival rate was 84.6% (99/117), and the fertilization rate following ICSI was 83.8% (83/99). We selected 30 embryos for transfer to 15 patients, 8 of whom became pregnant. The clinical pregnancy rate was 53.3% (8/15) and the implantation rate was 30.0% (9/30). Nine healthy live births resulted from 8 pregnancies. These results indicate that emergency oocyte vitrification is an effective rescue technique that can be applied clinically with acceptable pregnancy and live birth rates when testicular sperm extraction from the male partner failed on the day of oocyte retrieval. These results also highlight another important option for oocyte cryopreservation through the use of vitrification technology.

Expression and Potential Roles of HLA-G in Human Spermatogenesis and Early Embryonic Development

As one of the non-classical major histocompatibility complex(MHC)-1 antigens, Human Leukocyte Antigen G (HLA-G), has been suggested as a prognostic marker to identify the embryo developmental potential. In the present study, we investigated the potential roles of HLA-G in human spermatogenesis and early embryonic development. Quantitative real-time PCR analysis revealed that HLA-Gs expression was increased with increased Johnsen score in testicular tissues. There was no significant difference in HLA-G mRNA expression between testicular tissues with Johnsen score of 8–9 and normal sperm from ejaculated semen. HLA-G mRNA expression was detected in human zygotes, embryos and blastocysts but not in unfertilized oocytes. In testicular tissues where sperm was obtained by testicular sperm extraction (Johnsen score was 8 to 9), there were no correlations between HLA-G mRNA expression and fertilization, cleavage and high-quality embryo rates. At 48–72 h post-fertilization, HLA-G expression was higher in fast growing embryos. HLA-G specific siRNA injection into zygotes not only slowed down embryonic cleavage rate at 48 h post-fertilization, but also down-regulated the expression of embryo metabolism related gene (SLC2A1) and cell cycle-regulated gene (CCND2). Taken together, our findings suggested that HLA-G plays significant roles in human spermatogenesis and early embryonic development.

Developmental potential of clinically discarded human embryos and associated chromosomal analysis.

Clinically discarded human embryos, which are generated from both normal and abnormal fertilizations, have the potential of developing into blastocysts. A total of 1,649 discarded human embryos, including zygotes containing normal (2PN) and abnormal (0PN, 1PN, 3PN and ≥4PN) pronuclei and prematurely cleaved embryos (2Cell), were collected for in vitro culture to investigate their developmental potential and chromosomal constitution using an SNP array-based chromosomal analysis. We found that blastocyst formation rates were 63.8% (for 2Cell embryos), 22.6% (2PN), 16.7% (0PN), 11.2% (3PN) and 3.6% (1PN). SNP array-based chromosomal analysis of the resultant blastocysts revealed that the percentages of normal chromosomes were 55.2% (2Cell), 60.7% (2PN), 44.4% (0PN) and 47.4% (0PN). Compared with clinical preimplantation genetic diagnosis (PGD) data generated with clinically acceptable embryos, results of the SNP array-based chromosome analysis on blastocysts from clinically discarded embryos showed similar values for the frequency of abnormal chromosome occurrence, aberrant signal classification and chromosomal distribution. The present study is perhaps the first systematic analysis of the developmental potential of clinically discarded embryos and provides a basis for future studies.

miR-181a-5p suppresses invasion and migration of HTR-8/SVneo cells by directly targeting IGF2BP2

Pre-eclampsia is a pregnancy-related disease that may cause maternal, neonatal and fetal morbidity and mortality and exists in 3–5% of pregnancies worldwide. The discovery of dysregulated microRNAs and their roles in placental development has provided a new avenue for elucidating the mechanism involved in this pregnancy-specific disorder. Here, the roles of human miR-181a-5p, a microRNA that is increased in both the plasma and placenta of severe pre-eclamptic patients, in invasion and migration of trophoblasts were investigated. Ectopic-expression of miR-181a-5p impaired the invasion and migration of HTR-8/SVneo cells, whereas miR-181a-5p inhibition had the opposite effects. IGF2BP2, which harbors a highly conserved miR-181a-5p-binding site within its 3ʹ-UTR, was identified to be directly inhibited by miR-181a-5p. Moreover, siRNAs targeting IGF2BP2 imitated the effects of overexpressed miR-181a-5p on HTR-8/SVneo cell invasion and migration, whereas restoring IGF2BP2 expression by overexpressing a plasmid encoding IGF2BP2 partially reversed the studied inhibitory functions of miR-181a-5p. Thus, we demonstrated here that miR-181a-5p suppresses the invasion and migration of cytotrophoblasts, and its inhibitory effects were at least partially mediated by the suppression of IGF2BP2 expression, thus shedding new light on the roles of miR-181a-5p in the pathogenesis of severe pre-eclampsia.

Effects of Vitrification on Outcomes of In Vivo-Mature, In Vitro-Mature and Immature Human Oocytes.

Background/Aims: To observe the effects of vitrification on spindle, zona pellucida, embryonic aneuploidy and DNA injury in in vivo-maruted, in vitro-mature and immature human oocytes. Methods: Between January 2009 and February 2015, 223 immature oocytes from 450 infertile patients, and 31 in vivo-mature oocytes from 3 infertile couples were collected. Of the 223 immature oocytes, 113 were used for in vitro culture before vitrification. Some oocytes were randomly divided into in vivo-mature group (group A, n = 15), in vitro-mature group (group B, n = 88) and immature group (group C, n = 85), and then the oocytes with spindle in these three groups after freezing-thawing were selected to use for Polscope imaging, embryonic aneuploidy screening and embryo development evaluation. Other oocytes were randomly divided into group A (n = 16), group B (n = 25) and group C (n = 25) for detecting DNA injury. Results: After thawing, spindle occurrence rate, spindle Retardance value, and cleavage rate were significantly higher in groups A and B than in group C (all P < 0.05), but there were no statistical differences in fertility rate, high-quality embryo rate, blastulation rate and aneuploidy rate amongst the three groups (all P > 0.05). Zona pellucida density (ZPD) was significantly lower in group A than in groups B and C both before and after vitrification (all P < 0.05). ZPD was significantly higher after thawing than before vitrification (all P < 0.05), but zona pellucida thickness (ZPT) was not significantly changed in all the three groups (all P > 0.05). Rate of comet cells was significantly lower in group A than in groups B and C (all P < 0.01). Comet tail was significantly longer in group C than in groups B and A (all P < 0.05). Conclusion: In vivo- and in vitro-mature human oocytes are more suitable to vitrification than immature human oocytes. Spindle Retardance value has more predictive value for embryonic development potential than ZPD and ZPT.

Embryo developmental potential of microsurgically corrected human three-pronuclear zygotes

Abstract We explored the embryo development potential of human three-pronuclear (3PN) zygotes reduced to two-pronuclear (2PN) zygotes (3 → 2PN zygotes) by micropuncture. In this study, there were three groups, the 3 → 2PN group (338 zygotes), the non-corrected 3PN group (381 zygotes), and the normal 2PN group (359 zygotes). The first cleavage mode (2-cell cleavage or 3-cell cleavage), 6–8 cell embryogenesis rate, high-quality embryogenesis rate and Day 5/Day 6 blastulation rate were compared between the three groups. The success rate of enucleation was 92.9%. The 2-cell cleavage rate was significantly higher in the 3 → 2PN group (74.3%) than in the 3PN group (36.4%) (P < 0.05), but had no statistical difference compared with the 2PN group (86.0%) (P > 0.05). The 6–8 cell embryogenesis rate was significantly higher in the 3 → 2PN group (91.1%) as compared to the 2PN group (85.6%) (P < 0.05), but had no statistical difference compared with the 3PN group (95.0%) (P > 0.05). Total blastulation rate was significantly higher in the 2PN group (58.8%) as compared to the 3PN group (21.5%) (P < 0.01), and in the 3 → 2PN group as compared to the 3PN group (5.6%) (P < 0.01). Also D5 blastulation rate was significantly higher in the 2PN group (53.7%) as compared to the 3 → 2PN group (8.9%) (P < 0.01), and in the 3 → 2PN group as compared to the 3PN group (1.9%) (P < 0.01). In 3 → 2PN zygotes, the first cleavage mode is mainly 2 cells which is significantly higher than that in 3PN zygotes. Compared with 3PN zygotes, the embryo developmental potential of 3 → 2PN zygotes is improved, but still is lower than that in 2PN zygotes.

Role of PAFAH1B1 in human spermatogenesis, fertilization and early embryonic development

Spermatogenesis, fertilization and subsequent embryonic development are complex processes that require tight regulation. The PAFAH1B1 gene plays important roles in these reproductive events in mice, but its expression and roles in human reproduction have not been investigated. Expression analysis of testicular tissue by reverse transcription quantitative PCR and immunohistochemistry revealed varied expression levels among samples of different spermatogenic abilities (as assessed by the Johnsen score), with protein expression restricted to spermatogonia, spermatocytes and spermatids. Immunofluorescence on spermatozoa showed expression over the acrosome and midpiece regions of ejaculated samples, whereas a high proportion of percutaneous epididymal sperm aspiration-derived spermatozoa showed expression restricted to the midpiece. Analysis for PAFAH1B1 mRNA also revealed different expression levels among unfertilized oocytes, zygotes, cleavage stage embryos and blastocysts, with protein localized at the membrane level in oocytes and zygotes, and gradually distributing within the cytoplasm of cleavage stage embryos and blastocysts. Interestingly, microinjection of PAFAH1B1 siRNA into zygotes significantly (P = 0.024) increased fragmentation formation rates in subsequent embryonic development stages. Altogether, these are the first results to support a role for PAFAH1B1 in human spermatogenesis and early embryonic development.

Effects of frozen timing on the spindle density, the angle between the polar body and spindle, and embryo development of intracytoplasmic sperm injection in mouse mature oocytes

Better pregnancy outcomes can be obtained by human mature oocyte vitrification, but many problems remain to be resolved in human mature oocyte vitrification. Since mature oocyte development possesses its own maturity cycle, there should be the optimal timing for mature oocyte vitrification. The purpose of this study was to observe the effects of frozen timing on the spindle density, the angle between the polar body and spindle, and embryo development of intracytoplasmic sperm injection (ICSI) in vitrified mouse mature oocytes and explore its possible mechanism. Mouse oocytes were randomly divided into three groups according to different frozen timing including Groups A, B, and C in which oocytes were vitrified within 2 h after ovum pick‐up, and 3–4 and 5–6 h after ovum pick‐up, respectively. Spindle‐related parameters were measured, ICSI was performed. The spindle occurrence rate of vitrified‐thawed oocytes was 98.4% in Group A, 82.3% in Group B, and 75.8% in Group C, without statistical differences between pre‐vitrification and post‐thawing and among the three groups (P > 0.05). The angles between the polar body and spindle were larger after thawing than before vitrification (P < 0.01). The spindle retardance values were lower after thawing than before vitrification in Groups B and C (P < 0.05), but higher in Group A (P < 0.05). The spindle retardance values before vitrification were higher in Group B than in Groups A and C (P < 0.05), but the spindle retardance value, oocyte survival and two‐cell rate after thawing were higher in Group A than in Groups B and C (P < 0.05). There were no statistical differences in ICSI fertility rate between the three groups (P > 0.05). The damage on the spindle is the slightest and embryo quality is the highest in the mouse oocytes vitrified within 2 h after ovum pick‐up. The spindle retardance value is more valuable than the spindle occurrence rate in the evaluation of vitrified‐thawed oocyte quality, and is positively correlated with embryo quality.

Reduced microRNA‐188‐3p expression contributes to apoptosis of spermatogenic cells in patients with azoospermia

Human mutL homologl (MLH1) works coordinately in sequential steps to initiate repair of DNA mismatches, and aberrant MLH1 expression is related to spermatogenetic malfunction. In the present study, MLH1 expression in patients with azoospermia was investigated, and moderating effects of miR‐188‐3p on MLH1 expression and spermatogenesis were identified.

Mapping allele with resolved carrier status of Robertsonian and reciprocal translocation in human preimplantation embryos

Significance In in vitro fertilization, it is difficult, if not impossible, with current methods to determine whether an embryo carries a chromosomal translocation. We have established a method for diagnosing chromosome abnormality named “Mapping Allele with Resolved Carrier Status” (MaReCs), which enables simultaneous screening of chromosomal ploidy and translocation in an embryo by next-generation sequencing. We demonstrate and validate that MaReCs allows accurate selection of translocation-free embryos, preventing the transmission of chromosomal translocations to future generations. Reciprocal translocations (RecT) and Robertsonian translocations (RobT) are among the most common chromosomal abnormalities that cause infertility and birth defects. Preimplantation genetic testing for aneuploidy using comprehensive chromosome screening for in vitro fertilization enables embryo selection with balanced chromosomal ploidy; however, it is normally unable to determine whether an embryo is a translocation carrier. Here we report a method named “Mapping Allele with Resolved Carrier Status” (MaReCs), which enables chromosomal ploidy screening and resolution of the translocation carrier status of the same embryo. We performed MaReCs on 108 embryos, of which 96 were from 13 RecT carriers and 12 were from three RobT carriers. Thirteen of the sixteen patients had at least one diploid embryo. We have confirmed the accuracy of our carrier status determination in amniotic fluid karyotyping of seven cases as well as in the live birth we have thus far. Therefore, MaReCs accurately enables the selection of translocation-free embryos from patients carrying chromosomal translocations. We expect MaReCs will help reduce the propagation of RecT/RobT in the human population.