Guidong Yao

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.

Effect of paternal overweight or obesity on IVF treatment outcomes and the possible mechanisms involved

Leukocyte telomere lengths (LTLs) are shorter in obese compared with normal weight people. However, it is not known whether sperm telomere length (STL) is related to obesity. The aim of the study was to evaluate the impact of men’s body mass index (BMI) on STL, embryo quality, and clinical outcomes in couples undergoing IVF. In total, 651 couples were recruited, including 345 men with a normal BMI and 306 men with an overweight BMI (normal BMI group: 20–25 kg/m2; overweight BMI group: >28 kg/m2). We found that couples with male’s BMI over 28 kg/m2 exhibited a significantly lower fertilization rate, good-quality embryo rate and clinical pregnancy rate compared to their normal BMI counterparts. The mean STL in the overweight BMI group was also significantly shorter than that of the normal BMI group. The results also showed that individuals with higher BMI had higher ROS (Reactive oxygen species) content and sperm DNA fragmentation rate when compared with normal BMI individuals. Mitochondrial activity was also lower in the overweight BMI group than in the normal BMI group. This is the first report to find that STL is shorter in overweight/obese men, which may account for their poorer treatment outcomes in IVF cycles.

Blastocoele expansion degree predicts live birth after single blastocyst transfer for fresh and vitrified/warmed single blastocyst transfer cycles

OBJECTIVE To evaluate the independent effects of the degree of blastocoele expansion and re-expansion and the inner cell mass (ICM) and trophectoderm (TE) grades on predicting live birth after fresh and vitrified/warmed single blastocyst transfer. DESIGN Retrospective study. SETTING Reproductive medical center. PATIENT(S) Women undergoing 844 fresh and 370 vitrified/warmed single blastocyst transfer cycles. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Live-birth rate correlated with blastocyst morphology parameters by logistic regression analysis and Spearman correlations analysis. RESULT(S) The degree of blastocoele expansion and re-expansion was the only blastocyst morphology parameter that exhibited a significant ability to predict live birth in both fresh and vitrified/warmed single blastocyst transfer cycles respectively by multivariate logistic regression and Spearman correlations analysis. Although the ICM grade was significantly related to live birth in fresh cycles according to the univariate model, its effect was not maintained in the multivariate logistic analysis. In vitrified/warmed cycles, neither ICM nor TE grade was correlated with live birth by logistic regression analysis. CONCLUSION(S) This study is the first to confirm that the degree of blastocoele expansion and re-expansion is a better predictor of live birth after both fresh and vitrified/warmed single blastocyst transfer cycles than ICM or TE grade.

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.

Body mass index effects sperm quality: a retrospective study in Northern China

Excess weight and obesity have become a serious problem in adult men of reproductive age throughout the world. The purpose of this retrospective study was to assess the relationships between body mass index and sperm quality in subfertile couples in a Chinese Han population. Sperm analyses were performed and demographic data collected from 2384 male partners in subfertile couples who visited a reproductive medical center for treatment and preconception counseling. The subjects were classified into four groups according to their body mass index: underweight, normal, overweight, and obese. Of these subjects, 918 (38.3%) had a body mass index of >25.0 kg m−0 2 . No significant differences were found between the four groups with respect to age, occupation, level of education, smoking status, alcohol use, duration of sexual abstinence, or the collection time of year for sperm. The results clearly indicated lower sperm quality (total sperm count, sperm concentration, motile sperm, relative amounts of type A motility, and progressive motility sperm [A + B]) in overweight and obese participants than in those with normal body mass index. Normal sperm morphology and sperm volume showed no clear difference between the four groups. This study indicates that body mass index has a negative effect on sperm quality in men of subfertile couples in a Northern Chinese population. Further study should be performed to investigate the relationship between body mass index and sperm quality in a larger population.

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.

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.