A case report of a normal fertile woman with 46,XX/46,XY somatic chimerism reveals a critical role for germ cells in sex determination.

Individuals with 46,XX/XY chimerism can display a wide range of characteristics, varying from hermaphroditism to complete male or female, and can display sex chromosome chimerism in multiple tissues, including the gonads. The gonadal tissues of females contain both granulosa and germ cells. However, the specific sex chromosome composition of the granulosa and germ cells in 46,XX/XY chimeric female is currently unknown. Here, we reported a 30-year-old woman with secondary infertility who displayed a 46,XX/46,XY chimerism in the peripheral blood. FISH testing revealed varying degrees of XX/XY chimerism in multiple tissues of the female patient. Subsequently, the patient underwent preimplantation genetic testing (PGT) treatment, and 26 oocytes were retrieved. From the twenty-four biopsied mature oocytes, a total of 23 first polar bodies (PBs) and 10 second PBs were obtained. These PBs and two immature metaphase I (MI) oocytes only displayed X chromosome signals with no presence of the Y, suggesting that all oocytes in this chimeric female were of XX germ cell origin. On the other hand, granulosa cells obtained from individual follicles exhibited varied proportions of XX/XY cell types, and six follicles possessed 100% XX or XY granulosa cells. A total of 24 oocytes were successfully fertilized, and 12 developed into blastocysts, where 5 being XY and 5 were XX. Two blastocysts were transferred with one originating from an oocyte aspirated from a follicle containing 100% XY granulosa cells. This resulted in a twin pregnancy. Subsequent prenatal diagnosis confirmed normal male and female karyotypes. Ultimately, healthy boy-girl twins were delivered at full term. In summary, this 46,XX/XY chimerism with XX germ cells presented complete female, suggesting that germ cells may exert a significant influence on the sexual determination of an individual, which provide valuable insights into the intricate processes associated with sexual development and reproduction.

Oxygen concentration from days 1 to 3 after insemination affects the embryo culture quality, cumulative live birth rate, and perinatal outcomes.

PURPOSE: We aimed to compare embryo development, cumulative live birth rate (CLBR), and perinatal outcomes of embryos cultured in 20% and 5% oxygen from days 1 to 3 after insemination. METHODS: This retrospective study included patients who received in vitro fertilization (IVF) treatment between January 2015 and November 2019. Embryos of each patient were cultured at 20% or 5% oxygen from days 1-3 after insemination. The primary outcome was CLBR. Propensity score matching (PSM) was used to balance patients' baseline data in both oxygen groups. RESULTS: In total, 31,566 patients were enrolled. After PSM, the rate of high-quality day 3 embryos was significantly lower in the 20% than in the 5% oxygen group (0.49 ± 0.33 vs 0.51 ± 0.33; adjusted ß = -0.03; 95% confidence interval [CI], -0.03 to -0.02). The CLBR was significantly lower in the 20% than in the 5% oxygen group (58.6% vs. 62.4%; adjusted odds ratio = 0.85; 95% CI, 0.81-0.90). The birthweight and Z score of singletons were significantly higher in the 20% than in the 5% oxygen group (birthweight: 3.30 ± 0.50 vs. 3.28 ± 0.48; adjusted ß = 0.022; 95% CI, 0.004-0.040; Z score: 0.26 ± 1.04 vs. 0.22 ± 1.01; adjusted ß = 0.037; 95% CI, 0.001-0.074). CONCLUSION: Culturing embryos at atmospheric oxygen concentrations from days 1 to 3 compromises embryo quality, reduces CLBR, and affects birthweight. The 5% oxygen concentration is more suitable for embryo culture in IVF laboratories to achieve successful outcomes.

Reproductive risks and preimplantation genetic testing intervention for X-autosome translocation carriers.

RESEARCH QUESTION: What is the genetic cause of multiple congenital disabilities in a girl with a maternal balanced X-autosome translocation [t(X-A)]? Is preimplantation genetic testing (PGT), to distinguish non-carrier from euploid/balanced embryos and prioritize transfer, an effective and applicable strategy for couples with t(X-A)? DESIGN: Karyotype analysis, whole-exome sequencing and X inactivation analysis were performed for a girl with congenital cardiac anomalies, language impairment and mild neurodevelopmental delay. PGT based on next-generation sequencing after microdissecting junction region (MicroSeq) to distinguish non-carrier and carrier embryos was used in three couples with a female t(X-A) carrier (cases 1-3). RESULTS: The girl carried a maternal balanced translocation 46,X,t(X;1)(q28;p31.1). Whole-exome sequencing revealed no monogenic mutation related to her phenotype, but she carried a rare skewed inactivation of the translocated X chromosome that spread to the adjacent interstitial 1p segment, contrary to her mother. All translocation breakpoints in cases 1-3 were successfully identified and each couple underwent one PGT cycle. Thirty oocytes were retrieved, and 13 blastocysts were eligible for biopsy, of which six embryos had a balanced translocation and only four were non-carriers. Three cryopreserved embryo transfers with non-carrier status embryos resulted in the birth of two healthy children (one girl and one boy), who were subsequently confirmed to have normal karyotypes. CONCLUSIONS: This study reported a girl with multiple congenital disabilities associated with a maternal balanced t(X-A) and verified that the distinction between non-carrier and carrier embryos is an effective and applicable strategy to avoid transferring genetic and reproductive risks to the offspring of t(X-A) carriers.

The genetic cause of intellectual deficiency and/or congenital malformations in two parental reciprocal translocation carriers and implications for assisted reproduction.

PURPOSE: To elucidate the genetic cause of intellectual deficiency and/or congenital malformations in two parental reciprocal translocation carriers and provide appropriate strategies of assisted reproductive therapy (ART). MATERIALS AND METHODS: Two similar couples having a child with global developmental delay/intellectual disability symptoms attended the Reproductive and Genetic Hospital of CITIC-Xiangya (Changsha, China) in 2017 and 2019, respectively, in order to determine the cause(s) of the conditions affecting their child and to seek ART to have a healthy baby. Both of the healthy couples were not of consanguineous marriage, denied exposure to toxicants, and had no adverse life history. This study was approved by the Institutional Ethics Committee of the Reproductive & Genetic Hospital of CITIC-Xiangya, and written informed consent was obtained from the parents. Genetic diagnoses were performed by karyotype analysis, breakpoint mapping analysis of chromosomal translocation(s), single-nucleotide polymorphism (SNP) microarray analysis, and whole-exome sequencing (WES) for the two children and different appropriate reproductive strategies were performed in the two families. RESULTS: Karyotype analysis revealed that both patients carried parental reciprocal translocations [46,XY,t(7;16)(p13;q24)pat and 46,XY,t(13;17)(q12.3;p11.2)pat, respectively]. Follow-up breakpoint mapping analysis showed no interruption of associated genes, and SNP microarray analysis identified no significant copy number variations (CNVs) in the two patients. Moreover, WES results revealed that patients 1 and 2 harbored candidate compound heterozygous mutations of MCOLN1 [c.195G>C (p.K65N) and c.1061G>A (p.W354*)] and MCPH1 [c.877A>G (p.S293G) and c.1869_1870delAT (p.C624*)], respectively, that were inherited from their parents and not previously reported. Furthermore, the parents of patient 1 obtained 10 embryos during ART cycle, and an embryo of normal karyotype and non-carrier of observed MCOLN1 mutations according to preimplantation genetic testing for structural rearrangement and monogenic defect was successfully transferred, resulting in the birth of a healthy boy. The parents of patient 2 chose to undergo ART with donor sperm to reduce the risk of recurrence. CONCLUSIONS: Systematic genetic diagnosis of two carriers of inherited chromosomal translocations accompanied by clinical phenotypes revealed their cause of disease, which was critical for genetic counseling and further ART for these families.

Clinical outcomes following preimplantation genetic testing and microdissecting junction region in couples with balanced chromosome rearrangement.

PURPOSE: The purpose of this study is to summarize the clinical outcomes of apparently balanced chromosome rearrangement (ABCR) carriers in preimplantation genetic testing (PGT) cycles by next-generation sequencing following microdissecting junction region (MicroSeq) to distinguish non-carrier embryos from balanced carriers. METHODS: A retrospective study of 762 ABCR carrier couples who requested PGT for structural rearrangements combined with MicroSeq at the Reproductive and Genetic Hospital of CITIC-Xiangya was conducted between October 2014 and October 2019. RESULTS: Trophectoderm biopsy was performed in 4122 blastocysts derived from 917 PGT-SR cycles and 3781 blastocysts were detected. Among the 3781 blastocysts diagnosed, 1433 (37.9%, 1433/3781) were balanced, of which 739 blastocysts were carriers (51.57%, 739/1433) and 694 blastocysts were normal (48.43%, 694/1433). Approximately 26.39% of cycles had both carrier and normal embryo transfer, and the average number of biopsied blastocysts was 6.7. In the cumulative 223 biopsied cycles with normal embryo transfer, all couples chose to transfer the normal embryos. In the 225 cycles with only carrier embryos, the couples chose to transfer the carrier embryos in 169/225 (75.11%) cycles. A total of 732 frozen embryo transfer cycles were performed, resulting in 502 clinical pregnancies. Cumulatively, 326 babies were born; all of these babies were healthy and free of any developmental issues. CONCLUSION: Our study provides the first evaluation of the clinical outcomes of a large sample with ABCR carrier couples undergoing the MicroSeq-PGT technique and reveals its powerful ability to distinguish between carrier and non-carrier balanced embryos.

Novel homozygous variations in PLCZ1 lead to poor or failed fertilization characterized by abnormal localization patterns of PLCζ in sperm.

Total fertilization failure (TFF), which is the failure of fertilization in all oocytes, occurs in 1%-3% of intracytoplasmic sperm injection (ICSI) cycles. However, the sperm-related factors that cause fertilization failure in humans are still largely unknown. Here, we identified three novel homozygous variations in the PLCZ1 gene in a recessive inheritance pattern in three consanguineous families, which all located in a key catalytic domain, and predicted to modify its secondary structure and thus impair its hydrolytic activity. Moreover, immunofluorescent staining revealed that PLCζ in mutant sperm exhibited abnormal localization patterns. ICSI-AOA resulted in an increased rate of normal fertilization compared with previous ICSI cycles (75.0% vs 2.2%, P < .001). In summary, we identified three novel homozygous variations in PLCZ1 that led to poor or failed fertilization that could be overcame by ICSI-AOA.

The mitochondrial DNA copy number of cumulus granulosa cells may be related to the maturity of oocyte cytoplasm.

STUDY QUESTION: Is the mitochondrial DNA (mtDNA) copy number of cumulus granulosa cells (CGCs) related to the maturation of oocyte cytoplasm? SUMMARY ANSWER: Compared with the mtDNA copy number of CGCs from germinal vesicles (GV), CGCs from Metaphase I (MI) oocytes appear to have a lower mtDNA copy number. WHAT IS KNOWN ALREADY: The growth and development of CGCs and oocyte are synchronised. The interaction between CGCs and the oocyte provides the appropriate balance of energy, which is necessary for mammalian oocyte development. Moreover, in the oocyte-cumulus complex (OCC), mature oocytes with higher mtDNA copy numbers tend to have corresponding CGCs with higher mtDNA copy numbers. STUDY DESIGN, SIZE, DURATION: This is a prospective study of 302 OCCs obtained from 70 women undergoing in vitro fertilisation with intracytoplasmic sperm injection (ICSI) at the Reproductive and Genetic Hospital of CITIC-Xiangya, between 24 February 2018 and 21 December 2019. The CGCs were divided into three groups (GV, MI and MII stages) based on the maturation status of their corresponding oocyte. The sample sizes (n = 302) of CGCs in the three stages were 63 (CGCGV), 70 (CGCMI) and 169 (CGCMII), respectively. Some of the samples (n = 257) was used to quantify the mtDNA copy number, while the rest (n = 45) were used to analyse the expression level of mitochondrial genes. Furthermore, we retrieved 82 immature oocytes from among the 257 OCCs used for mtDNA copy numbers, including 36 GV oocytes and 46 MI oocytes, for analysis of oocyte mtDNA. PARTICIPANTS/MATERIALS, SETTING, METHODS: We selected genes with high consistency of real-time PCR results to accurately measure the mtDNA copy number by testing the efficacy and the reproducibility in whole genome amplification (WGA) samples from a human embryonic stem cell line. The CGCs of each oocyte were individually isolated. The mtDNA copy number and gene expression of the CGCs were assessed using real-time PCR techniques. Mitochondrial DNA copy number of the corresponding immature oocytes was also evaluated. MAIN RESULTS AND THE ROLE OF CHANCE: MT-ND1, MT-CO1 and ß-globin genes were chosen for the assessment of mtDNA content, and mRNA expressions of MT-ND1, MT-CO1, PGC-1α and TFAM were also measured. The genome of 257 CGCs and 82 immature oocytes were amplified according to the multiple displacement amplification (MDA) protocol, and RNA was extracted from 45 CGCs. Compared with CGCGV, CGCMI had a significantly lower mtDNA copy number. In the MT-ND1 assay, the CGCGV: CGCMI was [270 ± 302]: [134 ± 201], P = 0.015. In the MT-CO1 assay, CGCGV: CGCMI was [205 ± 228]: [92 ± 112], P = 0.026. There was no statistical difference in mtDNA between CGCGV and CGCMII. In the MT-ND1 assay, CGCGV: CGCMII was [270 ± 302]: [175 ± 223], P = 0.074. In the MT-CO1 assay, CGCGV: CGCMII was [205 ± 228]: [119 ± 192], P = 0.077. No statistical difference of mtDNA copy number was observed between CGCMI and CGCMII. In the MT-ND1 assay, CGCMI: CGCMII was [134 ± 201]: [175 ± 223], P = 0.422. In the MT-CO1 assay, CGCMI: CGCMII was [92 ± 112]: [119 ± 192], P = 0.478. To verify the reliability of the above results, we further analysed the mtDNA copy number of CGCs of 14 patients with GV, MI and MII oocytes, and the results showed that the mtDNA copy number of CGCMI may be lower. The mtDNA copy number of CGCGV and CGCMI was statistically different in the MT-ND1 assay where CGCGV: CGCMI was [249 ± 173]: [118 ± 113], P = 0.016, but in the MT-CO1 assay, CGCGV: CGCMI was [208 ± 199]: [83 ± 98], P = 0.109. There was no significant difference in mtDNA between CGCGV and CGCMII. In the MT-ND1 assay, CGCGV: CGCMII was [249 ± 173]: [185 ± 200], P = 0.096. In the MT-CO1 assay, CGCGV: CGCMII was [208 ± 199]: [114 ± 139], P = 0.096. There was also no significant difference in mtDNA between CGCMI and CGCMII. In the MT-ND1 assay, CGCMI: CGCMII was [118 ± 113]: [185 ± 200], P = 0.198. In the MT-CO1 assay, CGCMI: CGCMII was [83 ± 98]: [114 ± 139], P = 0.470. Moreover, there were no statistical differences in the expression levels of MT-ND1, MT-CO1, PGC-1α and TFAM between CGCGV, CGCMI and CGCMII (P > 0.05). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Due to the ethical issues, the study did not quantify the mtDNA content of MII oocytes. Thus, whether the change in mtDNA copy number in CGCs is related to the different developmental stages of oocytes has not been further confirmed. Moreover, the sample size was relatively small. WIDER IMPLICATIONS OF THE FINDINGS: The mtDNA copy number of CGCs decreases from the GV phase to the MI phase and stays steady from the MI to MII stage. At different stages of oocyte maturation, the mtDNA of CGCs may undergo self-degradation and replication to meet the energy requirements of the corresponding oocyte and the maturation of the oocyte cytoplasm. STUDY FUNDING/COMPETING INTEREST(S): Funding was provided by the National Key R&D Program of China (Grant 2018YFC1003100, to L.H.), the science and technology major project of the Ministry of Science and Technology of Hunan Province, China (grant 2017SK1030, to G.L.), the National Natural Science Foundation of China (grant 81873478, to L.H.), and Merck Serono China Research Fund for Fertility Experts (to L.H.). There is no conflict of interest.

New biallelic mutations in PADI6 cause recurrent preimplantation embryonic arrest characterized by direct cleavage.

PURPOSE: To investigate the pathogenesis of the recurrent preimplantation embryonic arrest characterized by direct cleavage. METHODS: Two affected individuals underwent time-lapse imaging to observe the cleavage behaviors in their final ICSI attempts. In addition, both patients were subjected to whole-exome sequencing. After the identification of possible causative genes, molecular modeling analyses were used to evaluate the possible effects of candidate mutations on protein secondary structure. RESULTS: All the bipronucleated (2PN) zygotes from both individuals presented multiple abnormal cleavage behaviors, particularly direct cleavage (DC) and subsequent cleavage arrest. Mutation analysis identified one new frameshift mutation c.1521dupC (p.S508Qfs*5) and two missense mutations c.A1117C and c.C1708T (p.T373P and p.R570C, respectively) of the PADI6 gene, which were in the protein-arginine deiminase (PAD) domain and highly conserved. CONCLUSION: This study expands the mutation spectrum of PADI6 and is the first to propose that the preimplantation embryonic arrest with concomitant abnormal cleavage behaviors, especially DC, maybe associated with PADI6 mutations.

ZP2 pathogenic variants cause in vitro fertilization failure and female infertility.

PURPOSE: The oocyte-borne genetic causes leading to fertilization failure are largely unknown. We aimed to identify novel human pathogenic variants (PV) and genes causing fertilization failure. METHODS: We performed exome sequencing for a consanguineous family with a recessive inheritance pattern of female infertility characterized by oocytes with a thin zona pellucida (ZP) and fertilization failure in routine in vitro fertilization. Subsequent PV screening of ZP2 was performed in additional eight unrelated infertile women whose oocytes exhibited abnormal ZP and similar fertilization failure. Expression of ZP proteins was assessed in mutant oocytes by immunostaining, and functional studies of the wild-type and mutant proteins were carried out in CHO-K1 cells. RESULTS: Two homozygous s PV (c.1695-2A>G, and c.1691_1694dup (p.C566Wfs*5), respectively) of ZP2 were identified in the affected women from two unrelated consanguineous families. All oocytes carrying PV were surrounded by a thin ZP that was defective for sperm-binding. Immunostaining indicated a lack of ZP2 protein in the thin ZP. Studies in CHO cells showed that both PV resulted in a truncated ZP2 protein, which might be intracellularly sequestered and prematurely interacted with other ZP proteins. CONCLUSION: We identified loss-of-function PV of ZP2 causing a structurally abnormal and dysfunctional ZP, resulting in fertilization failure and female infertility.

ZP1 mutations are associated with empty follicle syndrome: evidence for the existence of an intact oocyte and a zona pellucida in follicles up to the early antral stage. A case report.

Empty follicle syndrome (EFS) is the complete failure to retrieve oocytes after ovarian stimulation. Although LHCGR and ZP3 were identified as causative genes, it is still unclear what happens to these patients' oocytes, and the pathogenesis of EFS remains obscure. Here, we identified six novel ZP1 mutations associated with EFS and female infertility that was inherited recessively in five unrelated families. Studies in CHO-K1 cells showed that these mutations resulted in either degradation or truncation of ZP1 protein. Immunohistochemistry using ovarian serial sections demonstrated that all preantral follicles had normal architecture, but with a thin ZP, lacking ZP1, surrounding the growing oocytes. The antral follicles were also defective in normal cumulus-oocyte complex organisation, leading us to speculate that the lack of ZP1 might lead to oocyte degeneration or increased fragility of the oocyte during follicular puncture, ultimately resulting in EFS. To our knowledge, this is the first study that presents morphological evidence showing normal preantral folliculogenesis with abnormal ZP assembly in EFS patients. Our data provides a better understanding of the biological functions of ZP1 in human ZP assembly and folliculogenesis and gives new insights into the pathogenesis of EFS and possible therapeutic developments.

Analysis of molecular cytogenetic features and PGT-SR for two infertile patients with small supernumerary marker chromosomes.

RESEARCH QUESTION: Can preimplantation genetic testing for structural rearrangement (PGT-SR) with next-generation sequencing (NGS) be used to infertile patients carrying small supernumerary marker chromosomes (sSMCs)? DESIGN: In this study, two infertile patients carrying ring sSMCs were recruited. Different molecular cytogenetic techniques were performed to identify the features of the two sSMCs, followed by clinical PGT-SR cycles. RESULTS: The results of G-banding and FISH showed that patient 1's sSMC originated from the 8p23-p10 region, with a resulting karyotype of [ 47,XY, del(8)(p23p10), +r(8)(p23p10).ish del(8)(CEP8+,subtle 8p+,subtle 8q+),r(8)(CEP8+,subtle 8p-,subtle 8q-)[55/60].arr(1-22) ×2,(X,Y)×1]. The sSMC of patient 2 was derived from chromosome 3 and further microdissection with next-generation sequencing (MicroSeq) revealed it contained the region of chromosome 3 between 93,504,855 and 103,839,892 bp (GRCh37), which involved 52 known genes. So the karyotype of patient 2 was 47,XX, +mar.ish der(3)(CEP3+,subtle 3p-,subtle 3q-)[49/60].arr[GRCh37] 3q11.2q13.1(93,500,001_103,839,892) ×3(0.5). PGT-SR with NGS was performed to provide reproductive guidance for the two patients. For patient 1, four balanced euploid embryos and four embryos with partial trisomy/monosomy of (8p23.1-8p11.21) were obtained, and a balanced euploid embryo was successfully implanted and had resulted in a healthy baby. For patient 2, an embryo with monosomy of sex chromosomes and another embryo with a duplication at (3q11-q13.1), neither of which was available for implantation. CONCLUSIONS: The identification of the origins and structural characteristics of rare sSMCs should rely on different molecular cytogenetic techniques. PGT-SR is an alternative fertility treatment for these patients carrying sSMCs. This study may provide directions for the assisted reproductive therapy for infertile patients with sSMC.

Single embryo transfer by Day 3 time-lapse selection versus Day 5 conventional morphological selection: a randomized, open-label, non-inferiority trial.

STUDY QUESTION: Does single cleavage-stage (Day 3) embryo transfer using a time-lapse (TL) hierarchical classification model achieve comparable ongoing pregnancy rates (OPR) to single blastocyst (Day 5) transfer by conventional morphological (CM) selection? SUMMARY ANSWER: Day 3 single embryo transfer (SET) with a hierarchical classification model had a significantly lower OPR compared with Day 5 SET with CM selection. WHAT IS KNOWN ALREADY: Cleavage-stage SET is an alternative to blastocyst SET. Time-lapse imaging assists better embryo selection, based on studies of pregnancy outcomes when adding time-lapse imaging to CM selection at the cleavage or blastocyst stage. STUDY DESIGN, SIZE, DURATION: This single-centre, randomized, open-label, active-controlled, non-inferiority study included 600 women between October 2015 and April 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: Eligible patients were Chinese females, aged ≤36 years, who were undergoing their first or second fresh IVF cycle using their own oocytes, and who had FSH levels ≤12 IU/mL on Day 3 of the cycle and 10 or more oocytes retrieved. Patients who had underlying uterine conditions, oocyte donation, recurrent pregnancy loss, abnormal oocytes or <6 normally fertilized embryos (2PN) were excluded from the study participation. Patients were randomized 1:1 to either the cleavage-stage SET with a time-lapse hierarchical classification model for selection (D3 + TL) or blastocyst SET with CM selection (D5 + CM). All normally fertilized zygotes were cultured in Primo Vision. The study was conducted at a tertiary IVF centre (CITIC-Xiangya) and OPR was the primary outcome. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 600 patients were randomized to the two groups, among which 585 (D3 + TL = 290, D5 + CM = 295) were included in the Modified-intention-to-treat (mITT) population and 517 (D3 + TL = 261, D5 + CM = 256) were included in the PP population. In the per protocol (PP) population, OPR was significantly lower in the D3 group (59.4%, 155/261) than in the D5 group (68.4%, 175/256) (difference: -9.0%, 95% CI: -17.1%, -0.7%, P = 0.03). Analysis in mITT population showed a marginally significant difference in the OPR between the D3 + TL and D5 + CM groups (56.6 versus 64.1%, difference: -7.5%, 95% CI: -15.4%, 0.4%, P = 0.06). The D3 + TL group resulted in a markedly lower implantation rate than the D5 + CM group (64.4 versus 77.0%; P = 0.002) in the PP analysis, however, the early miscarriage rate did not significantly differ between the two groups. LIMITATIONS, REASONS FOR CAUTION: The study lacked a direct comparison between time-lapse and CM selections at cleavage-stage SET and was statistically underpowered to detect non-inferiority. The subject's eligibility criteria favouring women with a good prognosis for IVF weakened the generalizability of the results. WIDER IMPLICATIONS OF THE FINDINGS: The OPR from Day 3 cleavage-stage SET using hierarchical classification time-lapse selection was significantly lower compared with that from Day 5 blastocyst SET using conventional morphology, yet it appeared to be clinically acceptable in women underwent IVF. STUDY FUNDING/COMPETING INTEREST(S): This study is supported by grants from Ferring Pharmaceuticals and the Program for New Century Excellent Talents in University, China. TRIAL REGISTRATION NUMBER: ChiCTR-ICR-15006600. TRIAL REGISTRATION DATE: 16 June 2015. DATE OF FIRST PATIENT'S ENROLMENT: 1 October 2015.

Prevalence and authenticity of de-novo segmental aneuploidy (>16 Mb) in human blastocysts as detected by next-generation sequencing.

RESEARCH QUESTION: What is the prevalence and authenticity of de-novo segmental aneuploidies (>16 Mb) detected by next-generation sequencing (NGS) in human preimplantation blastocysts? DESIGN: Between April 2013 and June 2016, 5735 blastocysts from 1854 couples (average age 33.11 ± 5.65 years) underwent preimplantation genetic testing for chromosomal structural rearrangement (PGT-SR) or for aneuploidy (PGT-A) using NGS on trophectoderm (TE) biopsy samples. The prevalence of de-novo segmental aneuploidy was calculated from these results. Forty blastocysts with de-novo segmental aneuploidy detected by NGS, which had been donated for research, were warmed for further fluorescence in-situ hybridization (FISH) analysis to confirm their authenticity. RESULTS: The frequency of de-novo segmental aneuploidies in blastocysts was 10.13% (581/5735); the phenomenon was not related to maternal age and occurred on all chromosomes. Of the 40 donated blastocysts, 39 were successfully warmed and fixed for FISH analysis at the single-cell level. The de-novo segmental aneuploidies identified by NGS were confirmed by FISH in all 39 blastocysts. However, the de-novo segmental aneuploidies in these blastocysts were not all pure patterns, with 66.67% (26/39) of blastocysts exhibiting mosaic patterns varying from 8.30% to 92.86% of cells with de-novo segmental aneuploidy. The concordance rate between NGS and FISH in TE and inner cell mass (ICM) samples was 47.69% (31/65). CONCLUSIONS: De-novo segmental aneuploidy above 16 Mb occurred in blastocysts and could be detected by NGS, while some aneuploidies existed as mosaics in both TE and ICM.

The clinical and neonatal outcomes after stimulation of immotile spermatozoa using SperMagic medium.

To evaluate the efficiency and safety of SperMagic medium on stimulating the immotile spermatozoa in testicular sperm extraction (TESE) and absolute asthenozoospermia, 96 patients with TESE and 106 patients with absolute asthenozoospermia were enrolled in this study. The motile spermatozoa were detected in 47 TESE patients and 68 absolute asthenozoospermia and these patients were assigned to control group. The immotile spermatozoa in 49 TESE patients and 34 absolute asthenozoospermia were stimulated with SperMagic medium. Patients were treated by standard intracytoplasmic sperm injection (ICSI). There were no significant differences in fertilisation, cleavage, implantation, pregnancy, live birth and neonatal outcomes. SperMagic medium does not increase incidence of adverse neonatal outcomes and is a reliable tool for selection of viable spermatozoa in ICSI.

Polar body transfer restores the developmental potential of oocytes to blastocyst stage in a case of repeated embryo fragmentation.

PURPOSE: We aimed to determine the developmental potential of human reconstructed oocytes after polar body genome transfer (PBT) and to report the case of a woman with multiple cycles of severe embryo fragmentation. METHODS: Fresh and cryopreserved first polar bodies (PB1s) were transferred to enucleated metaphase II oocytes (PB1T), while fresh PB2s were removed from fertilized oocytes and used instead of the female pronucleus in donor zygotes. Reconstructed oocytes underwent intracytoplasmic sperm injection (ICSI) and were cultured to blastocyst. Biopsied trophectoderm cells of PBT-derived blastocysts were screened for chromosomes by next-generation sequencing (NGS). Then, cryopreserved PB1T was carried out in one woman with a history of several cycles of extensive embryo fragmentation, and the blastocysts derived from PB1T were screened for aneuploidy but not transferred to the patient. RESULTS: There were no significant differences in the rates of normal fertilization and blastocyst formation between fresh and cryopreserved PB1T and control oocytes. Of the three fresh and three cryopreserved PB1T-derived blastocysts, two and one blastocysts exhibited normal diploidy respectively. In contrast, 17 PB2 transfers yielded 16 two pronuclei (2PN) zygotes with one normal and one small-sized pronucleus each and no blastocyst formation. In the female patient, 18 oocytes were inseminated by ICSI in the fourth cycle and the PB1s were biopsied. Although the embryos developed from the patient's own oocytes showed severe fragmentation, the oocytes reconstructed after PB1T produced three chromosomally normal blastocysts. CONCLUSIONS: Normal blastocysts can develop from human reconstructed oocytes after PB1T. The application of the first PB transfers may be beneficial to patients with a history of poor embryo development and excessive fragmentation.

Abnormalities in centrosome number in human embryos and embryonic stem cells.

Chromosomal abnormalities are common in human embryos. Previous studies have suggested links between centrosome number and chromosome abnormalities, but information regarding abnormalities in centrosome number in human embryos is limited. We analyzed abnormalities in centrosome number in human embryos and embryonic stem cells (hESCs). Following normal fertilization, supernumerary centrosomes were present at rates of 7.3% in two-pronucleus (2PN)-stage zygotes and 6.5% in first-cleavage zygotes. Supernumerary centrosomes were also detected in 24.4% of blastomeres from 60% of embryos derived from 2PN zygotes. Conversely, in mono- (1PN) and tri-pronucleus (3PN) zygotes, the frequency of abnormal centrosome number increased substantially at first cleavage. Rates in blastomeres of Day-3 embryos, however, were about the same between embryos derived from 1PN and 2PN zygotes, whereas abnormalities in centrosome number were higher in those from 3PN zygotes. By comparison, the rate of abnormal centrosome numbers in hESCs was 1.5-11.2%. Thus, abnormalities in centrosome number existed in human zygotes and cleaved embryos-especially those resulting from aberrant fertilization-but the frequency of such abnormalities was lower in hESCs derived from these embryos. These findings identify a source of the chromosomal instability in human embryos and hESCs, and highlight new safety issues for human assisted reproductive technology. Mol. Reprod. Dev. 83: 392-404, 2016. © 2016 Wiley Periodicals, Inc.

A novel modified ultra-long agonist protocol improves the outcome of high body mass index women with polycystic ovary syndrome undergoing IVF/ICSI.

In an attempt to evaluate the effectiveness of a novel modified ultra-long agonist (ULA) protocol on polycystic ovary syndrome (PCOS) patients undergoing in vitro fertilisation (IVF)/intracytoplasmic sperm injection (ICSI), a retrospective study of 499 women employed with either ULA or conventional long agonist (LA) protocol was analyzed. In high BMI group (>25 kg/m²), the ULA protocol yielded significant higher clinic pregnancy rate (PR) (70.2% versus 50.8%, p < 0.05), implantation rate (52.7% versus 35.7%, p < 0.05) and live birth rate (63.8% versus 39.0%, p < 0.05) when compared with LA protocol. In low BMI group (≤25 kg/m²), the ULA protocol also demonstrated a higher clinic PR (70.8% versus 59.5%, p < 0.05) whereas implantation rate and live birth rate are comparable. Within ULA protocol, the clinic PR, implantation rate and live birth rate are similar between high and low BMI patients. Similarly, the clinic PR and live birth rate demonstrated no significant difference within LA group but there is a significant lower implantation rate (35.7% versus 63.9%, p < 0.05) observed in high BMI patients. No difference in miscarriage rate and severe OHSS rate was found among all groups. In conclusion, ULA protocol benefits the IVF outcomes of PCOS patients with high BMI status.

Karyotype determination and reproductive guidance for short stature women with a hidden Y chromosome fragment.

Two unrelated couples came to the Reproductive and Genetic Hospital of Citic-Xiangya to ask for reproductive guidance. One couple had an affected son and the other couple had secondary infertility. Conventional GTG banding showed that the women in both couples had a 46,X,add(X)(p22) karyotype. Further molecular cytogenetic studies showed that both women had a 46,X,der(X)t(X;Y)(p22;q11.2) karyotype and that the affected boy had inherited the derivative X chromosome, which resulted in an Xp contiguous gene syndrome. After an assessment of reproductive risk, the first couple conceived naturally and opted for prenatal diagnosis (PND) by amniocentesis. No abnormal karyotypes were found for the twin pregnancy and healthy twin girls were born after a full-term normal pregnancy. The second couple chose to undergo IVF with preimplantation genetic diagnosis (PGD). Two PGD cycles were performed by fluorescence in-situ hybridization. In the first PGD cycle, all three embryos had abnormal hybridization signals. In the second cycle, a male embryo with normal hybridization signals was transferred into the womb and a normal pregnancy was achieved. The results show the importance of detecting the derivative chromosome followed by PND or PGD if a woman carries an Xp;Yq translocation.

ERBB2-induced inflammation in lung carcinogenesis.

ERBB2/HER2/NEU, a member of the epidermal growth factor receptor family, is overexpressed in more than 25 % of non-small cell lung cancer and is considered to be a significant and independent prognostic factor in lung cancer. Here we generated a lung specific HER2 overexpressing transgenic mouse model. In this model, HER2 was driven by the human surfactant protein-C promoter to investigate the role of the HER2 oncogene in pulmonary carcinogenesis and progression. Notably, significant pathological changes, including lymphocyte infiltration and mesenchymal cells hyperplasia, were found in the lung tissue of transgenic mice aged from 4 to 12 months. The occurrence and severity of those lesions increased as the mice aged. Some inflammatory factors, such as tumor necrosis factor, interleukin 1 and interleukin 6, were upregulated in lung tissue of transgenic mice compared with that of wild-type mice, implying that long-term HER2 overexpression could induce serious lung inflammation and some precancerous lesions. This model would be useful for studying the mechanism of HER2 involvement in lung carcinogenesis and for understanding the relationship between carcinogenesis and inflammation.

Dynamic distribution of NuMA and microtubules in human fetal fibroblasts, developing oocytes and somatic cell nuclear transferred embryos.

BACKGROUND: The nuclear mitotic apparatus (NuMA) plays a central role in the assembly and maintenance of spindle poles. Somatic cell nuclear transfer (SCNT) studies on non-human primates have shown that meiotic spindle removal during enucleation causes depletion of NuMA and the minus-end-directed motor protein (HSET) from the ooplasm, and this in turn leads to failure of embryo development. To determine whether NuMA from somatic cells could compensate for NuMA loss during enucleation, the distribution of NuMA and microtubule organization were investigated in human fibroblasts, developing oocytes and SCNT embryos. METHODS: Human fetal fibroblasts, oocytes at various maturation stages and human embryos reconstructed by different SCNT methods were analyzed for NuMA and α-tubulin using immunofluorescent confocal microscopy. RESULTS: NuMA was detected in interphase nuclei of fibroblasts and oocytes. During mitosis and meiosis, NuMA relocated to the domain surrounding the two spindle poles. During the enucleation process, NuMA was removed along with the meiotic spindle. At 2 h after injection into a donor cell, transitory bipolar spindles were organized and NuMA was detected in the reformed poles. NuMA could be detected spreading uniformly across the nucleoplasm of one pseudo-pronucleus in SCNT embryos but was excluded from the nucleolus. Regardless of the method used for SCNT (enucleation-injection or injection-pronuclei enucleation), NuMA aggregated and relocated to the reformed spindle poles at metaphase of the first mitotic event. At interphase, NuMA relocated throughout the nucleus in developmentally arrested SCNT embryos. CONCLUSIONS: Our results show that donor cell nuclei contain NuMA, which might contribute to the maintenance of spindle morphology in SCNT embryos. Normal spindle and NuMA expression were found in human SCNT embryos at different developmental stages.