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.

Neonatal outcomes of live births after blastocyst biopsy in preimplantation genetic testing cycles: a follow-up of 1,721 children.

OBJECTIVE: To investigate whether blastocyst biopsy in preimplantation genetic testing (PGT) increases the risk of adverse neonatal outcomes. DESIGN: Retrospective cohort study. SETTING: University-affiliated center. PATIENTS: Live births after blastocyst biopsy combined with frozen ET (PGT group) and frozen blastocyst transfer after in vitro fertilization or intracytoplasmic sperm injection (control group). INTERVENTION(S): Blastocyst biopsy. MAIN OUTCOME MEASURE(S): Gestational age (GA), birth weight (BW), and rates of preterm birth (PB), very preterm birth (VPB), extreme preterm birth (EPB), low birth weight (LBW), very low birth weight (VLBW), and macrosomia. RESULT(S): No significant differences were observed in the sex ratio, GA, PB, VPB, EPB, BW, or rates of LBW, VLBW, and macrosomia between the PGT and control groups for either singletons or twins. However, the cesarean section rate of the PGT group was significantly higher than that of the control group for twins (adjusted odds ratio, 2.383 [1.079, 5.259]). Regarding fluorescence in situ hybridization-PGT neonates, neonatal outcomes, including GA, BW, and rates of PB, VPB, LBW, and VLBW, did not differ between the different groups of biopsied cells (≥10 group and <10 group) for either the grade B or grade C trophectoderm score subgroups; however, in the grade B trophectoderm score subgroup, the rate of boy babies in the ≥10 group was significantly higher than that in the <10 group (83.3% vs. 40.9%). The association between the number of biopsied cells and GA/BW was not statistically significant. CONCLUSION(S): Blastocyst biopsy may not add additional risk to neonatal outcomes when compared with a control group.

Clinical and neonatal outcomes of patients of different ages following transfer of thawed cleavage embryos and blastocysts cultured from thawed cleavage-stage embryos.

BACKGROUND: Frozen-thawed embryo transfer (FET) has become a routine procedure in assisted reproductive technology (ART). In FET, although blastocysts cultured from thawed cleavage-stage embryos are associated with better perinatal outcomes. it may increase cycle cancellation due to no suitable embryo to transfer. The overall clinical outcomes following transfer of thawed cleavage-stage FET and blastocysts cultured from thawed cleavage-stage embryos in young and advanced age patients remains unclear. Therefore, we aimed to identify the optimal FET strategy in young and advanced age women who undergo FET. METHODS: This retrospective study included 16,387 thaw cycles. We retrospectively analyzed data of couples who had completed the first FET cycle. Two FET strategies were studied: transfer of thawed cleavage-stage embryos (strategy A) or blastocysts cultured from thawed cleavage-stage embryos (strategy B). The clinical and neonatal outcomes of two FET strategies were compared in young (<35 years) and advanced (≥35 years) age women. RESULTS: In young women, the clinical outcomes per transfer cycle were better in strategy B than strategy A. While the clinical pregnancy (59.29%, 52.60%) and live birth rates (49.37%, 43.88%) per thaw cycle were significantly higher in strategy A than in B. In women of advanced age, the clinical outcomes per transfer cycle were still better in strategy B than in A, and the clinical pregnancy (36.44%, 39.66%) and live birth rates (25.70%, 30.00%) per thaw cycle were significantly higher in strategy B than in A. CONCLUSIONS: FET of blastocysts cultured from cleavage-stage embryos showed higher efficiency for per transfer cycle whether in younger or advanced age women. Whereas, when cycle cancellations due to no suitable embryo to transfer were considered, cleavage-stage FET was found to be more suitable for younger women, while FET of blastocysts cultured from cleavage-stage embryos was better suited for women of advanced age.

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.

Inner cell mass incarceration in 8-shaped blastocysts does not increase monozygotic twinning in preimplantation genetic diagnosis and screening patients.

BACKGROUND: The use of assisted reproductive technology (ART) has been reported to increase the incidence of monozygotic twinning (MZT) compared with the incidence following natural conception. It has been hypothesized that splitting of the inner cell mass (ICM) through a small zona hole may result in MZT. In this study, using a cohort of patients undergoing preimplantation genetic diagnosis/screening (PGD/PGS), we compared the clinical and neonatal outcomes of human 8-shaped blastocysts hatching with ICM incarceration with partially or fully hatched blastocysts, and attempted to verify whether this phenomenon increases the incidence of MZT pregnancy or negatively impact newborns. METHODS: This retrospective study included 2059 patients undergoing PGD/PGS between March 1, 2013, and December 31, 2015. Clinical and neonatal outcomes were only collected from patients who received a single blastocyst transfer after PGD/PGS (n = 992). A 25- to 30-µm hole was made in the zona of day 3 embryos by laser. The blastocysts were biopsied and vitrified on day 6. The biopsied trophectoderm (TE) cells were analyzed using different genetic methods. One tested blastocyst was thawed and transferred to each patient in the subsequent frozen embryo transfer cycle. All the biopsied blastocysts were divided into three types: 8-shaped with ICM incarceration (type I), partially hatched without ICM incarceration (type II), and fully hatched (type III). ICM/TE grading, clinical and neonatal outcomes were compared between the groups. RESULTS: The percentage of grade A ICMs in type I blastocysts (22.2%) was comparable to that in type III blastocysts (20.1%) but higher than that in type II blastocysts (4.5%). The percentage of grade A TEs in type I blastocysts (4.2%) was comparable to that in type II (3.6%) but lower than that in type III (13.5%). There were no significant differences in clinical pregnancy, MZT pregnancy, miscarriage, live birth, MZT births, and neonatal outcomes between the groups. CONCLUSIONS: Compared to partially and fully hatched blastocysts, 8-shaped blastocysts with ICM incarceration showed relatively higher ICM and lower TE grades. ICM incarceration in 8-shaped blastocysts does not increase the incidence of MZT and has no negative effects on newborns in PGD/PGS patients.

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.

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.

A comparative analysis of the zona pellucida birefringence of fresh and frozen-thawed human embryos.

The cryopreservation of human embryos is thought to induce alteration in the glycoprotein matrix and lead to zona change. However, this assumption has been full of controversies till now. The objective of this study was to evaluate the effect of cryopreservation on zona pellucida of human embryos. Fresh (n=106, from 40 patients) and frozen-thawed embryos (n=123, from 40 patients) were obtained from consenting patients who received conventional IVF and ICSI treatment. The birefringence of zona pellucida in human fresh and frozen-thawed embryos was imaged and quantitatively analyzed using polarized light microscopy before embryo transfer. There was no significant difference in retardance and thickness of the zona pellucida multilaminar structure between the two groups. Pregnancy and implantation rates of transferred fresh and frozen-thawed embryos were also compared. No significant difference was found in the rates of clinical pregnancy (47.5 vs 37.5%) and implantation (24.5 vs 23.2%) between the two groups. This study suggests that there is no significant change in the zona pellucida birefringence of human embryos before and after cryopreservation.

Analysis of the first mitotic spindles in human in vitro fertilized tripronuclear zygotes after pronuclear removal.

Tripronuclear zygotes (3PN) occur in about 5% of cases in human IVF programmes. Human 3PN zygotes derived from a conventional IVF programme may contain not only the extra male pronucleus but also a supplementary centriole. Researchers have tried to restore diploidy by removing the extra male pronucleus of the tripronuclear zygote. However, it is still unknown whether the procedure can remove the supernumerary centriole. The objective of this study was to evaluate the safety of this manipulation by analysing the first mitotic spindles of 3PN zygotes that have undergone extra pronuclear removal. A controlled trial was conducted using human 3PN zygotes from conventional IVF treatment. In the experimental group, the assumed extra male pronuclei in the 3PN zygotes were removed. The first cleavage patterns and in vitro development were observed in both groups; polarized light microscopy and immunocytochemistry were used to analyse the first mitotic spindles. The blastocyst formation rate was significantly higher (P = 0.007) in the pronuclear-removed group (16.0%) than in the control group (4.5%). No significant differences were found between the groups in the first cleavage patterns and the distributions of the first mitotic spindle structure. This study suggests that, after extra pronuclei are removed, the developmental potential of human 3PN zygotes is improved. However, the abnormal patterns in the first mitosis are not corrected by this removal.

[Influence of patient age and the number of good-quality-embryos transferred on multiple gestation in in vitro fertilization and embryo transfer].

OBJECTIVE: To observe the influence of patient's age, and the number of transferred-good-quality-embryos on multiple gestation rates in in vitro fertilization and embryo transfer (IVF-ET) cycles. METHODS: In this retrospective study, a total of 4,395 patients who transferred fresh embryo between Jan 2004 and Nov 2006 was analyzed. According to the age, the patients were divided into 2 groups: aged < 35 (3,442 cycles) or aged >or= 35 (953 cycles). We regularly transferred 2 - 3 embryos. If the patients had only one embryo, one was transferred. And those patients who had only 2 embryos, even if they were more than 35 years old or it would be the second time for them to transfer, were transferred 2 embryos. The influence of female age and the number of good quality embryos transferred on the multiple gestation rates in IVF-ET cycle was analyzed. RESULTS: (1) The multiple gestation rate of the groups of 1 good quality embryo, 2 good quality embryos, or 3 good quality embryos transferred were 21.08% (35/166), 31.41% (413/1315), and 42.37% (75/177), respectively in women aged < 35, with a significant difference between them. The pregnancy rates of these groups were 29.64% (166/560), 51.63% (1,315/2,547), and 52.84% (177/335), respectively; there were no significant differences between 2 good quality embryos transferred group and 3 good quality embryos transferred group. (2) The multiple gestation rates of the groups of 1 good quality embryo, 2 good quality embryos, or 3 good quality embryos transferred were 19.51% (8/41), 20.65% (19/92), and 40.66% (74/182), respectively, in women aged >or= 35; there were no significant differences between 1 good quality embryo transferred group and 2 good quality embryos transferred group. The pregnancy rates of these groups were 19.07% (41/215), 33.70% (92/273), and 39.14% (182/465), respectively; there were no significant differences between 2 good quality embryos transferred group and 3 good quality embryos transferred group. (3) The pregnancy rate of the patients aged < 35 [48.17% (1,658/3,442)] was significantly higher than in women aged >or= 35 [33.05% (315/953)]. CONCLUSION: The transfer of 2 good quality embryos results in similar pregnancy rates and significantly reduced multiple gestation rates when compared to the transfer of 3 good quality embryos in women regardless of their ages.

[Research on the selection of embryos for transfer using a combined grading for embryo growth rate and morphology and zygote pronuclear morphology].

OBJECTIVE: To evaluate the value of using a combined grading for embryo growth rate and morphology and zygote pronuclear morphology, and to select embryos for transfer in clinical in-vitro fertilization (IVF)/intra-cytoplasmic sperm injection (ICSI). METHODS: In this retrospective study, a total of 2714 normal zygotes (2 pronuclei) from 434 treatment cycles with IVF/ICSI was analyzed between May 2003 and December 2003. These zygotes were divided into two groups (synchronous group and nonsynchronous group) according to the developmental synchrony of pronuclei. The developmental potential of zygotes from these two groups was observed. Embryos for transfer were selected initially by embryo growth rate and morphology, secondarily by zygote grade. The clinical pregnancy rates and implantation rates were compared according to whether the embryos from synchronous group were transferred. RESULTS: There were 2714 normal zygotes, the good-quality embryos (> or = 6 cells, grade I - II) rate of the synchronous group (41.88%, 743/1774) was significantly higher (P < 0.001) than the nonsynchronous group (33.94%, 319/940). Totally 395 cycles transferred at least one embryo from synchronous group, the clinical pregnancy rate was 47.85% (189/395) and implantation rate was 27.49% (273/993). There was no significant difference from 39 cycles which did not transfer embryos from nonsynchronous group. The clinical pregnancy rate was 43.59% (17/39) and implantation rate was 25.00% (21/84). CONCLUSION: The data indicate that there are no significant differences in pregnancy rates and implantation rates between different zygote grade when selecting embryos for transfer by combined grading.

[Preliminary exploration of the influence factors on amplification of single cell duplex-nested PCR].

OBJECTIVE: To explore the influence factors on amplification of single cell duplex-nested PCR. METHODS: The mutational loci region CD41-42 and IVS-II 654 of beta-globin gene were amplified by duplex-nested PCR with different combination of primers concentration, different Taq DNA polymerases, different neutralization buffers and with or without predenaturation at 98 degrees C before the PCR amplification in single lymphocyte or single blastomere, thus, to investigate the influence of these factors on the amplification efficiency of PCR. RESULTS: TaKaRa EX Taq was the most efficient Taq DNA polymerase among different Taq DNA polymerases; primer pair R1+F1 at final concentration of 0.25 micromol/L and R2+F2 at 0.3 micromol/L were the most efficient ones in amplification among different combinations of primers concentrations; the amplification efficiency in neutralization buffer-1 (200 mmol/L Tricine) was obviously higher than that of neutralization buffer-2 (900 mmol/L Tris-HCl, pH 8.3/300 mmol/L KCl/200 mmol/L HCl)(P<0.05); there were no remarkable differences of the amplification efficiency while using whether predenaturation at 98 degrees C before the single cell PCR amplification or not (P>0.05). CONCLUSION: There were remarkable differences of the amplification efficiency of single cell duplex-nested PCR while using different combination of primers concentrations, different Taq DNA polymerases, different neutralization buffers. However, predenaturation at 98 degrees C before the single cell PCR amplification could not improve the PCR amplification efficiency.

[Diagnosing achondroplasia by single cell nested-PCR].

OBJECTIVE: To research on the reliability of diagnosing achondroplasia (ACH) on single cell level and to provide a basis for preimplantation genetic diagnosis(PGD). METHODS: The high-frequency mutation region G380R of fibroblast growth factor receptor 3(FGFR3) gene was amplified by nested-PCR with single lymphocyte and single blastomere. The products of PCR were digested by restriction enzyme Bfm I, then the digested products were detected by 10% polyacrylamida gel electrophoresis(PAGE). RESULTS: The amplification success rate, allele dropout rate and correct diagnosis rate of single lymphocyte's PCR were 90.4%, 8.2% and 91.8%,respectively. The amplification success rate of single blastomere was 75.4%. CONCLUSION: The diagnosis of ACH by single cell nested-PCR is comparatively stable and reliable.