What was the fate of human embryos following long-term cryopreservation (≥12 years) and frozen embryo transfer?

STUDY QUESTION: Do human embryos survive long-term cryopreservation (CP) (≥12 years) and implant after frozen embryo transfer (ET)? SUMMARY ANSWER: Human embryos remain usable after long-term CP. WHAT IS KNOWN ALREADY: Several cohort studies have reported the live birth rate or neonatal outcomes of human embryos after CP for up to 5 years. Only a few case reports have described successful live births from human embryos after long-term CP up to 12 years. STUDY DESIGN, SIZE, DURATION: This retrospective observational study in China included 20 patients (128 embryos) from March 2016 to April 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: Twenty patients who had at least one live birth during their previous IVF/ICSI treatments and had surplus embryos cryopreserved were observed. Data concerning frozen embryo recovery, pregnancy and obstetric outcomes following frozen ET were recorded. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 128 embryos of 20 patients were observed. The embryo storage duration was 12.0-17.1 years, with a mean of 13.9 ± 1.73 years. In all, 115 embryos were thawed to transfer, with a survival rate of 74%. Sixty embryos were further cultured, which resulted in 20 blastocysts with a blastocyst formation rate of 33%. There were 21 cleavage-stage embryos and 13 blastocysts transferred in a total of 12 and 11 cycles, respectively, which resulted in one biochemical pregnancy, one first trimester miscarriage, two ectopic pregnancies, three singletons and one case of twins, with a clinical pregnancy rate of 25% (D3 ET) and 36% (blastocyst transfer) and a live birth rate of 17% (D3 ET) and 27% (blastocyst transfer). Two of the four patients who had live birth developed gestational diabetes mellitus. One of the five live births was a preterm delivery. LIMITATIONS, REASONS FOR CAUTION: The sample size was small due to the unique study population, and all the embryos underwent slow freezing. The fate of long-term cryopreserved embryos after vitrification is still unclear. WIDER IMPLICATIONS OF THE FINDINGS: The results provide evidence to support the use of embryos after extended CP to preserve patients' fertility. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the National Key Research and Development Programme of China (2016YC1000205) and the Guangzhou Scientific Programme (201508020006). None of the authors has any conflicts of interest to declare.

Pregnancy derived from human zygote pronuclear transfer in a patient who had arrested embryos after IVF.

Nuclear transfer of an oocyte into the cytoplasm of another enucleated oocyte has shown that embryogenesis and implantation are influenced by cytoplasmic factors. We report a case of a 30-year-old nulligravida woman who had two failed IVF cycles characterized by all her embryos arresting at the two-cell stage and ultimately had pronuclear transfer using donor oocytes. After her third IVF cycle, eight out of 12 patient oocytes and 12 out of 15 donor oocytes were fertilized. The patient's pronuclei were transferred subzonally into an enucleated donor cytoplasm resulting in seven reconstructed zygotes. Five viable reconstructed embryos were transferred into the patient's uterus resulting in a triplet pregnancy with fetal heartbeats, normal karyotypes and nuclear genetic fingerprinting matching the mother's genetic fingerprinting. Fetal mitochondrial DNA profiles were identical to those from donor cytoplasm with no detection of patient's mitochondrial DNA. This report suggests that a potentially viable pregnancy with normal karyotype can be achieved through pronuclear transfer. Ongoing work to establish the efficacy and safety of pronuclear transfer will result in its use as an aid for human reproduction.

[Effect of domestic highly purified urinary follicle stimulating hormone on outcomes of in vitro fertilization-embryo transfer in controlled ovarian stimulation].

OBJECTIVE: To investigate the effect of domestic urine-derived high-purity follicle- stimulating hormone (HP-FSH, Lishenbao) on the outcome of in vitro fertilization(IVF) embryo transfer (ET) in controlled ovarian stimulation (COS). METHODS: From 1 September 2010 to 31 March 2011, total of 3178 infertility patients from 14 Reproductive Center with IVF or intracytoplasmic sperm injection (ICSI) indications who accepted first IVF or ICSI cycle were studied retrospectively. Their causes of infertility include all infertility factors except ovulatory dysfunction infertility and uterine factor infertility. The only long luteal phase gonadotropin-releasing hormone agonist (GnRH-a) protocol was included. Patients were divided into 2 groups according to the type of follicle-stimulating hormone (FSH) agents used: 1932 cases in HP-FSH group and 1246 cases in recombinant FSH (rFSH)group. Patients in both groups were combined with human menopausal gonadotropin (hMG) at doses of 150 U when follicle with diameter reached to 14-16 mm. When 3 dominate follicle with diameter reached 18 mm, hCG at dose of 5000 to 10 000 U or recombinant hCG at dose of 250 µg was administered by intramuscular injection. After 34 to 36 hours, oocytes were obtained guided by ultrasound, then IVF-ET were underwent in their Reproductive Center. The primary endpoint was comparison of live birth rate between the two groups. The secondary endpoints were comparisons of clinical pregnancy rate, miscarriage rate, and implantation rate, as well as COS and IVF outcome between the two groups. RESULTS: (1) There were significantly differences in baseline characteristics of the patients between two groups. The mean age was elder(32 ± 4 versus 30 ± 4, P < 0.01) , the infertility duration was longer (5 ± 4 versus 5 ± 3, P < 0.01) , and antral follicle count (AFC) was less (11 ± 5 versus 13 ± 7, P < 0.01) in patients of HP-FSH group compared with those in patients of rFSH group. (2) As compared with rFSH, the total doses of gonadotropin needed was (2348 ± 1011) U in HP-FSH group versus (2022 ± 659) U in rFSH group, the number of oocytes 13 ± 6 in HP-FSH group and 14 ± 7 in rFSH group, the rate of embryo frozen cycle of 66.30% (1281/1932) in HP-FSH group and 74.88% (933/1246) in rFSH group, which all reached statistical difference (P < 0.01). However, there were no significant different implantation rate [30.49% (1111/3644) versus 32.45% (737/2271)] between two groups. The other clinical parameters did not show significant difference, including clinical pregnancy rate per started cycle [41.61% (804/1932) versus 41.97% (523/1246) ] , clinical pregnancy rate per ET cycle[46.58% (804/1726) versus 48.47% (523/1079)], live birth rate per started cycle[34.21% (661/1932) versus 34.19% (426/1246)], live birth rate per ET cycle [38.30% (661/1726) versus 39.48% (426/1079)], miscarriage rate[13.6% (109/804) versus 16.4% (86/523)], and moderate/severe ovarian hyperstimulation syndrome (OHSS) rate [5.80% (112/1932) versus 7.78% (97/1246)](P > 0.05).(3) Treatment cost: the cost of gonadotropins needed for the patients in HP-FSH group was lower than that in rFSH group (4005 ± 1650 versus 6482 ± 2095, P < 0.01). CONCLUSION: In IVF/ICSI treatment cycles, domestic HP-FSH has similar live birth rate and lower financial burden when compared with rFSH.

Preimplantation genetic diagnosis for α-and ß-double thalassemia.

PURPOSE: To evaluate the use of multiple displacement amplification (MDA) for preimplantation genetic diagnosis (PGD) of α- and ß-double thalassemia. METHOD: Whole genome of a single cell was directly amplified using MDA and its products were used as templates in fluorescent gap polymerase chain reaction (PCR) analysis of α-thalassemia and in PCR-reverse dot blot analysis, singleplex fluorescent PCR of ß-28 and CD17 mutation and HumTH01 for ß-thalassemia. RESULTS: 1) MDA from single cell could produce enough DNA templates for the detection of both α and ß-thalassemia; 2) The established MDA-PGD protocol for α- and ß-double thalassemia was successfully applied in PGD of six embryos, among which, three were transferred, but no pregnancy ensued. CONCLUSIONS: The use of MDA as a universal step allows for the simultaneous diagnosis of two or more hereditary defects.

[Clinical analysis of 100 preimplantation genetic diagnosis cycles].

OBJECTIVE: To investigate influence of chromosomal translocations on early embryo development and to evaluate the efficacy and feasibility of preimplantation genetic diagnosis (PGD) techniques through clinical analysis on PGD cycles. METHODS: Embryo development, efficacy of PGD and clinical outcome of 100 cycles were studied retrospectively, including 23 cycles with Robertsonian translocations, 19 cycles with reciprocal translocations, and 58 cycles for α-Thalassaemia. RESULTS: Among 354 embryos biopsied by PGD for translocations, 321 (90.7%) presented fluorescence in situ hybridization (FISH) results. The rate of normal/balanced embryos in the Robertsonian translocation was 38.3% (64/167), which was significantly higher than 20.8% (32/154) in the reciprocal translocation group. Amplification was achieved in 443 blastomeres from 537 embryos in Thalassaemia group, which given to an amplification efficiency rate of 82.5% (443/537). Totally, 140 normal homozygous, 112 heterozygotes and 155 affected homozygous embryos were identified, while 36 embryos had uncertain result. The successful diagnostic rate was 75.8% (407/537). After 3 days in the translocation groups, the rate of normal and/or balanced translocations in biopsed embryos with ≥7 cells was 34.4% (77/224), which was significantly higher than 19.6% (19/97) of biopsed embryos with <7 cells. After 4 days, the compaction rate in normal/balanced embryos was 59.4% (57/96), which was significantly higher than 34.2% (77/225) in imbalanced embryos significantly. Seventy-five embryos transferred in 37 cycles with translocations group led to clinical pregnancy rate of 27.0% (10/37), and 170 embryos transferred in 58 cycles with Thalassaemia got a clinical pregnancy rate of 43.1% (25/58). CONCLUSIONS: PGD can provide management efficiently for both chromosome translocations and Thalassaemia. Translocations might have slightly negative impact on embryo development before implantation.

Improved nude mouse models for green fluorescence human endometriosis.

AIM: To establish an improved noninvasive fluorescent animal model for endometriosis. MATERIAL AND METHODS: Adenovirus encoding enhanced green fluorescent protein (Ad-eGFP) was used to transfect primary culture endometrial glandular cells and stromal cells (purified cell transfection and mixed injection, Group 1) as well as endometrial fragments (tissues transfection and injection, Group 2). Transfection results were compared between the cells and tissues in vitro. The GFP-transfected cells suspension of Group 1 or endometrial fragments of Group 2, with similar weight, were injected into nude mice subcutaneously and noninvasively observed every 5 days until day 15 (Subgroup 1, N = 5), day 20 (Subgroup 2, N = 5) or day 25 (Subgroup 3, N =11). The positive rates and duration times of the fluorescent lesions were calculated. RESULTS: After 18 h of incubation, glandular cells and stromal cells all had higher GFP-positive rates. In vivo imaging showed that the GFP positive rates of Group 1 were significantly higher than those of Group 2. The fluorescent-positive durations of Groups 1 and 2 were 23.636 ± 4.523 days and 5.909 ± 5.394 days, respectively (P < 0.001). In vivo analysis demonstrated that on days 15, 20, and 25, there were more typical lesions and fluorescent-positive lesions formed in Group 1 and that the lesion weight in Group 1 was greater. The structures of the lesions were all identified as human origin. CONCLUSION: A noninvasive animal model for endometriosis created by subcutaneous injection of an Ad-eGFP-transfected endometrial glandular and stromal cells suspension had higher a positive rate, longer duration time of fluorescent imaging and greater lesion weight.

[Evaluation of the fidelity of multiple displacement amplification from small number of cells].

OBJECTIVE: To evaluate the fidelity of multiple displacement amplification (MDA) from small number of cells (1-10 cells) by 10K 2.0 SNP mapping array. METHODS: A fibroblast cell line (Tri-18; GM02732, 47, XY, +18) was used as the template, and 6 groups were set up in the study. Groups A and B were positive and negative control, respectively; groups C-F were experimental groups involving the MDA products from 1, 2, 5 and 10 cells respectively. In combination of single nucleotide polymorphism (SNP) array, the product of each group was assessed based on the genome coverage, loss of heterozygosity (LOH) rate and allele dropout (ADO) rate. RESULTS: The nonspecific product of negative control presented an average call rate of 3.2%. The genome coverage of the MDA product increased from 86.4% to 96.4% with the increasing number of template from 1 to 10 cells, while the LOH rate and ADO rate decreased significantly (P<0.05). CONCLUSION: MDA is a highly efficient and reliable method for whole genome amplification. The fidelity of MDA will be improved significantly with the increasing number of template cells. 10K 2.0 SNP mapping array is a quick, accurate and comprehensive method to evaluate the fidelity of amplified DNA products, but the ADO SNPs should be distinguished from those of preferential amplification from the LOH loci to avoid errors.

Evaluation of genome coverage and fidelity of multiple displacement amplification from single cells by SNP array.

The scarce amount of DNA contained in a single cell is a limiting factor for clinical application of preimplantation genetic diagnosis mainly due to the risk of misdiagnosis caused by allele dropout and the difficulty in obtaining copy number variations in all 23 pairs of chromosomes. Multiple displacement amplification (MDA) has been reported to generate large quantity of products from small amount of templates. Here, we evaluated the fidelity of whole-genome amplification MDA from single or a few cells and determined the accuracy of chromosome copy number assessment on these MDA products using an Affymetrix 10K 2.0 SNP Mapping Array. An average coverage rate (86.2%) from single cells was obtained and the rates increased significantly when five or more cells were used as templates. Higher concordance for chromosome copy number from single cells could be achieved when the MDA amplified product was used as reference (93.1%) than when gDNA used as reference (82.8%). The present study indicates that satisfactory genome coverage can be obtained from single-cell MDA which may be used for studies where only a minute amount of genetic materials is available. Clinically, MDA coupled with SNP mapping array may provide a reliable and accurate method for chromosome copy number analysis and most likely for the detection of single-gene disorders as well.

Expression of certain HLA-I types in cleavage-stage embryos.

This study examined the expression of human leukocyte antigen (HLA)-G and HLA-I (which includes HLA-A, -B, -C, -E and -F, but is without HLA-G) in the cleavage embryo and its supernatant, and related the results to embryo development including growth rate and grade. In total, 136 day-3 cleavage embryos were used for detection of HLA-G and 24 embryos for HLA-I without HLA-G by immunohistochemistry. The expression of HLA-I was examined by western blot in the lysates of a further 63 day-3 cleavage embryos; soluble HLA-I in the culture supernatant of embryos with detectable HLA-I was also examined by western blot. It was found that 90 of 136 (66.2%) cleavage embryos expressed HLA-G, whereas 23 of 24 (95.8%) embryos expressed HLA-I without HLA-G. HLA-G expression typically showed an even and symmetrical pattern of distribution in each blastomere. HLA-I without HLA-G in cleavage-stage embryos is typically scattered around the blastomere surface. The expression of HLA-G but without HLA-I in cleavage-stage embryos was significantly associated with embryo grade (P < 0.001) and cell number (P = 0.03). In conclusion, HLA-I is expressed on day-3 cleavage embryos, and HLA-G expression on preimplantation embryos is related to embryo development, including embryo growth rate and embryo grade.

Preimplantation genetic diagnosis for alpha-thalassaemia in China.

PURPOSE: To report the usage of PGD for alpha-thalassaemia with the - -(SEA) genotype. METHOD: A PGD protocol using fluorescent gap PCR was performed for 51 cycles on 43 couples with the - -(SEA) genotype. Allele drop-out and amplification failure rates were retrospectively analyzed. RESULTS: A total of 472 embryos were biopsied. Amplification was achieved in 390 blastomeres, accounting for an amplification rate of 82.6%. In total, 120 wild-type, 94 heterozygotes and 140 homozygous mutant embryos were diagnosed. The successful diagnosis rate was 75.0%. The ADO rate in 49 blastomeres from six donated embryos was 16.4%. One hundred and fifty four embryos were transferred, resulting in 25 clinical pregnancies with an implantation rate of 24.0%. CONCLUSIONS: Single-round fluorescent gap PCR is a feasible and effective strategy in the PGD for alpha-thalassaemia with the - -(SEA) genotype.

Bulk vitrification of human embryonic stem cells.

BACKGROUND: The traditional vitrification method cannot keep up with the increased culture and propagation efficiency required to cryopreserve large quantities of vigorously proliferating human embryonic stem (HES) cells. In this study, we describe a newly invented vitrification carrier for cryopreserving large amount of HES cells and evaluate whether this bulk vitrification (BV) method is as effective as the popular open-pulled straw (OPS) vitrification method. METHODS: HES cell clumps were harvested after passage and transferred to a cell strainer; only those clumps with a diameter more than 70 microm were included in the study and randomly selected to be cryopreserved by the BV method, OPS vitrification or slow freezing method. HES cell survival, growth and pluripotency were analyzed after thawing. RESULTS: Bulk vitrification method with cell strainer could cryopreserve 136 +/- 23.4 cell clumps at one time (round), which was 30 times as high as those for OPS method (4 +/- 1.5). After thawing, bulk-vitrified HES cells exhibited high survival rate up to 94.3%, comparable with the OPS method. All surviving cell clumps generated HES cell colonies. Teratomas comprising all three primordial germ layers were formed in severe combined immunodeficient mice after subcutaneous injection of post-thawed, bulk-vitrified HES cell clumps, confirming pluripotency. CONCLUSIONS: This new BV method could cryopreserve a large quantity of HES cell clumps at one time, which not only would satisfy routine cryopreservation of HES cell during daily culture process but also guarantee researchers have large quantity of efficiently cryopreserved HES cells ready for a scheduled study at any time.

Effects of cilostamide and forskolin on the meiotic resumption and embryonic development of immature human oocytes.

BACKGROUND: In an attempt to allow for acquisition of oocyte cytoplasmic maturation, PDE3 specific inhibitor, cilostamide and adenylate cyclase activator, forskolin were used to extend pre-maturation culture of immature human oocytes. METHODS: Cumulus-oocyte complexes retrieved from unstimulated ovaries were continuously cultured under 20 microM cilostamide or 50 microM forskolin, alone or in combination for 6, 12, 24 or 48 h, respectively. Levels of intercellular gap junction communication (GJC) and maturational status were examined at these designated time points. Metaphase II oocytes obtained following 54 h biphasic culture (with meiotic inhibitors from 0 to 24 h, no meiotic inhibitors from 24 to 54 h) were subject to intracytoplasmic sperm injection and embryos were cultured for five more days. RESULTS: Both cilostamide and forskolin delayed spontaneous meiotic progression after continuous culture with immature human oocytes. Combined treatment of cilostamide and forskolin significantly lowered the rates of germinal vesicle breakdown (GVBD) at 6, 12, 24 or 48 h after meiotic inhibitory culture, when compared with the control (all P < 0.05). A delay of 6 h for the loss of GJC was also observed under the combined treatment of cilostamide and forskolin. The fertilization rate was significantly higher under the combined treatment of cilostamide and forskolin than that of the control. Although the rates of oocyte maturation and embryo cleavage were similar among groups, there was a slight but non-significant increase in blastocyst formation rate with the treatment of cilostamide and forskolin. CONCLUSIONS: Combined treatment of cilostamide and forskolin positively influences oocyte developmental competence by exhibiting a synergistic effect on the prevention of GJC loss and resumption of meiosis.

[Variance of serum prolactin in controlled ovarian stimulation].

OBJECTIVE: To investigate the variance of peripheral blood prolactin (PRL) in controlled ovarian stimulation. METHODS: Seventy-two patients, with totally 106 cycles receiving a long protocol of gonadotropin-releasing hormone agonist combined with gonadotropin (Gn) were randomly enrolled in this retrospective study. During controlled ovarian stimulation, peripheral blood hormones were measured by chemiluminescent microparticle immunoassay. RESULTS: Prolactin was positively correlated with estradiol (r = 0.5897, P < 0.01) while there was no significant correlation between luteinizing hormone and PRL Progesterone had a positive relation with prolactin (r = 0.1412, P < 0.01). CONCLUSIONS: During controlled ovarian stimulation, prolactin secretion is not affected by Gn but may be stimulated by estradiol. Progesterone has a positive relation with prolactin.

[Efficiency comparison between two preimplantation genetic diagnostic methods for chromosomal translocation carriers].

OBJECTIVE: To compare the diagnostic efficiency between blastomere preimplantation genetic diagnosis (PGD) and polar body PGD for chromosomal translocation carriers. METHODS: Group A had 8 cycles using whole painting probes for the first polar body diagnosis, while group B had 29 cycles using two subtelomeric probes and one centromeric probe for the blastomere diagnosis. RESULTS: The fertilization rate of group A was significantly lower than group B [66.1% (72/109) vs 85.2% (304/357), P < 0.05]. There was no significant difference in the successful biopsy rate between two groups. However, group A had a significantly higher loss rate during fixation and higher no signal rate after fluorescence in situ hybridization [FISH, 9.6% (12/104) vs 1.6% (4/252), 11.2% (10/89) vs 3.0% (7/233)]. Totally, the diagnostic efficiency in group A (72.5%, 79/109) was significantly lower than that in group B (89.8%, 230/256, P < 0.05). Although both the clinical pregnancy rate (3/7) and implantation rate (22.2%, 4/18) of group A were higher, the differences were not statistically significant (P > 0.05). CONCLUSION: Both methods can be used efficiently in the PGD for chromosomal translocation carriers. Blastomere PGD has a higher diagnostic rate.

[Analysis of chromosome mosaicism in preimplantation embryos by using 2 sequential rounds of fluorescence in situ hybridization].

OBJECTIVE: To investigate the mechanism and factors affecting mosaicism in human preimplantation embryos by using 2 sequential rounds of fluorescence in situ hybridization(FISH). METHODS: Totally 51 normal fertilized embryos, which were not suitable for embryo transfer and cryopreservation, were analyzed on day 3 after fertilization by using two sequential rounds of FISH. Chromosomes 13, 16, 18, 21, 22, X and Y were analyzed. RESULTS: Among 51 embryos, 16 (31.4%) were mosaic, 12 (23.5%) were chaotic, and the remaining were either normal (27.5%) or non-mosaic abnormal (17.6%). The incidence of mosaic embryos was related to embryo developmental stage, for the incidence of mosaicism increased from 12.5% in embryos

[The clinical application of whole chromosome painting probes in preimplantation genetic diagnosis for translocation carriers].

OBJECTIVE: To make preimplantation genetic diagnosis (PGD) for female translocation carriers by analyzing first polar bodies (1PBs) with whole chromosome painting probe (WCP). METHODS: WCP was used in fluorescence in situ hybridization (FISH) analysis of 1PBs for four female Robertsonian carriers presented for PGD with 45 XX, der(13;14)(q10;q10) karyotype. All the patients underwent ovarian stimulation and during 6 h after oocyte retrieval 1PBs were biopsied and WCP were used in FISH. On day 3 after fertilization embryos diagnosed as normal or balanced were transferred. RESULTS: A total of 61 oocytes were collected in 4 PGD cycles. Of the 54 matured oocytes, 50 were biopsied and 45 were fixed successfully. Results were obtained in 40 1PBs. Overall, 74.1% (40/54) oocytes were diagnosed. The fertilization rate and good embryo rate were 64.8% (35/54) and 65.7% (23/35) respectively. Two clinical pregnancies were obtained. One patient delivered a normal female baby with karyotype 46, XX in June 2006. For another patient, the fetus spontaneously aborted at 9th week of pregnancy with karyotype of 45, X confirmed by amniotic villus diagnosis. CONCLUSION: WCP can differentiate normal, balanced and unbalanced oocytes accurately and can be used as an efficient PGD method for female carriers of translocation.

[Meiotic spindle location and embryo development of in vivo and in vitro matured human oocytes].

OBJECTIVE: To analyze the relationship between meiotic spindle location and embryo developmental potential of in vivo and in vitro matured human oocytes. METHODS: One hundred and thirty-four in vivo matured oocytes and 45 in vitro matured oocytes were observed with polscope at the time of intracytoplasm sperm injection (ICSI). RESULTS: Meiotic spindle was detected in 83.6% (112/134) and 82.2% (37/45) in in vivo and in vitro matured oocytes respectively. In vivo matured oocytes which showed a minimal angle (0-5 degrees ) between the meiotic spindle and the first polar body had a higher fertilization rate (93.3%) than the others. The frequency of the oocytes which had a 0-5 degrees spindle angle in in vivo and in vitro matured oocytes was 22.4% and 17.8%, respectively, and that of oocytes which had a 6 degrees - 45 degrees, 46 degrees-90 degrees and > 90 degrees spindle angle was 55.2% vs 51.1%, 3.0% vs 8.9%, and 3.0% vs 4.4%. No significant difference was found between them. No relationship was found between the position of meiotic spindle and embryo quality. CONCLUSIONS: There is some relationship between the angle of the meiotic spindle with the first polar body and fertilization rate. No significant difference is found in the position of the meiotic spindle between in vivo and in vitro matured human oocytes.

[Characteristics of IGF-II gene imprinting in twin placentas].

OBJECTIVE: To compare insulin-like growth factor II (IGF-II) gene imprinting in twin placentas with singleton ones and to determine whether imprinting was influenced by assisted reproductive technology, zygosity and fetal sex. METHODS: One hundred and sixty cases of twin placentas and 42 cases of singleton ones were recruited. Allele-specific IGF-II expression was determined by reverse transcription-PCR combined with analysis of an Apa I-sensitive restriction fragment length polymorphism. RESULTS: Although the incidence of IGF-II imprinting loss was higher in normal twin placentas than in singleton ones (20.6% vs 8.7%), there was no statistical significance. There were no significant differences between twins conceived by assisted reproductive technology and those conceived spontaneously (17.9% vs 24.4%), and between dizygotic and monozygotic twins (22.4% vs 16.7%). The incidence of IGF-II imprinting loss in placenta of female twins was statistically higher than that of male ones (26.4% vs 9.8%). CONCLUSION: The risk of IGF-II gene imprinting loss is higher in female twins and has no relationship with assisted reproductive technology and zygosity.

[Sperm sex chromosome analysis and preimplantation genetic diagnosis of patients with sex chromosome anomalies].

OBJECTIVE: To investigate the constitution of abnormal spermatozoa from patients with sex chromosome anomalies. METHODS: Triple color fluorescence in situ hybridization (FISH) was used to determine the sex chromosome constitution of spermatozoa from three patients with sex chromosome anomalies (case 1:46,XY/47,XXY, case 2:45,XO/46,X,Yqh-, case 3:47,XXY). The preimplantation genetic diagnosis (PGD) was performed to case 2. RESULTS: An increased ratio (2.05 vs 1) of X-bearing to Y-bearing spermatozoa was only observed in case 2, who also had an increased incidence of total abnormal spermatozoa (29.71%). An increased incidence of total abnormal spermatozoa (4.91%) was also observed in case 3. Among the constitution of abnormal spermatozoa, case 2 had the increased proportions of XY18 disomy, O18 monosomy and XO monosomy, while case 3 had an increase proportion of XY18 disomy (1.87%). PGD was performed to case 2 and one embryo with XX1818 was selected for implanting. CONCLUSION: Using FISH to detect the sperm sex chromosomes in patients with sex chromosome anomalies can provide the useful information to evaluate the risk of sex chromosome anomalies in preimplantation embryos.