人类常规体外受精中3原核(3PN)受精卵及胚胎的染色体组成

目的:分析人类常规体外受精(in vitro fertilization,IVF)中3原核(tripronuclear,3PN)受精卵及胚胎的染色体组成。方法:收集IVF来源的3PN受精卵及胚胎,与秋水仙素共培养14～20 h后制备染色体。结果:共收集到46枚3PN受精卵,277枚3PN来源的胚胎,核型分析显示:受精卵多为三倍体,胚胎可为近单倍体、近二倍体、近三倍体、多倍体等。结论:人类IVF来源3PN受精卵多为三倍体,3PN胚胎染色体紊乱。     

Chromosome analysis of single- and multipronucleated human zygotes proceeded after the intracytoplasmic sperm injection procedure

Purpose: Fertilization of an egg by injection of a single spermatozoon into the cytoplasm has been shown to be an effective procedure to obtain a pregnancy in human in vitro fertilization. This, as one of the most invasive micromanipulation techniques, has generated concern about inducing embryo abnormalities. The objective of this study was to obtain insight into the chromosomal constitution of zygotes proceeded after the intracytoplasmic sperm injection procedure. Methods: For this purpose the first cleavage division of 33 single- and 16 multipronucleated zygotes proceeded after the intracytoplasmic sperm injection procedure was cytogenetically analyzed. Results: Chromosome spreading permitted adequate karyotyping in 28 (84.8%) single-pronucleated zygotes. Among these, 19 (67.9%) were haploid, 5 (17.9%) hypohaploid (n=20–22), and 4 (14.3%) hyperhaploid (n=24–25). The overall rate of aneuploidy found here for single-pronucleated zygotes was 32.1%. In the 23 (82.1%) analyzed single-pronucleated zygotes, besides single mitotic complements, an intact sperm head or sperm nuclei structure has been found, indicating the maternal origin of these chromosomes. Conclusions: Ten digynic zygotes with three pronuclei and six zygotes with more than three pronuclei were identified after injecting a single spermatozoon, representing 3.2% of all the fertilized oocytes. Aneuploid chromosome complements were found in three of seven tripronuclear zygotes and each one exhibited a hypo-, hyper-, and haploid complement (23,X, 21,X,–A,–B, 25,X,+A.+B; 23,Y, 22,X,–D, 24,X,+D; 23,X, 22,X,–G, 24,X,+G). The absolute difference in the number of chromosomes between each of these two imbalanced corresponding haplotypes was the same and this difference was caused by the chromosomes belonging to the same groups of karyotype. Of the remaining four tripronuclear zygotes, three had triploid and one had diploid numbers of chromosomes. Furthermore, five zygotes having more than three pronuclei at the first cleavage division displayed severely depleted chromosome complements. The majority of imbalanced multipronuclear zygotes was found after delay of the microinjection, suggesting that aging of oocytes might be the reason for their abnormal chromosomal arrangements.     

体外受精后多原核受精卵的移植价值

目的:对体外受精周期中产生多原核受精卵运用荧光原位杂交技术(FISH)进行非整倍体率检测,分析其移植价值。方法:应用FITC、Texas red标记的X/Y双色染色体着丝粒部位探针对体外受精(IVF)和单精子显微注射(ICSI)周期中的三原核受精卵(3PN)进行荧光原位杂交和分析。结果:分析有杂交信号的3PN卵子68个,IVF后3PN的三倍体率为92.31%,二倍体率为3.85%,单倍体率为3.85%;共存在3种三倍体核型:XXX、XXY、XYY。ICSI后3PN 的三倍体率为70.83%,二倍体率为25%,单倍体率为4.17%;共存在2种三倍体核型:XXX、XXY。结论:IVF后3PN卵子中多余的原核多为精子来源,没有移植价值;ICSI后3PN中多余的原核非精子来源,ICSI后3PN中二倍体率与IVF相比显著增高,在患者ICSI后无2PN卵子时可考虑行植入前遗传学诊断。     

Chromosomal preparations of human triploid zygotes and embryos fertilized in vitro

Forty-eight zygotes with more than two pronuclei were identified after in vitro fertilization, representing 6.1% of all fertilized oocytes. The chromosome preparations from pronuclear stage to the cleaved human embryos were examined. Prophase was found in eight out of ten zygotes. The spreading of chromosomes allowed an adequate counting in only two cases. Six of the eight preparations displayed a late prophase. In this stage each haploid group of chromosomes can be analysed separately. Kariogamy usually occurred 4 to 5 h after the pronuclei had disappeared, and polyploid number of chromosomes were found in well-spread metaphases. The chromosomal preparations were made for eleven human embryos arising from zygotes with three pronuclei. Out of ten preparations, where the chromosomes could be counted, seven embryos (70%) contained hypodiploidic groups of chromosomes. In two of the cases, however, triploid metaphases were found, and in the last one a triploid/diploid mosaicism.     

Rapid chromosome detection in human gametes,zygotes, and preimplantation embryos using the PRINS technique

Purpose: The analysis of the chromosomal constitution of human gametes and embryos is of particular importance for investigation of aneuploidy occurrence and diagnostic purposes. The PRINS method constitutes an alternative to FISH for in situ chromosomal identification. We have adapted this method to human gametes, zygotes, and preimplantation embryos. Results: Chromosome-specific labeling was obtained in gametes, zygotes, and isolated blastomeres. Simultaneous detection of two or three chromosomes can be completed in less than 3 hr using fluorochrome-labeled nucleotides. Conclusions: The PRINS technique appears to be more efficient than FISH for detection and discrimination of -satellite DNA sequences. The present study demonstrates the usefulness of PRINS for chromosomal screening and preimplantation diagnosis.     

IVF和ICSI中多原核受精卵的发育及遗传多态性分析

目的:了解IVF和ICSI治疗周期中多原核受精卵的发育情况及其基因座遗传多态性。方法:分别收集IVF和ICSI治疗中的废弃多原核(≥3PN)受精卵315个和67个,进行体外培养,观察比较其发育能力,并复合扩增多原核来源的1株胚胎干细胞和2个囊胚细胞DNA的15个短串联重复序列(STR)基因座,利用3100遗传分析仪对其进行STR基因座多态性检测。结果:IVF组和ICSI组的多原核受精卵卵裂率无显著性差异(P>0.05),但IVF组胚胎停育率和囊胚形成率显著低于ICSI组(P<0.01)。STR基因座多态性检测显示,IVF组3PN受精卵来源的胚胎干细胞为典型的三倍体特征,但ICSI组4PN受精卵来源的2个囊胚未表现多倍体特征。结论:多原核受精卵有继续发育能力,其中IVF组多原核来源胚胎干细胞表现遗传物质倍性改变,而ICSI组多原核来源囊胚无遗传物质倍性变化。     

Cytogenetic analysis of human embryos and embryonic stem cells derived from monopronuclear zygotes

Purpose

By comparing the chromosomal constitution among the arrested cleavage-stage embryos, blastocysts and human embryonic stem cells (hESCs) which are all derived from monopronuclear (1PN) zygotes, it is aimed to determine whether chromosomally normal embryos can be reliably selected by blastocyst culture.

Methods

After 1PN zygotes are sequentially cultured for 5 days, the blastocysts and arrested cleavage-stage embryos were analyzed by fluorescence in situ hybridization (FISH) with probes for chromosomes 18, X and Y; G-banding analysis was adopted to analyze the karyotype of 1PN hESCs.

Results

The diploid rate of blastocysts was 74.6%, which was significantly (P?<?0.001) higher than that of arrested cleavage-stage embryos (31.6%), and the diploid rate of hESCs was 97.0%, which was significantly (P < 0.01) higher than that of blastocysts; the haploid embryos were excluded by blastocyst culture; nevertheless, there still existed such chromosomal abnormalities as mosaic and monosomic in blastocysts and trisomy in hESCs.

Conclusions

Blastocyst culture is an effective method to select against chromosomal abnormalities, especially the haploids in 1PN embryos; however, development to the blastocyst stage is not a reliable marker for mosaicism or aneuploidy.     

体外受精中3原核胚胎的发育及可利用价值的探讨

目的:探讨体外受精(IVF)中异常的3原核(PN)胚胎的发育及可利用价值。方法:收集IVF治疗周期中废弃的3PN受精卵204个进行体外培养,观察其发育能力,并与同周期的1 138个2PN受精卵进行比较;采用胚胎植入前遗传学筛查(PGS)技术对由3PN发育成的19枚囊胚进行非整倍体分析。结果:3PN组和2PN组的卵裂率无统计学差异(P0.05);但3PN组囊胚形成率显著低于2PN组[9.6%(19/97)vs 37.9%(204/342),P0.01]。整倍体分析显示,10.5%(2/19)的3PN来源的囊胚为正常二倍体核型。结论:3PN受精卵有继续发育能力;囊胚培养和高通量测序可作为有效筛选异常PN受精卵中正常核型胚胎的一种方法。     

Preimplantation genetic diagnosis by fluorescence in situ hybridization: clinical possibilities and pitfalls

Preimplantation genetic diagnosis using the fluorescence in situ hybridization technique (FISH) is being used widely to prevent the transmission of sex-linked diseases, to screen for translocations, and for aneuploidy screening in specific in vitro fertilization (IVF) patient groups, along with FISH analysis of spermatozoa in infertile men. In this study, we aim to critically analyze our clinical results in patients at risk of transmitting sex-linked diseases (n = 55), in carriers of translocations (n = 43), in women who have recurrent miscarriage (two or more miscarriages) (n = 128), recurrent IVF failure (three or more failed IVF attempts) (n = 47), and patients of advanced maternal age (37 years old or older) (n = 79). The use of the FISH technique in carriers of sex-linked diseases and translocation patients prevents transmission of these conditions and provides good IVF outcome. In patients with recurrent miscarriage, implantation failure, and advanced maternal age, a high incidence of embryos with abnormal chromosomes 13,16,18,21,22, X, and Y was observed (range 69-75%), as expected. In those three groups of patients, the selection of euploid embryos for transfer resulted in good pregnancy rates with a low incidence of miscarriage. Limitations and pitfalls of this technique are also discussed.     

Pronuclear removal of tripronuclear zygotes can establish heteroparental normal karyotypic human embryonic stem cells

Purpose

This study aimed to derive heteroparental normal karyotypic human embryonic stem cells (hESCs) from microsurgically corrected tripronuclear (3PN) zygotes.

Methods

After sequential culture for 5–6 days, embryos developed from microsurgically corrected 3PN zygotes were analyzed by fluorescence in situ hybridization (FISH) using probes for chromosomes 17, X and Y. Intact 3PN zygotes from clinical in vitro fertilization (IVF) cycles were cultured as the control group. The inner cell mass (ICM) of blastocysts that developed from microsurgically corrected 3PN zygotes was used to derive hESC lines, and the stem cell characteristics of these lines were evaluated. G-banding analysis was adopted to identify the karyotype of the hESC line, and the heteroparental inheritance of the hESC line was analyzed by DNA fingerprinting analysis.

Results

The blastocyst formation rate (13.5 %) of the microsurgically corrected 3PN zygotes was significantly higher (P?<?0.05) than that of intact 3PN zygotes (8.7 %). The diploid rate of the blastocysts (55.0 %) was significantly higher (P?<?0.05) than that of the arrested cleavage-stage embryos (18.4 %) in microsurgically corrected 3PN zygotes. The triploid rate of the microsurgically corrected 3PN zygotes (5.7 %) was significantly lower (P?<?0.01) than that of intact 3PN zygotes (19.4 %). Furthermore, we established one heteroparental normal karyotypic hESC line from the microsurgically corrected tripronuclear zygotes.

Conclusions

Pronuclear removal can effectively remove the surplus chromosome set of 3PN zygotes. A combination of pronuclear removal and blastocyst culture enables the selection of diploidized blastocysts from which heteroparental normal karyotypic hESC lines can be derived.

     

Reproductive oocyte/embryo genetic analysis: comparison between fluorescence in-situ hybridization and comparative genomic hybridization

Multiple displacement amplification (MDA) renders an increased quantity of genomic DNA available for comparative genomic hybridization (CGH), enabling it to be used to identify genomic imbalances in human blastomeres. The karyotypic lineage of 57 blastocysts derived from 11 ovum donors following ovarian stimulation was examined. CGH was performed on all first polar bodies, and linearly on corresponding second polar bodies and blastomeres. A diploid karyotype was propagated from the prefertilized oocyte to the embryo in 25 (44%) sets of analyses. In 32/57 sets (56%), aneuploidy was detected in the post-fertilized zygotes/embryos and in nine (28%) of such cases the aneuploidy was 'chaotic' (> or =3 chromosomes). In 4/57 cases (7%) mitotic aneuploidy was observed. CGH and fluorescence in-situ hybridization (FISH) were concurrently performed on two blastomeres removed from each of 44 embryos obtained from four patients. In 43 (98%) of these embryos there was a direct karyotypic correlation between nine-probe commercial FISH and CGH. CGH identified > or =15% more chromosomal abnormalities than through FISH alone. The linear propagation using MDA-CGH, of the same karyotypic abnormalities that affected the oocyte of origin, in the corresponding embryos, coupled with the fact that CGH confirmed the aneuploidies identified through FISH, validates the accuracy and reliability of CGH technology.     

Simultaneous Detection of Chromosomes X, Y, 13, 18, and 21 by Fluorescence In Situ Hybridization in Blastomeres Obtained from Preimplantation Embryos

Purpose. Simultaneous fluorescence in situ hybridization (FISH) was used in a preimplantation genetic diagnosis program to determine which embryos were normal for chromosomes X, Y, 13, 18, and 21. Methods: Single blastomeres were disrupted and attached to glass slides using acetic acid and ethanol. Using a ratio mixture of chromosome enumeration DNA probes in combination with locus-specific identifier DNA probes, FISH was performed for the identification of chromosomes X, Y, 13, 18, and 21. Results: Fourteen couples enrolled in IVF produced 134 embryos for biopsy. Blastomeres subjected to five-color FISH revealed that 22% of embryos were normal for chromosomes X, Y, 13, 18, and 21. In addition, 52% were abnormal and no results could be detected for 25%. Twelve couples underwent embryo transfer, two couples did not receive embryos due to lack of any normal embryos, and three couples became pregnant. Conclusions: The simultaneous detection of five-color FISH is a feasible method to detect aneuploidy in preimplantation embryos from women of advanced maternal age.     

Discordance among blastomeres renders preimplantation genetic diagnosis for aneuploidy ineffective

Purpose: To investigate the contribution of discordance among blastomeres from the same embryo in the interpretation of blastomeres biopsied from day 3 embryos. Methods: 228 IVF embryos had two blastomeres removed and fluorescent in situ hybridization (FISH) was used to detect aneuploidy of chromosomes 13, 15, 16, 18, 21, 22, X and Y. Of the 228 embryos, 102 had complete FISH results for both blastomeres. Results: When the 2 blastomeres of 102 embryos with successful FISH results were compared, 26 (25.5%) were concordant for all 8 chromosomes and 76 (74.5%) were discordant for one or more chromosomes. Among the 102 embryos, 12 (12%) were disomy in both blastomeres and 37 (36%) were disomic in all 8 chromosomes in one of the two blastomeres. Conclusion: Discordance among blastomeres from the same embryo appears to present a significant problem in interpreting results of embryos biopsied on day 3 and analyzed by FISH especially when most PGD’s are done on single blastomeres.     

Pregnancy after vitrification of pronuclear stage oocytes biopsied for polar body aneuploidy screening

This paper reports the case of a patient undergoing IVF and polar body analysis for aneuploidy screening because of advanced maternal age. A total of nine fertilized oocytes were obtained from the patient's third treatment cycle. Three supernumerary pronuclear stage oocytes were identified as suitable after aneuploidy screening for five chromosomes and vitrified. Because the fresh cycle did not result in a pregnancy, vitrified pronuclear stage oocytes were warmed for transfer in an artificial cycle. All three zygotes survived warming and were transferred to the patient's uterus 2 days later as 4-, 5- and 6-cell stage embryos. After 2 weeks, a positive pregnancy test indicated successful implantation. The gestation is developing normally and is presently (end of September 2007) in the 22nd week.     

Incidence of Chromosomal Abnormalities from a Morphologically Normal Cohort of Embryos in Poor-Prognosis Patients

Purpose: Preimplantation genetic diagnosis of aneuploidy was performed on the embryos yielded by 70 poor-prognosis patients, with the aim of transferring those with a normal chromosomal complement, thus possibly increasing the chances of pregnancy. Methods: Multicolor fluorescence in situ hybridization (FISH) was applied for the simultaneous detection of chromosomes X, Y, 13, 16, 18, and 21. Inclusion criteria were (1) a maternal age of 36 years or older (n = 33), (2) three or more previous in vitro fertilization cycles (n = 20), and (3) an altered karyotype (n = 17). Results: A total of 412 embryos underwent FISH, resulting in 234 (57%) that were chromosomally abnormal. Euploid embryos were available for transfer in 59 patients, generating 19 pregnancies (32%), with an implantation rate of 19.9%. Conclusions: High rates of chromosomally abnormal embryos in poor-prognosis patients can determine repeated in vitro fertilization failures when embryo selection is performed on the basis of morphological criteria alone. Hence, the FISH analysis could represent the prevailing approach for the identification of embryos possessing full potential for developing to term.     

Relationship between embryo quality and aneuploidies

Many high-grade embryos selected for transfer according to their morphological evaluation were detected to have chromosomal abnormalities after aneuploidy screening for infertility by preimplantation genetic diagnosis (PGD). The aim of this study was to detect if there is any correlation between embryo quality and genetic status. The chromosomal status of the day three embryos was studied by multicolour fluorescence in-situ hybridization for chromosomes 13, 18, 21, X and Y. PGD was performed on 132 patients for 1107 embryos. The correlation between embryo quality and aneuploidy was analysed. The analysis showed that a large proportion of normal embryos (50.7%, n = 280) were grade I. In addition, a considerably high proportion of aneuploid embryos (36.1%, n = 83) were evaluated as grade I. There was a significant relationship between PGD results and embryo grades (P = 0.001). Of the 69 polyploid embryos, 21.7% were grade I and 37.8% were grade II. Of the 83 haploid embryos, 27.8% were grade I and 34.9% were grade II. Euploidy was positively related to morphological grade of embryo (P = 0.001). It was also possible for chromosomally abnormal embryos to have a good developmental potential, and they could be selected for embryo transfer unless the PGD procedure was applied.     

Increased chromosome X, Y, and 18 nondisjunction in sperm from infertile patients that were identified as normal by strict morphology: implication for intracytoplasmic sperm injection.

OBJECTIVE: To determine the incidence of nondisjunction for chromosomes X, Y, and 18 using fluorescence in situ hybridization (FISH) on morphologically normal sperm from infertile men who are candidates for ICSI. DESIGN: After standard hematoxylin staining, sperm with normal morphology were identified using Kruger's strict morphology criteria. The location of each normal-appearing sperm was recorded using an electronic microstage locator. Slides were subsequently subjected to FISH for detection of chromosomes X, Y, and 18 (control probe). Nuclei were relocated and analyzed under the fluorescent microscope. SETTING: University-affiliated IVF and intracytoplasmic sperm injection program. PATIENT(s): Men classified as infertile on the basis of abnormal strict morphology (<4% by Kruger's criteria). For controls, normal fertile men (n=6) were also analyzed. INTERVENTION(S): Semen smears were obtained retrospectively from infertile (n=8) and fertile (n=6) men. MAIN OUTCOME MEASURE(s): Ploidy of each cell was determined according to the number of signals detected for each probe. RESULT(S): Approximately 100-150 morphologically normal sperm were identified and located in each case. Subsequent FISH analysis of these normal sperm showed aneuploidy to range from 1.8% to 5.5% in the infertile group as compared with 0 to 2.6% among the control fertile group. Statistically significant differences in the incidence of aneuploidy for the sex chromosomes as well as for all three (X, Y, and 18) chromosomes was observed. CONCLUSION(S): Although 95% to 98% of the sperm were found to be normal for X, Y, and 18, our findings show that infertile couples undergoing ICSI are likely to be at an increased risk for having a genetically abnormal conceptus as compared with the fertile controls. These results demonstrate that normal morphology is not an absolute indicator for the selection of genetically normal sperm. Hence, observed pregnancy failures among ICSI patients may in part be due to the selection of aneuploid sperm.     

Preimplantation diagnosis for aneuploidies in patients undergoing in vitro fertilization with a poor prognosis: identification of the categories for which it should be proposed

OBJECTIVE: To verify whether advantages can derive from the implementation of preimplantation genetic diagnosis for aneuploidy in patients with a poor prognosis of full-term pregnancy, compared with conventional treatment procedures. DESIGN: A randomized, controlled study. SETTING: Reproductive Medicine Unit of the Società Italiana Studi Medicina della Riproduzione, Bologna, Italy. PATIENT(S): In a total of 262 stimulated cycles, women presented with the following poor-prognosis indications: maternal age of > or =36 years (n = 157), > or =3 previous IVF failures (n = 54), and an altered karyotype (n = 51). After giving consent, 127 patients underwent preimplantation genetic diagnosis for aneuploidy, whereas 135 controls underwent assisted zona hatching. INTERVENTION(S): Analysis of chromosomes XY, 13, 14, 15, 16, 18, 21, and 22 was carried out with the fluorescence in situ hybridization technique in a blastomere biopsied from day 3 embryos. Assisted zona hatching was performed on day 3 embryos from the control group. MAIN OUTCOME MEASURE(S): Embryo morphology and chromosomal status, number of transferred embryos, clinical pregnancies, implantation rates, and abortions. RESULT(S): In the study group, 717 embryos were analyzed by fluorescence in situ hybridization, and 60% were chromosomally abnormal. A mean of 2.3+/-0.9 euploid embryos were transferred in 99 cycles, resulting in 37 clinical pregnancies (37%) and a 22.5% ongoing implantation rate. In the control group, 126 cycles were performed with 3.2+/-1.3 embryos transferred, yielding 34 clinical pregnancies (27%) and a 10.2% ongoing implantation rate. CONCLUSION(S): The advantage of selecting embryos with a normal chromosome complement has an immediate impact on the ongoing implantation rate, especially in patients aged > or =38 years and carriers of an altered karyotype.     

Morphological and cytogenetic analysis of intact oocytes and blocked zygotes

We examined cytological and cytogenetic parameters of 1076 oocytes and 385 zygotes that failed to develop post in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Out of 1076 oocytes, 894 (83%) arrested oocytes showed a first polar body and were thus assumed arrested at metaphase II while the remainder showed no polar body. In the group of oocytes with a polar body, 20.5% had an abnormal karyotype. Cytologically, premature sperm chromosome condensation was noted in 28.3% of uncleaved oocytes. This high PCC can be explained by the different grades of oocyte maturity from one center to another. Oocytes from older women showed no increased aneuploidy but did show increased premature chromosome condensation.Analysis by classical technique of 220 uncleaved zygotes showed 91 with highly condensed chromosomes, 53 with asynchrony of condensation, 31 with pulverized chromosomes, and 45 arrested at the first somatic metaphase. Out of 385 arrested zygotes, 165 were explored by in situ hybridization. FISH using a set of 7 chromosome-specific probes showed aneuploidy in the chromosomes analyzed (13, 16, 18, 21, 22, X, Y) in 21.8% of blocked zygotes (19-25% depending on morphology). Extrapolating to other chromosomes, we expect that a vast majority of blocked zygotes and oocytes probably carry chromosome abnormalities. These data demonstrate the contributions of chromosome disorder in early embryo development blocking and implantation failure. Certainly, the issue of cytoplasm and nuclear immaturity and their relation to each other and to chromosome abnormalities provides a fertile area for future investigation in ART.