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PubMed1989

Stochastic model for multipotent hemopoietic progenitor differentiation.

In this study, the authors propose a stochastic model for multipotent hemopoietic progenitor differentiation, which assumes that there is a fixed probability (P) that a progenitor with a potential for differentiation along a particular lineage maintains the potential in each cell division in each daughter cell, and this differentiation process of each lineage proceeds independently. To examine the applicability of this model, a sequential micromanipulation of paired progenitors was carried out and followed by cytological examination of the cells contained in the colonies derived from these progenitors; then calculation was made of the ratio of the number of paired colonies containing cell(s) with a particular lineage to the number of paired colonies in which only one colony contained cell(s) with the lineage at the first and second cell division. The ratios were similar at the first and second cell division within each lineage. Furthermore, the frequences of each lineage in multilineage hemopoietic colonies were calculated using the P values obtained from these micromanipulation experiments. The expected frequencies were similar to those in the actual experiments. These results suggested that the stochastic model was applicable to multipotent hemopoietic progenitor differentiation.

Cell Differentiation↗
PubMed1990

Zona drilling enhances fertilization by mouse caput epididymal sperm.

Spermatozoa from the caput epididymis are known to be much less capable of fertilization when compared to sperm from more distal segments of the epididymis. The purpose of this study was to determine if two micromanipulative techniques, zona drilling (ZD) and a modification of partial zona dissection (PZD), could be used to enhance fertilization with caput epididymal sperm. A mouse in vitro fertilization model was used. Inseminating oocytes with 500-1,000 sperm/oocyte from the cauda epididymis as a control resulted in fertilization of 98 of 300 (32.6%) oocytes. Of those fertilized, 47 developed to the blastocyst stage (47.9%). Caput sperm fertilized 13 of 116 (11.2%) nonmanipulated oocytes. Only 1 of 13 developed into a blastocyst, while with oocyte ZD, caput sperm fertilized 24 of 144 (16.7%) oocytes, 50% of those fertilized developing to blastocyst (P = 0.0129). When modified PZD was performed on oocytes, only one of 23 was fertilized, with no blastocyst development. These results indicate that acid Tyrode ZD enhances both fertilization and early embryonal development when caput epididymal sperm are used for insemination. These mouse studies suggest that ZD or other micromanipulation techniques may prove clinically useful in men with proximal epididymal obstruction where only caput sperm are available.

Animals↗
PubMed1993

The cleavage rate of digynic triploid mouse embryos during the preimplantation period.

Triploidy is a lethal condition in mammals, with most dying at some stage between implantation and term. In humans, however, a very small proportion of triploids are liveborn but display a wide range of congenital abnormalities. In particular, the placentas of human diandric triploid embryos consistently display "partial" hydatidiform molar degeneration, while those of digynic triploids generally do not show these histopathological features. In mice, the postimplantation development of diandric and digynic triploid embryos also differs. While both classes are capable of developing to the forelimb bud stage, no specific degenerative features of their placentas have been reported. Diandric triploid mouse embryos are morphologically normal while digynic triploid mouse embryos consistently display neural tube and occasionally cardiac abnormalities. Previously it was shown that the preimplantation development of micromanipulated diandric triploid mouse embryos was similar to developmentally matched diploid control embryos. In this study, the preimplantation development of micromanipulated digynic triploid mouse embryos is analysed and compared with that of diandric triploid mouse embryos in order to determine whether there is any difference in cleavage rate between these two classes of triploids. Standard micromanipulatory procedures were used to insert a female or a male pronucleus into a recipient diploid 1-cell stage embryo. The karyoplast was fused to the cytoplasm of the embryo by electrofusion. These tripronucleate 1-cell stage embryos were then transferred to pseudopregnant recipients and, at specific times after the HCG injection to induce ovulation, the embryos were recovered and total cell counts made. These results were plotted and regression lines drawn.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Microfluidics device for single cell gene expression analysis in Saccharomyces cerevisiae.

We have measured single-cell gene expression over time using a microfluidics-based flow cell which physically traps individual yeast using microm-sized structures (yeast jails). Our goal was to determine variability of gene expression within a cell over time, as well as variability between individual cells. In our flow cell system, yeast jails are fabricated out of PDMS and gene expression is visualized using fluorescently-tagged proteins of interest. Previously, single-cell yeast work has been done using micromanipulation on agar, or FACS. In the present device agar is eliminated, resulting in a superior optical system. The flow of media through the flow cell washes daughter cells away, eliminating the need for micromanipulation. Unlike FACS, the described device can track individual yeast over a time course of many hours. The flow cells are compatible with the needs of quantitative fluorescence microscopy, and allow simultaneous measurements to be done on a large number of individual yeast. We used these flow cells to determine the expression of HSP104-GFPand RAS2-YFP, genes known to affect yeast life span. The results demonstrate inter-cell variation in expression of both genes that could not have been detected without this single-cell analysis.

Bacterial Proteins↗
PubMed2002

A comparison of hydraulic and laser capture microdissection methods for collection of single B cells, PCR, and sequencing of antibody VDJ.

During the development of B lymphocytes, a series of gene rearrangements assemble the sequences that encode immunoglobulin heavy and light chains (VDJ). Earlier studies of VDJ sequence diversification during expansion of cells in splenic or appendix germinal centers used hydraulic micromanipulation (HM) to collect single B cells for PCR amplification of rearranged antibody heavy and light chain genes. PCR products were directly sequenced without a cloning step. Hydraulic micromanipulation is a very tedious method. Once capability to collect single cells by laser capture microdissection (LCM) was developed, we modified previous tissue staining and fixation methods so that we could collect cells from a given stained tissue section by HM and LCM and directly compare our success rates using these two methods. Cells were alkaline lysed and after two rounds of nested PCR products were recovered and directly sequenced. Because each rearrangement of genomic DNA that occurs to form the immunoglobulin heavy-chain-encoding sequence in developing B cells is unique, this system allowed us to verify our success rate in recovering single lymphocytes from tissue sections and amplifying a single allele. The methods developed have now made LCM an efficient alternative to HM for the collection of single B cells.

Animals↗
PubMed1989

Tension, microtubule rearrangements, and the proper distribution of chromosomes in mitosis.

The basis for stable versus unstable kinetochore orientation was investigated by a correlated living-cell/ultrastructural study of grasshopper spermatocytes. Mal-oriented bivalents having both kinetochores oriented to one spindle pole were induced by micromanipulation. Such mal-orientations are stable while the bivalent is subject to tension applied by micromanipulation but unstable after tension is released. Unstable bivalents always reorient with movement of one kinetochore toward the opposite pole. Microtubules associated with stably oriented bivalents, whether they are mal-oriented or in normal bipolar orientation, are arranged in orderly parallel bundles running from each kinetochore toward the pole. Similar orderly kinetochore microtubule arrangements characterized mal-oriented bivalents fixed just after release of tension. A significantly different microtubule arrangement is found only some time after tension release, when kinetochore movement is evident. The microtubules of a reorienting kinetochore always include a small number of microtubules running toward the pole toward which the kinetochore was moving at the time of fixation. All other microtubules associated with such a moving kinetochore appear to have lost their anchorage to the original pole and to be dragged passively as the kinetochore proceeds to the other pole. Thus, the stable anchorage of kinetochore microtubules to the spindle is associated with tension force and unstable anchorage with the absence of tension. The effect of tension is readily explained if force production and anchorage are both produced by mitotic motors, which link microtubules to the spindle as they generate tension forces.

Animals↗
PubMed1997

CO2 laser in the diagnosis and treatment of early cancer of the vocal fold.

A total of 74 patients underwent cordectomy using CO2 laser for either diagnosis or treatment of an early cancer of the vocal fold. Type I cordectomy consisted in the resection of the entire epithelium, while leaving the vocal ligament intact. Type II cordectomy involved removal of the vocal fold from the vocal process to the anterior commissure and passing through the inferior thyroarytenoid muscle. Type IIIA required vocal fold resection along the internal side of the thyroid ala, while type IIIB included removal of the anterior commissure. Type I cordectomies were carried out with an Acuspot micromanipulator, which provided a 250-micron-diameter beam for a working distance of 400 mm, and in the shot-by-shot cutting mode with 3 W power superpulse. This cordectomy was carried out in 39 patients and a dysplasia or an early carcinoma were detected in 45.9% of cases. Type II and type III procedures were performed with the Microslad micromanipulator having a 700-micron-diameter beam in the continuous cutting mode, 7 W power superpulse. Fifteen cases were treated by type II cordectomy, of which 3 T1aN0M0 cases underwent postoperative radiotherapy due to insufficient resections and 2 cases with T1bN0M0 tumors later underwent reconstructive laryngectomy. A type III cordectomy was used for 14 cases of T1aN0M0 carcinomas and 3 cases of severe dysplasia. The margins of resection were found to be positive histologically in 23.5% of these cases, making frozen section examinations mandatory at time of surgery. Results of all procedures showed that voice was best after a type I cordectomy where only the epithelium was resected. In the type II and type III cordectomies, the quality of voice depended on the development of a fibrous fold and the absence of anterior synechia in the healed larynx.

Adult↗
PubMed1993

Chromosomal constitution of mouse blastocysts derived from oocytes inseminated by multiple sperm insertion into the perivitelline space.

PURPOSE: Our purpose was to evaluate the rate of chromosomal aberrations in mouse blastocysts obtained after microinjection of multiple spermatozoa under the zona pellucida of mature oocytes. Without detecting the appearance of pronuclei, the microinjected mouse oocytes containing two polar bodies were cultivated to the blastocyst stage and then analyzed cytogenetically. RESULTS: A chromosome study was carried out in a total of 109 blastocysts derived after microinjection of motile spermatozoa into the perivitelline space. Fifty-five blastocysts (50.5%) exhibited normal diploid chromosome complements, 30 (27.5%) showed different forms of mosaicism, and 24 (22%) exhibited haploidy caused by parthenogenetic activation. Compared to in vivo and in vitro control groups there was a significant increase in the parthenogenesis and mosaic forms of embryos produced by micromanipulation (P < 0.001). A total of 360 well-spread metaphases of 103 blastocysts was analyzed to determine whether the micromanipulation procedure increased the chance of aneuploidy. Aneuploid numbers of chromosomes were absent in all the metaphases analyzed. CONCLUSION: Mosaicism and parthenogenesis appear to be increased significantly following microinjection of multiple spermatozoa under the zona pellucida of mouse oocytes, and there was no evidence of aneuploidy.

Animals↗
PubMed1987

A method for recording isometric tension development by isolated cardiac myocytes: transducer attachment with fibrin glue.

The purpose of this study was to develop a method for attachment of single isolated cardiac myocytes to a transducer for recording isometric tension development. Cardiac myocytes were isolated from the hearts of the toad, Bufo marinus or ferrets by enzymatic digestion with collagenase. The method that we used provided a 60-80% yield of Ca++-tolerant cells. A suspension of cells was placed into a superfusion chamber coated with bovine thrombin. Two glass microtools - each attached to a micromanipulator - were brought into proximity with the ends of a single myocyte; one of the microtools was attached to the element of a low-level force transducer. Human fibrinogen was loaded into a fine-tipped glass micropipette mounted on a micromanipulator. Small amounts of fibrinogen were pressure-ejected from the pipette at each junction between the microtool and the end of the myocyte. The fibrin that formed produced a stable attachment of the ends of the myocyte to the microtools. The myocyte could subsequently be stretched and a length-tension curve recorded. We have used this method to record concentration-dependent tension development in response to the Ca++-ionophore, A23187, and potassium depolarization. Our results indicate that fibrin glue may facilitate the study of the mechanical properties of isolated myocytes.

Animals↗
PubMed2001

Y specific sequence gene analysis of single fetal nucleated erythroblasts from the peripheral blood of pregnant women.

The single cell isolation technique was used to detect fetal nucleated erythroblasts (FNRBCs) at a single cell level from the peripheral blood of pregnant women in order to investigate the feasibility of this method for noninvasive prenatal diagnosis. Single fetal nucleated erythroblasts were isolated from the peripheral blood samples from 51 pregnant women (14 to 26 weeks of gestation) by micromanipulation techniques after density gradient centrifugation. Nested polymerase chain reaction method was used to amplify the SRY gene. It was found that the concordance rate of amplification results with real fetal sex was 82.61%. The sensitivity and specificity were 80% and 87.50% respectively. It was suggested that it is feasible and promising in non invasive prenatal diagnosis to detect fetal nucleated erythroblasts at a single cell level by using micromanipulation techniques.

Adult↗
PubMed1993

Treatment of male infertility and idiopathic failure to fertilize in vitro with under zona insemination and direct egg injection.

OBJECTIVE: Failure to fertilize eggs in vitro may be countered by micromanipulation of gametes to place selected spermatozoa underneath the zona pellucida of the egg or directly into the egg, thereby improving chances of fertilization and production of viable embryos. Analysis of our clinical data for assisted fertilization was undertaken to assess those factors of relevance in this therapy, and a description of our procedures are given. STUDY DESIGN: Retrospective analysis of 85 cycles (73 couples) of in vitro fertilization and embryo transfer performed at a private infertility clinic, in which micromanipulation for assisted fertilization was used to overcome either severe male factor infertility or idiopathic failure to fertilize, was performed. RESULTS: In 60 cycles where only embryos from under zona insemination were available for uterine transfer, 15 singleton and two twin pregnancies occurred (28.3% viable pregnancy rate per transfer, 14.1% embryonic implantation). In 14 of these cycles embryos arose only after repeated under zona insemination adding more spermatozoa; this accounted for four of the singleton and one of the twin pregnancies (38.5% pregnancy rate, 22.2% embryonic implantation). No embryos arose from partial zona dissection performed in five cycles on sibling eggs. However, in 16 cycles conventional insemination yielded fertilization in six cycles, and mixed transfer of these embryos and sibling embryos from under zona insemination gave rise to one pregnancy from four transfers (pregnancy rate 25%, embryonic implantation 7.1%). Likewise, in nine cycles donor spermatozoa yielded fertilization in eight cycles, and mixed transfer with sibling embryos fertilized by under zona insemination with partner's spermatozoa gave rise to two pregnancies from five transfers (pregnancy rate 40%, embryonic implantation 15.8%). Fertilization and pregnancy rates did not differ whether couples suffered either from male factor infertility or from previous idiopathic fertilization failure. Direct egg injection of a single spermatozoon into 105 eggs gave an 88.6% egg survival and 32.3% fertilization. Mixed transfers with sibling embryos from conventional and under zona insemination yielded one triplet, one twin, and three singleton pregnancies. CONCLUSIONS: Overall, a 24.7% (21/85) viable pregnancy rate per cycle initiated occurred when only embryos from assisted fertilization were available. This strongly indicates that assisted fertilization made a real contribution in cases where either insufficient spermatozoa were available for conventional insemination or in cases where previous fertilization failure had arisen. The wide range of seminal parameters were found to be unhelpful in defining chances of success with assisted fertilization.

Female↗
PubMed1986

Personal computer-controlled microsurgery of fertilized eggs and early embryos.

The microsurgery of mouse and rat eggs and early embryos was attempted using a micromanipulator driven by three pulse motors. The pulse signals that regulate the three pulse motors for the X, Y, and Z axes were controlled according to the personal computer programs produced on the basis of the displayed data. As a result, the following was found. 1) The computer-controlled operation was possible in the X and Y plane on a specimen previously suctioned and retained by a holding pipet. A microinjection pipet was inserted into the male pronucleus of a fertilized egg and the morula was bisected using a microblade; these microtools were moved horizontally. 2) A more complicated micromanipulation in two dimensions (X and Z axes), which is very difficult manually, was possible by using this system. 3) microsurgery (microinjection of a fluorescent material (FITC) into the male pronucleus, enucleation of a fertilized egg, and vertical or horizontal bisection of morulae) was carried out successfully by a student who had no practical experience in this field. These facts suggest that the system markedly facilitates microsurgery, without need for full training in the manual procedures.

Journal Article↗
PubMed1987

Development of one or two blastomeres from eight-cell mouse embryos to term in the presence of parthenogenetic eggs.

This study was undertaken to develop a new technique to produce identical offspring by aggregating a quarter or eighth embryo with a parthenogenetically activated egg in the mouse. One or two blastomeres from 8-cell embryos were aggregated with a parthenogenetic 4-cell egg from which one or two blastomeres had been removed. After micromanipulation and culture for 2 d in vitro, the morphologically normal blastocysts were transferred to the uterus of recipient females. The success rate in micromanipulation of eggs was 93 to 100%: aggregation of blastomeres occured about 60% of the time and the proportion of live young after transfer of aggregated eggs was 11 to 33% for the quarter and 2 to 24% for the eighth egg. The proportion of chimaeras as judged by coat color was 10% for the quarter and 20% for the eighth egg. However, GPI-1 analysis and progeny testing could not detect a parthenogenetic contribution in all offspring. The mean number of young obtained from one embryo was 1.7 for the quarter and 1.6 for the eighth embryo. The maximal number of young obtained from splitting one 8-cell embryo into quarters was three and into eighths was four. The mice of each set derived from a single embryo were of the same sex. Our study clearly demonstrates that the parthenogenone can assist development of the quarter and eighth mouse embryo to term. The proportion of chimaeras is low compared with that obtained when two fertilied eggs are combined.

Journal Article↗
PubMed1994

In vitro fertilization rate of horse oocytes with partially removed zonae.

Frozen-thawed ejaculated stallion spermatozoa were preincubated for 3 h in BO medium containing 5 mM caffeine and then treated with 0.1 micro M calcium ionophore A23187 for 60 sec. Aliquots of the sperm suspension (final concentration 1-2 x 10(7)/ml) were added to the oocytes which had been matured in vitro for 32 h. In Experiment 1, there were 3 groups of oocytes; cumulus intact, denuded zona-intact, and zona-free. Cumulus cells were removed with 0.5% hyaluronidase and the zona pellucida with 0.1% protease. The oocytes were fixed 20 h after insemination with acetic acid:ethanol (1:3) and stained with 1% orcein. The sperm penetration rate of zona-free oocytes was 83%, whereas the sperm penetration rate was very low (1 to 3%) in the cumulus-enclosed or zona-intact oocytes. In Experiment 2, denuded zona-intact oocytes were placed in PBS supplemented with 10% fetal bovine serum 1 h before the end of in vitro maturation. The zona pellucida was micromanipulated with a metal microblade under x 100 magnification within 20 min of treatment with 0.3 M sucrose. For partial zona dissection, a slit in the zona pellucida was made. For partial zona removal, oocytes were transferred to protein-free PBS to fix the oocytes on the bottom of the Petri-dish and to remove a piece of the zona pellucida. Micromanipulated oocytes were subjected to in vitro fertilization as described above. Zona-intact and zona-free oocytes treated with sucrose solution for 20 min were used as controls. The penetration rates were 4 (2/57), 12 (7/58), 52 (31/60), and 86% (44/51) for zona-intact, partially zona dissected, partially zona removed, and zona-free oocytes, respectively. Proportions of oocytes with monospermic penetration were 100 (2/2), 57 (4/7), 58 (18/31), and 34% (15/44), respectively. In Experiment 3, sperm penetration and male pronucleus formation in the partially zona removed oocytes were examined at 2.5 to 20.0 h of insemination. Sperm penetration started 2.5 h post-insemination (22%, 11/49), and increased to 38% (21/55) at 5 h, to 46% (23/50) at 10 h, and to 56% (27/48) at 20 h. The transformation of sperm heads into male pronuclei was first observed 10 h post insemination. These results indicate that assisted fertilization techniques may be a useful tool for achieving fertilization and embryo production in vitro in horses.

Journal Article↗
PubMed1995

Adapters for combined intrapipette pressure pulses and patch pipette step movements during 'blind' cell search in brain slices.

A procedure is described for 'blind' cell search in brain slices based on pressure pulses instead of steady-state pressure applied to the patch pipette during its stepwise movements. For reproducibility of the pressure/movement pattern during the cell search, we have developed two adapters, one for electrically and the other for hydraulically driven micromanipulators which generate pressure pulses synchronized with patch-pipette step movements. Both adapters increase the intrapipette pressure prior to a step movement of the pipette, maintain the pressure during the pipette movement, and release it between steps, thus minimizing the possibility of 'blowing-away' the cells during the search. The hydraulic micromanipulator adapter converts this into a stepping one. Both adapters also allow simultaneous recording of pipette step movements and of intrapipette pressure. The use of these adapters allows standardization of the 'blind' cell search and greatly increases the success rate of cells detection.

Animals↗
PubMed2007

Reconstruction of ooplasm recipient oocytes with frozen-thawed donor microcytoplasts and influence on the microtubular spindle.

OBJECTIVE(S): To cryopreserve micromanipulated ooplast segments (microcytoplasts) from mouse oocytes, compare microcytoplast and parent or recipient oocyte fusion performed within or without the zona pellucida, compare electrofusion of fresh or frozen oocyte with frozen-thawed microcytoplasts, and assess spindle integrity after reconstruction of oocytes. DESIGN: Prospective experimental study. SETTING: University-based experimental laboratory. ANIMAL(S): Mouse (MII) oocytes obtained after superovulation (n = 363). INTERVENTION(S): Micromanipulation of oocytes (n = 363) into microcytoplasts (n = 181), cryopreservation of microcytoplasts along with parent and sibling control oocytes (n = 182), reconstruction by electrofusion of microcytoplast and parent or recipient oocyte performed with (group A, n = 35) or without a zona pellucida (group B, n = 32), comparison of electrofusion of fresh oocyte (group C, n = 40) or frozen oocyte (group D, n = 36) with frozen-thawed microcytoplasts fused within zona, and assessment of spindle morphology of reconstructed oocyte. MAIN OUTCOME MEASURE(S): Post-thaw survival, success of fusion, and spindle integrity as assessed by immunostaining. RESULT(S): Higher success of post-thaw fusion was seen in group A (91.4%) compared with group B (56.2%). The post-thaw fusion of microcytoplasts with either fresh or frozen oocytes was not significantly different. Spindle integrity was 82.5% in group C as compared with 47.2% in group D. CONCLUSION(S): Microcytoplasts created from oocytes can be successfully cryopreserved, thawed, and used to reconstruct oocytes with intact spindles.

Animals↗
PubMed2005

Diagnosis of human cytomegalovirus intrauterine infection using fetal cells from maternal blood.

OBJECTIVE: The sensitivity and specificity for the noninvasive prenatal diagnosis of human cytomegalovirus intrauterine infection were estimated by using isolating single fetal cells from maternal peripheral blood. METHODS: Micromanipulation techniques were employed to isolate single fetal nucleated erythroblasts from 273 maternal blood samples. SRY gene and HCMV-DNA in single fetal cells were detected by multiple primed in situ labeling (PRINS) from 76 HCMV-DNA positive samples of maternal peripheral blood. 273 samples of maternal peripheral blood were tested for SRY gene and HCMV-DNA in single fetal cells by primed extension preamplification (PEP) and polymerase chain reaction (PCR). RESULTS: The detection rate of fetal cells from maternal blood was 100% with micromanipulation techniques. The sensitivity of PRINS for SRY gene detection was 97.56% and its specificity was 100%. The sensitivity and specificity of PEP and PCR for SRY gene detection were 97.39% and 99.17%, respectively. The sensitivity of PRINS for HCMV-DNA detection was 92.68% and the specificity was 100%. The sensitivity and specificity of PEP and PCR for HCMV-DNA detection were 95.12%and 100%, respectively. CONCLUSION: The technique for noninvasive prenatal detection of intrauterine infection of HCMV using single fetal cells from maternal peripheral blood by using PRINS and PEP and PCR is more reliable than the CMV-DNA detection in peripheral maternal blood, amniocentesis or percutaneous umbilical blood sampling.

Cytomegalovirus↗
PubMed1999

Fetal cell recycling: diagnosis of gender and RhD genotype in the same fetal cell retrieved from maternal blood.

OBJECTIVE: Our aim was to develop a new technique, which we have termed fetal cell recycling, that combines the 2 powerful methods of fluorescence in situ hybridization and polymerase chain reaction to maximize the genetic information available from a small number of fetal nucleated erythrocytes obtained noninvasively from the blood of pregnant women. STUDY DESIGN: Blood samples were obtained from 4 Rh-negative women after elective termination of pregnancy at 7 to 17 weeks' gestation. Fetal nucleated erythrocytes were separated by flow sorting with antibody to the gamma chain of fetal hemoglobin. Fluorescence in situ hybridization with chromosome-specific probes was used to diagnose fetal gender. After fluorescence in situ hybridization analysis the fetal nucleated erythrocytes were recycled by a micromanipulation technique and deoxyribonucleic acid diagnosis was performed with polymerase chain reaction amplification of the RhD gene. RESULTS: Among the 4 case patients we detected a total of 101 fetal nucleated erythrocytes. All targeted cells were successfully retrieved with a micromanipulator. In each case we successfully performed both fluorescence in situ hybridization and polymerase chain reaction analysis. The predicted fetal gender and Rh status corresponded to the results obtained from fetal tissue. CONCLUSIONS: Fetal cell recycling combines the powers of highly sensitive molecular methods to maximize the genetic information available from a single fetal cell. This technique will permit noninvasive diagnosis of recessively inherited single-gene disorders.

Aneuploidy↗