FERTILITY AND STERILITY Copyright c 1992 The American Fertility Society
Vol. 58, No.6, November 1992
Printed on acid-free paper in U.S.A.
Human embryo biopsy on the 2nd day after insemination for preimplantation diagnosis: removal of a quarter of embryo retards cleavage Juan J. Tarin, Ph.D.* Joe Conaghan, B.Sc. Robert M. L. Winston, M.D. Alan H. Handyside, Ph.D. Institute of Obstetrics and Gynaecology, Royal Postgraduate Medical School, Hammersmith Hospital, London, United Kingdom
Objective: To assess any reduction in viability and development in vitro after biopsy of a quarter of the cells of human embryos on day 2 after insemination. Design: A prospective study in which normally fertilized surplus embryos of good morphology with two to eight cells approximately 48 hours after insemination were randomly allocated to a control or biopsied group, respectively. Setting: In vitro fertilization (IVF) unit and laboratories of the Hammersmith Hospital, Institute of Obstetrics and Gynaecology, London University. Patients, Participants: One hundred twenty-nine embryos from 28 infertile IVF patients. Interventions: Follicular aspiration by ultrasound-guided transvaginal puncture and embryo biopsy by micromanipulative procedures. Main Outcome Measure(s): Pyruvate uptake and cell number at the blastocyst stage. Results: Embryo biopsy did not have an adverse effect on either the proportion developing to the blastocyst stage (50% [32 of 64] and 47.7% [31 of 65] for the control and biopsied groups, respectively) or embryo viability, measured indirectly through pyruvate uptake. However, the proportion of embryos that reached the morula stage after day 4 (retarded embryos) was significantly higher (44%, 11 of 25 versus 8.7%, 2 of 23) in the biopsied group. The total number of cells (29.6 ± 3.1 versus 62.4 ± 4.7), numbers of inner cell mass (7.7 ± 2.2 versus 24.5 ± 1.4) and trophectoderm (24.0 ± 5.2 versus 45.0 ± 6.4) cells, and the inner cell mass:trophectoderm ratio (34.7 ± 7.9 versus 59.5 ± 11.7) were strikingly reduced at the blastocyst stage in the biopsied group. This reduction was greater in embryos that reached the morula stage after day 4. Conclusions: More investigation is needed to assess whether the detrimental effects observed were because of the biopsy method used in this study or to a high sensitivity of human embryos at early stages to manipulation in vitro. Fertil Steril 1992;58:970-6 Key Words: Embryo biopsy, embryo manipulation, in vitro fertilization, preimplantation diagnosis
In general, preimplantation diagnosis of genetic defects, especially by deoxyribonucleic acid (DNA) analysis, requires a cell or cells to be removed from each embryo. Alternatively, the first polar body could be biopsied and analyzed (1). However, the fact that only maternal genetic defects can be de-
Received April 13, 1992; revised and accepted July 14, 1992.
* Reprint requests and present address: Juan J. Tarin, Ph.D., Instituto Valenciano de Infertilidad, Guardia Civil 23, 46020, Spain.
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tected and the considerable reduction in the number of embryos available for transfer as result of the possible crossing over between homologous chromosomes raise serious doubts about the clinical application of this approach. To obtain embryonic material, biopsies could be performed at any stage between the two-cell and expanded blastocyst stages. However, embryo biopsy at the two-cell and blastocyst stages cannot be considered at the present time as feasible procedures for preimplantation diagnosis because first, the reFertility and Sterility
moval of half the embryo at the two-cell stage may be harmful to the subsequent in vitro and/or in vivo development (2) and second, a low pregnancy rate (PR) after in vitro fertilization (IVF) and embryo transfer at the blastocyst stage has been reported (3-4). These limitations could be overcome if intermediate cleavage stages were used for preimplantation diagnosis. In fact, biopsy of human embryos at the eight-cell stage and the removal of one or two cells does not adversely affect their preimplantation development in vitro (5) and in vivo (6). Nevertheless, after this approach, embryos must bebiopsied and transferred on day 3, which is the latest day embryos have been routinely transferred without affecting PRo This allows a maximum of approximately 12 hours in which to biopsy the embryos and carry out the genetic analysis before transfer later the same day, which may not be enough for some methods of genetic analysis. The use ofthe polymerase chain reaction for DNA analysis requires approximately 5 hours from receipt of the cell to direct visualization of amplified fragment (7), and the sex of embryos biopsied early on day 3 can be identified in time to select female embryos for transfer in couples at risk of having children with X-linked disease later the same day (6). In this case, male embryos were identified by the presence of an amplified fragment specific for a repeated sequence on the Y -chromosome. For the detection of specific mutations in the amplified fragment, however, further analysis may often be necessary by, for example, restriction enzyme digestion or hybridization to allele-specific oligonucleotides, which would take longer. Also, the diagnosis of chromosomal abnormalities by karyotype analysis requires cells to be arrested in metaphase generally by overnight incubation in spindle inhibitors (8). Similarly, the use of nonisotopic or conventional fluorescent methods for detecting in situ hybridization has reduced the time for analysis in comparison with the use of radiolabeled probes, but they still require 24 hours for completion (9). This time limitation could be overcome if biopsy was performed at early stages on day 2 after insemination. However, several authors have suggested that mouse (10) and bovine (Loskutoff et aI., unpublished data, 1991) embryos at early stages may be more sensitive than later stages to manipulation in vitro. In addition, Somers et al. (11) have shown in the mouse a reduction in the inner cell mass: trophectoderm ratio at the blastocyst stage after removal of one cell at the four-cell stage (3/4 embryos). This reduction may result in an insufficient total Vol. 58, No.5, November 1992
number of inner cell mass cells and a reduced capacity to further development of embryos after implantation because fetuses are derived from the inner cell mass (12). Therefore, before attempting diagnosis and transfer of human embryos biopsied on day 2 after insemination, a study assessing any damage on embryo viability and development in vitro is needed. In the present study, several parameters were evaluated in control and biopsied embryos on day 2 after insemination as follows: [1] the percentage of embryos reaching the blastocyst stage to assess any block to embryo development; [2] daily pyruvate uptake as an indirect measure of embryo viability (13); [3] the total number of cells at the blastocyst stage to evaluate any effect on cleavage rate; and [4] the allocation of cells to the trophectoderm and inner cell mass of blastocysts because of the importance of number of inner cell mass cells for fetal development (12). MATERIALS AND METHODS Patients and Protocols
This study is composed of 28 infertile women who, between March 25 and May 23, 1991, underwent IVF -treatment at Hammersmith Hospital. After approval of the project by the Voluntary Licensing Authority for Human In-Vitro Fertilization and Embryology and the Ethics Committee of the Royal Postgraduate Medical School and after obtaining patients' consent, normally fertilized surplus day 2 embryos of good morphology were studied. The IVF protocol has been described by Rutherford et al. (14). Briefly, pituitary desensitization with D-Ser(TBU)6ethylamide-Iuteinizing hormone-releasing hormone (buserelin acetate, Suprefact; Hoechst United Kingdom Ltd, London, United Kingdom) was achieved and maintained during superovulation with human menopausal gonadotropin (Pergonal; Serono Laboratories Ltd, Welwyn Garden City, United Kingdom). A dose of 10,000 IU human chorionic gonadotropin (hCG, Pregnyl; Organon Laboratories Ltd, Cambridge, United Kingdom) was administered when at least three follicles had attained a minimum mean diameter of 17 mm with consistent serum estradiol levels. Oocyte collection was performed 34 hours later. Oocytes and embryos were cultured individually in 1 mL of Earle's balanced salt solution (EBSS; GIBCO, Paisley, United Kingdom), supplemented with 0.47 mM sodium pyruvate, antibiotics, and 10% (vol/vol) heat-inactivated maternal serum in a gas Tarin et al.
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phase of 5% CO 2 , 5% O2 , and 90% N 2 • Oocytes were inseminated at 40 hours after hCG administration and checked for the presence of pronuclei and polar bodies 16 to 18 hours later. After a further 24 hours in culture, two, or exceptionally, three normally fertilized embryos were selected for transfer on the basis of morphological grade and stage of development.
differentially labeled but simply fixed and labeled in bisbenzimide (Hoechst 33342; Sigma Chemical Co., St. Louis, MO) in absolute alcohol at 4°C for 30 minutes. After this time, the labeled blastocysts were washed in absolute alcohol at 4°C for another 30 minutes. The methods used for counting nuclei by fluoresence microscopy are described by Hardy et al. (15).
Embryo Biopsy
One hundred twenty-nine human embryos with two to eight cells approximately 48 hours after insemination were studied. Only normally fertilized embryos of good morphology, graded I and II according to Hardy et al. (15), were suitable for inclusion in the study. Each patient had one half of her spare embryos with three to eight cells randomally allocated for biopsy (n = 65), and the remaining embryos served as controls (n = 57). However, all of two-cell embryos (n = 7) were allocated to the control group because the aim of the present work was to biopsy a quarter of the embryo cells. Further culture of biopsied and control embryos as well as the biopsy procedure were as described by Hardy et al. (5), although some modifications were introduced. Embryos were biopsied in 30 ILL droplets of Hepes-buffered medium M2 supplemented with 4 mg/mL bovine serum albumin (BSA; ICN ImmunoBiologicals, Bucks, United Kingdom) in a Petri dish under silicone oil (BDH, Poole, United Kingdom). Control embryos were exposed to identical conditions to the biopsied group in adjacent droplets. The internal diameter of the holding, acid Tyrode's and sampling micropipettes was 20, 5 and 35 ILm, respectively. One (1/4) or two (2/8) blastomeres were removed according to the embryo stage and/or ease of biopsy. Biopsied and unmanipulated control embryos were cultured until day 6 after insemination in 30-ILL droplets of EBSS (n = 97) or in 5 ILL of a modified T6 medium (HLT6) (n = 32), containing 0.47 mM pyruvate, 1 mM glucose, 5 mM lactate, and 4 mg/mL BSA (16) under silicone oil. Both media were kept at 4°C so that the embryos could be transferred to a new relatively fresh droplet every 24 hours. Labeling of Blastocyst Nuclei
Differential labeling of trophectoderm and inner cell mass nuclei of 36 blastocysts on day 6 after insemination was attempted with polynucleotide-specific fluorochromes using the modification of the method of Handyside and Hunter (17) described by Hardy et al. (15). In 21 blastocysts, after removing the zona pellucida (ZP), the blastocysts were not 972
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Measurement of Pyruvate Uptake
After biopsy, 16 control and 16 biopsied embryos from six patients were washed individually through three droplets of HL T6 before culture in 5 ILL-droplets under silicone oil. Droplets of medium alone incubated adjacent to the embryo containing droplets acted as controls. After daily culture, spent, as well as control droplets from the embryos that reached the blastocyst stage, were diluted 200 times with bidistilled water and frozen immediately at -70°C. The pyruvate assay was based on the conversion of pyruvate to lactate, catalyzed by lactate dehydrogenase (LDH), coupled to the oxidation of the reduced form of nicotinamide-adenine dinucleotide (NADH). The decrease in florescence of NADH, as the reaction goes to completion, was measured using a Technicon AutoAnylizer (New York, NY) with fluorometric attachment and was directly proportional to the concentration of pyruvate in the assay mixture. All of the samples were assayed for pyruvate levels in a single run on the analyzer, and pyruvate depletion by each embryo was calculated by comparing levels in spent medium to that of the control droplets. Statistical Analysis
For comparison ofthe means, Student's t-test was applied. Chi-squared test, with continuity correction when needed, was used for frequency comparisons. Significance was considered as P ::; 0.05. Cleavage index was defined as follows: [(no. of blastomeres on day X + 1 - no. of blastomeres on day X)/no. of blastomeres on day X] X 100. RESULTS
Normally fertilized surplus embryos approximately 48 hours after insemination ranged between the two- and eight-cell stages. The percentage of embryos in each stage was 5.4% (7/129),14.7% (19/ 129), 55% (71/129), 14% (18/129), 7.8% (10/129), 0.8% (1/129), and 0.8% (1/129), respectively. Because embryos from each IVF patient were randomFertility and Sterility
ally allocated to the control or biopsied group, the number of embryos at each stage was not significantly different between these groups.
i
60
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50
. . CXJflRl.
.A
. . BIOPSED
E E
.&
~
Development In Vitro
The numbers ofbiopsied and control human embryos on day 2 that reached the blastocyst stage by day 5 or 6 in different culture media are presented in Table 1. Because embryo development did not vary significantly with the kind (HLT6 or EBSS) and/or amount (5 or 30 JLL) of medium used, the observations are presented together. Thirty-two of 64 control (50%) and 31 of 65 biopsied embryos (47.7%) reached the blastocyst stage. Embryo stage at the time of biopsy did not affect their development capability in vitro either, although among control embryos, the proportion developing to blastocyst tended to be higher in those at more advanced stages at the time of biopsy. Overall, the proportion of blastocysts that hatched from the ZP by day 6 in the biopsied group (10 of 31,32.3%) was significantly (P < 0.01) higher than the control group (1 of 32, 3.1%). Pyruvate Uptake In Vitro
The pyruvate uptake by six control and seven biopsied embryos at four- and five-cell stage that developed to the blastocyst stage is shown in Figure 1. In the control group, pyruvate utilization increased from 21.2 ± 1.9 to 52.3 ± 6.3 pmoljembryo per hour by day 4.5 before dropping to 20.5 ± 4.5 pmoljembryo per hour on day 5.5. The biopsied embryos showed a similar pattern, increasing from 19 ± 1.92 to 42 ± 2.20 pmoljembryo per hour from day 2.5 to day 4.5 before declining to 27.3 ± 6.9 pmolj embryo per hour on day 5.5. The reduction in the biopsied group on days 2.5, 3.5, and 4.5 was 10.3%, 10.4%, and 18.4%, respectively. No significant dif-
Table 1 Development of Human Embryos Biopsied on Day 2 After Insemination in Different Culture Media No. of blastocysts
Control HLT6 EBSS Total Biopsied HLT6 EBSS Total
40
C
No. of embryos
Day 5
Day 6
Total
16 48 64
4 (25.0)* 14 (29.2) 18 (28.1)
2 (12.5) 12 (25.0) 14 (21.9)
6 (37.5) 26 (54.2) 32 (50.0)
16 49 65
5 (31.3) 15 (30.6) 20 (30.8)
2 (12.5) 9 (18.4) 11 (16.9)
7 (43.8) 24 (49.0) 31 (47.7)
* Values in parentheses are percents. Vol. 58, No.5, November 1992
Ii::::) w
~
II:
30
20
:0IL
10 2
3
4
5
6
DAY POST-INSEMINATION
Figure 1 Pyruvate uptake by six control and seven biopsied embryos that developed to the blastocyst stage.
ferences were found either between control and biopsied embryos or with the 75% expected in the biopsied group. Although pyruvate dropped for both biopsied and control embryos over the last 24 hours in culture, the most significant drop was by those embryos that had reached the blastocyst stage by day 5 in culture. The uptakes for these embryos were markedly lower for biopsied and control embryos (20.9 and 16.7 pmoljembryo per hour) than for embryos that reached blastocyst on day 6 (37.8 and 24.2 pmolj embryo per hour, respectively). Total Number of Cells and Numbers of Trophectoderm and Inner Cell Mass Cells
Accurate counts of the numbers of trophectoderm and inner cell mass cells both in control and biopsied embryos were obtained in blastocysts from embryos originally at four- (n = 37) or five-cell (n = 11) stages. Among the other cleavage stages (2- [n = 2], 3- [n = 8], 6- [n = 4], 7- [n = 1], and 8-cell [n = 1] stage), only control or biopsied embryos could be successfully labeled, respectively. Total number of cells, numbers of trophectoderm and inner cell mass cells, and inner cell mass:trophectoderm ratios did not vary significantly between four- and five-cell embryos and are presented together in Table 2. Biopsied embryos had significantly fewer total cells and trophectoderm and inner cell mass cells than the control group. The numbers were not uniformly reduced (52.6%, 46.7%, and 68.6%, respectively) and were disproportionately reduced with respect to the values expected on the basis of a 25% reduction in cell mass. The ratio of inner cell mass:trophectoderm cells was strikingly decreased in the biopsied embryos compared with the controls. Two populations of four- or five-cell embryos were evident in terms of their rate of development and Tarin et aI.
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Table 2
Numbers of Trophectoderm and Inner Cell Mass Cells in Human Blastocysts After Biopsy at the Four or Five-Cell Stage Trophectoderm
Inner cell mass
Inner cell mass: trophectoderm ratio
Total
%
Control' Morula stage Morula stage Biopsied:j: Morula stage Morula stage Expected:j::j: Morula stage Morula stage
45.0 ± 6.4 (28 to 55) t 50.7 ± 4.3 (42 to 55) 28 24.0 ± 5.2 (11 to 45) § 37.0 ± 6.1 (25 to 45) 14.3 ± 1.8 (11 to 19) 33.8 ± 4.8 (21.0 to 41.3) 38.0 ± 3.3 (31.5 to 41.3) 21.0
on day 4 after day 4 on day 4 after day 4 on day 4 after day 4
24.5 ± 1.4 (21 to 28) 24.3 ± 2.0 (21 to 28) 25 7.7 ± 2.2 (3 to 18) II'~ 11.7 ± 4.1 (4 to 18) § 4.8 ± 1.2 (3 to 8) 18.4 ± 1.1 (15.8 to 21.0) 18.3 ± 1.5 (15.8 to 21.0) 18.8
59.5 ± 11.7 (38.2 to 89.3) 49.5 ± 8.7 (38.2 to 66.7) 89.3 34.7 ± 7.9 (9.8 to 72.0) 36.9 ± 18.4 (9.8 to 72.0) 33.0 ± 6.8 (25.0 to 53.3) 59.5 ± 11.7 (38.2 to 89.3) 49.5 ± 8.7 (38.2 to 66.7) 89.3
62.4 ± 4.7 (26 to 85) 63.3 ± 5.1 (26 to 85) 53 29.6 ± 3.1 (14 to 58) ··,tt 40.8 ± 4.3 (30 to 58) § 22.1 ± 1.5 (14 to 27) 47.7 ± 3.8 (19.5 to 63.8) 47.5 ± 3.8 (19.5 to 63.8) 39.8
• The number of trophectoderm and inner cell mass cells are based on 3 and 1 embryos, whereas the total number of cells in 11 and 1 embryos for the normal and retarded embryos, respectively. The total number of cells was determined in four differentially and eight uniformly labled blastocysts). t Values are means ± SE with ranges in parentheses. :j: The number of trophectoderm and inner cell mass cells are based on three and four embryos, whereas the total number of cells in six and nine embryos for the normal and retarded embryos, respectively. The total number of cells was determined in seven differentially and eight uniformly labeled blastocysts). § P < 0.05, significantly different from control group. II P < 0.0005, significantly different from control group. ~ P < 0.01, significantly different from expected group . •• P < 0.0001, significantly different from control group. tt P < 0.001, significantly different from expected group. :j::j: Values are calculated as 75% of the control embryos.
whether the morula stage was reached on or after day 4 after insemination (Table 3). Ninety-one percent of control embryos that developed to blastocyst by day 5 or 6 reached the morula stage on day 4 after insemination, whereas the rest did not reached this stage until later. A similar dichotomy in developmental rate was observed in the biopsied embryos. However, the number of retarded embryos was significantly (P < 0.05) greater. The retardation in embryo development was mainly because of a significant (P < 0.001) decrease in the cleavage index over the period between days 3 and 4. The percentage of blastocysts on day 5 or 6 did not show significant differences either between embryos that reached the morula stage on or after day 4 after insemination or between control and biopsied groups. The total number of cells, numbers of trophectoderm and inner cell mass cells, and inner cell mass:
trophectoderm ratio of both populations of embryos is shown in Table 2. Biopsied embryos that reached the morula stage by day 4 had a significantly (P < 0.05) smaller total number of cells and numbers of inner cell mass cells than the control group. Furthermore, the total and inner cell mass numbers were disproportionately reduced (35.5% and 51.9%, respectively), whereas the number of trophectoderm cells was close to the value expected on the basis of a 25% reduction in cell mass. Therefore, the inner cell mass:trophectoderm ratio was lower in the biopsied than in control group and lower than predicted. Similarly, the retarded population ofbiopsied embryos had a relatively smaller total number of cells and numbers of trophectoderm and inner cell mass cells, and a lower inner cell mass:trophectoderm ratio than control or expected values, although the differences with respect to the expected values
Table 3 Cleavage Index of Human Embryos Whereas Developed to Blastocyst Stage by Day 5 or 6 After Biopsy at the Four- or Five-Cell Stage Cleavage index No. of embryos Control Morula Morula Biopsied Morula Morula
stage on day 4 stage after day 4 stage on day 4 stage after day 4
23 21 (91.3)' 2 (8.7) 25 14 (56.0) 11 (44.0) §
Day 2 to 3
Day 3 to 4
Day 5
Day 6
64.1 ± 7.9t 25.0 ± 0.0
:j: 0.0 ± 0.0
12 (57.1) 2 (100.0)
9 (42.9) 0(0.0)
:j: 10.5 ± 5.711
10 (71.4) 5 (45.5)
4 (28.6) 6 (54.5)
67.8 ± 11.9 53.7 ± 9.5
• Values in parentheses are percents. t Values are means ± SE. :j: Unknown value because of the impossibility of counting the number of blastomeres at the morula stage.
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No. of blastocysts
§ Value significantly different from control group (P < 0.05). II Value significantly different from day 2 to 3 group (P < 0.001).
Fertility and Sterility
were higher than embryos that reached the morula stage on day 4. The reduction in the total number of cells and numbers of trophectoderm and inner cell mass cells was 58.3%, 48.9%, and 80.8%, respectively. DISCUSSION
The present study shows that biopsy of a quarter of human embryo on the 2nd day after insemination has no effect on the proportion developing to the blastocyst stage by day 5 or 6. Similar results have been found after biopsy of one or two blastomeres from eight-cell human embryos on day 3 (5). However, in the mouse, conflicting results have been published. Wilton and Trounson (18) reported that single-cell biopsy at the four-cell stage had no effect on the proportion of embryos reaching the blastocyst stage, whereas Krzyminska et al. (10) found a lower proportion of blastocysts in 3/4 embryos than in intact-controls or 6/8 embryos, suggesting that the developmental potential of embryos is affected by the cleavage stage at the time of biopsy. The overall pattern of pyruvate uptake for both control and biopsied embryos was similar to that reported previously (16), and pyruvate consumption dropped sharply as expected on day 5.5, when the embryos preference is expected to switch from pyruvate to glucose. No clear relationship W/lS demonstrated between pyruvate uptake and cellular mass. Although the pyruvate uptake by manipulated embryos was lower than control embryos, it did not reflect the expected 25% reduction. Similar results have been obtained after biopsy of human embryos at eight-cell stage (5). Furthermore, although pyruvate dropped for both biopsied and control embryos over the last 24 hours in culture, the most significant drop was by those embryos that had reached the blastocyst stage by day 5 in culture. These results suggest that the culture medium is unable to sustain the metabolic requirements of the embryo once the blastocyst stage is reached. Two populations of four- or five-cell human embryos were evident according to their developmental rate and whether they reached the morula stage on or after day 4 after insemination. In both populations, the cleavage rate was retarded after biopsy, although the retardation was more manifest in embryos that reached the morula stage after day 4. This effect was evidenced by a smaller total number of cells at the blastocyst stage in comparison with the 75% expected values. These results contrast with the proportional reduction in the total number of cells at the blastocyst stage found in seven of eight Vol. 58, No.5, November 1992
and six of eight human biopsied embryos (5) and suggest that as in the mouse (10) and cow (Loskutoff et aI., unpublished data, 1991), human embryos at early stages may be more sensitive than later stages to manipulation in vitro. This effect may be caused by embryo exposure to acid Tyrode's solution because exposure of human oocytes is detrimental to later pre implantation development after fertilization (19). The reduction in cell numbers at the blastocyst stage in biopsied embryos (whether or not they reached the morula stage on day 4 after insemination) was disproportionately distributed between the trophectoderm and inner cell mass. In particular, a reduced inner cell mass to trophectoderm ratio was observed in both populations. This result contrasts with the proportional reduction in the number of trophectoderm and inner cell mass cells and unaltered inner cell mass:trophectoderm ratio found in 7/8 and 6/8 human embryos biopsied on day 3 after insemination (5), but it is in agreement with previous studies that analyzed these parameters in 3/4 (11) and 1/2 mouse embryos (20). In the mouse, the allocation of embryonic cells to the trophectoderm and inner cell mass occurs during the fourth (21) and fifth cleavage divisions (22). However, little is known about allocation to the trophectoderm and inner cell mass in human embryos. Hardy et al. (15) estimated by extrapolating from trophectoderm and inner cell mass numbers in day 5 to 7 human blastocysts that there would be approximately 6 and 12 cells allocated to the inner cell mass at the 16- and 32-cell stages, respectively. In the present study, it is possible that after biopsy either less inner cell mass cells were allocated at the morula stage and cleavage overall was retarded, or the same proportion of inner cell mass cells was allocated but division of only within the inner cell mass was retarded. Although it is not possible from the present data to discriminate between these alternatives, the fact that in the more retarded biopsied embryos the inner cell mass cells:trophectoderm ratio was reduced with respect to embryos that reached the morula stage on day 4 supports the first hypothesis. In conclusion, the present paper shows that the biopsy of a quarter of embryo on day 2 following the aspiration (after zona drilling with acid Tyrode's solution) method does not have an adverse effect either on the proportion developing to the blastocyst stage or on embryo viability, measured indirectly through pyruvate uptake. In addition, it appears that zona drilling with acid Tyrode's assists hatching and could therefore facilitate implantation (23). However, Tarin et al.
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there is clear evidence of retardation of cleavage and an altered inner cell mass:trophectoderm ratio. In the human, it is not known whether retardation of cleavage and a reduction in the inner cell mass: trophectoderm ratio will affect implantation or development in vivo. However, studies using the mouse as a model suggest that the proportion of biochemical pregnancies may be increased. In this species, a decrease in the total number of cells at the blastocyst stage does not affect implantation and initiation of a decidual reaction in the uterine stroma (12) but is positively correlated with a decrease in the proportion of inner cell mass (11, 20) and reduced development in vivo (12, 24). Unfortunately, it is not possible to determine from the present data whether the retardation of cleavage and reduction in the inner cell mass:trophectoderm ratio were because of the aspiration (after zona drilling with acid Tyrode's solution) method used or to a high sensitivity of human embryos at early stages to manipulation in vitro. Further work is needed to discriminate between both alternatives.
REFERENCES 1. Verlinsky Y, Ginsberg N, Lifchez A, Valle J, Moise J, Strom CM. Analysis of the first polar body: preconception genetic diagnosis. Hum Reprod 1990;5:826-9. 2. Nijs M, Camus M, Van Sterteghem AC. Evaluation of different biopsy methods of blast orne res from 2-cell mouse embryos. Hum Reprod 1988;3:999-1003. 3. Dawson KJ, Rutherford AJ, Winston NJ, Subak-Sharpe R, Winston RML. Human blastocyst transfer, is it a feasible proposition? Hum Reprod 1988;145(Suppl):44-5. 4. Bolton VN, Wren ME, Parsons JH. Pregnancies after in vitro fertilization and transfer of human blastocyst. Fertil Steril 1991;55:830-2. 5. Hardy K, Martin KL, Leese HJ, Winston RML, Handyside AH. Human pre implantation development in vitro is not adversely affected by biopsy at the 8-cell stage. Hum Reprod 1990;5:708-14. 6. Handyside AH, Kontogianni EH, Hardy K, Winston RML. Pregnancies from biopsied human preimplantation embryos sexed by Y -specific DNA amplification. Nature 1990;344:76870. 7. Handyside AH, Penketh RJA, Winston RML, Pattinson JK, Delhanty JDA, Tuddenham EGD. Biopsy of human preimplantation embryos and sexing by DNA amplification. Lancet 1989;1:347-9. 8. Roberts C, Lutjen J, Krzyminska D, O'Neill C. Cytogenetic analysis of biopsied preimplantation mouse embryos: implications for prenatal diagnosis. Hum Reprod 1990;5:197-202.
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9. Griffin DK, Handyside AH, Penketh RJA, Winston RML, Delhanty JDA. Fluorescent in situ hybridization to interphase nuclei of human preimplantation embryos with X and Y chromosome specific probes. Hum Reprod 1991;6:101-5. 10. Krzyminska DB, Lutjen J, O'Neill C. Assessment of the viability and pregnancy potential of mouse embryos biopsied at different preimplantation stages of development. Hum Reprod 1990;5:203-8. 11. Somers GR, Trounson AO, Wilton LJ. Allocation of cells to the inner cell mass and trophectoderm of 3/4 mouse embryos. Reprod Fertil Dev 1990;2:51-9. 12. Zhouji W, Trounson AO, Dziadek M. Developmental capacity of mechanically bisected mouse morulae and blastocysts. Reprod Fertil Dev 1990;2:683-91. 13. Gardner DK, Leese HJ. Assessment of embryo viability prior to transfer by the noninvasive measurement of glucose uptake. J Exp Zool 1987;242:103-5. 14. Rutherford AJ, Subak-Sharpe R, Dawson K, Margara RA, Franks S, Winston RML. Dramatic improvement in IVF success following treatment with LHRH agonist. Br Med J 1988;296:1765-8. 15. Hardy K, Handyside AH, Winston RML. The human blastocyst: cell number, death and allocation during late preimplantation development in vitro. Development 1989;107:597604. 16. Hardy K, Hooper MAK, Handyside AH, Rutherford AJ, Winston RML, Leese HJ. Noninvasive measurement of glucose and pyruvate uptake by individual human oocytes and preimplantation embryos. Hum Reprod 1989;4:188-91. 17. Handyside AH, Hunter S. A rapid procedure for visualising the inner mass and trophectoderm nuclei of mouse blastocysts in situ using polynucleotide-specific fluorochromes. J Exp Zool 1984;231:429-34. 18. Wilton LJ, Trounson AO. Biopsy of pre implantation mouse embryos: development of micromanipulated embryos and proliferation of single blastomeres in vitro. Bioi Reprod 1989;40:145-52. 19. Malter HE, Cohen J. Partial zona dissection of the human oocyte: a non traumatic method using micromanipulation to assist zona pellucida penetration. Fertil Steril 1989;51:13948. 20. Rands GF. Cell allocation in half- and quadruple-sized preimplantation mouse embryos. J Exp ZooI1985;236:67 -70. 21. Handyside AH. Immunofluorescence techniques for determining the number of inner and outer blastomeres in mouse morulae. J Reprod Immunol 1981;2:339-50. 22. Balakier H, Pedersen RA. Allocation of cells to inner cell mass and trophectoderm linages in preimplantation mouse embryos. Dev Bioi 1982;90:352-62. 23. Cohen J, Elsner C, Kort H, Malter H, Massey J, Mayer MP, et al. Impairment of the hatching process following IVF in the human and improvement of implantation by assisting hatching using micromanipulation. Hum Reprod 1990;5:713. 24. Van Steirteghem A, Liu J, Van den Abbeel E, Liebaers I, Devroey P. In vitro fertilization and pre implantation diagnosis. In: Verlinsky Y, Kuliev A, editors. Preimplantation genetics. New York and London: Plenum Press, 1991:15564.
Fertility and Sterility
Vol. 58, No.6, November 1992
Printed on acid-free paper in U.S.A.
Human embryo biopsy on the 2nd day after insemination for preimplantation diagnosis: removal of a quarter of embryo retards cleavage Juan J. Tarin, Ph.D.* Joe Conaghan, B.Sc. Robert M. L. Winston, M.D. Alan H. Handyside, Ph.D. Institute of Obstetrics and Gynaecology, Royal Postgraduate Medical School, Hammersmith Hospital, London, United Kingdom
Objective: To assess any reduction in viability and development in vitro after biopsy of a quarter of the cells of human embryos on day 2 after insemination. Design: A prospective study in which normally fertilized surplus embryos of good morphology with two to eight cells approximately 48 hours after insemination were randomly allocated to a control or biopsied group, respectively. Setting: In vitro fertilization (IVF) unit and laboratories of the Hammersmith Hospital, Institute of Obstetrics and Gynaecology, London University. Patients, Participants: One hundred twenty-nine embryos from 28 infertile IVF patients. Interventions: Follicular aspiration by ultrasound-guided transvaginal puncture and embryo biopsy by micromanipulative procedures. Main Outcome Measure(s): Pyruvate uptake and cell number at the blastocyst stage. Results: Embryo biopsy did not have an adverse effect on either the proportion developing to the blastocyst stage (50% [32 of 64] and 47.7% [31 of 65] for the control and biopsied groups, respectively) or embryo viability, measured indirectly through pyruvate uptake. However, the proportion of embryos that reached the morula stage after day 4 (retarded embryos) was significantly higher (44%, 11 of 25 versus 8.7%, 2 of 23) in the biopsied group. The total number of cells (29.6 ± 3.1 versus 62.4 ± 4.7), numbers of inner cell mass (7.7 ± 2.2 versus 24.5 ± 1.4) and trophectoderm (24.0 ± 5.2 versus 45.0 ± 6.4) cells, and the inner cell mass:trophectoderm ratio (34.7 ± 7.9 versus 59.5 ± 11.7) were strikingly reduced at the blastocyst stage in the biopsied group. This reduction was greater in embryos that reached the morula stage after day 4. Conclusions: More investigation is needed to assess whether the detrimental effects observed were because of the biopsy method used in this study or to a high sensitivity of human embryos at early stages to manipulation in vitro. Fertil Steril 1992;58:970-6 Key Words: Embryo biopsy, embryo manipulation, in vitro fertilization, preimplantation diagnosis
In general, preimplantation diagnosis of genetic defects, especially by deoxyribonucleic acid (DNA) analysis, requires a cell or cells to be removed from each embryo. Alternatively, the first polar body could be biopsied and analyzed (1). However, the fact that only maternal genetic defects can be de-
Received April 13, 1992; revised and accepted July 14, 1992.
* Reprint requests and present address: Juan J. Tarin, Ph.D., Instituto Valenciano de Infertilidad, Guardia Civil 23, 46020, Spain.
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tected and the considerable reduction in the number of embryos available for transfer as result of the possible crossing over between homologous chromosomes raise serious doubts about the clinical application of this approach. To obtain embryonic material, biopsies could be performed at any stage between the two-cell and expanded blastocyst stages. However, embryo biopsy at the two-cell and blastocyst stages cannot be considered at the present time as feasible procedures for preimplantation diagnosis because first, the reFertility and Sterility
moval of half the embryo at the two-cell stage may be harmful to the subsequent in vitro and/or in vivo development (2) and second, a low pregnancy rate (PR) after in vitro fertilization (IVF) and embryo transfer at the blastocyst stage has been reported (3-4). These limitations could be overcome if intermediate cleavage stages were used for preimplantation diagnosis. In fact, biopsy of human embryos at the eight-cell stage and the removal of one or two cells does not adversely affect their preimplantation development in vitro (5) and in vivo (6). Nevertheless, after this approach, embryos must bebiopsied and transferred on day 3, which is the latest day embryos have been routinely transferred without affecting PRo This allows a maximum of approximately 12 hours in which to biopsy the embryos and carry out the genetic analysis before transfer later the same day, which may not be enough for some methods of genetic analysis. The use ofthe polymerase chain reaction for DNA analysis requires approximately 5 hours from receipt of the cell to direct visualization of amplified fragment (7), and the sex of embryos biopsied early on day 3 can be identified in time to select female embryos for transfer in couples at risk of having children with X-linked disease later the same day (6). In this case, male embryos were identified by the presence of an amplified fragment specific for a repeated sequence on the Y -chromosome. For the detection of specific mutations in the amplified fragment, however, further analysis may often be necessary by, for example, restriction enzyme digestion or hybridization to allele-specific oligonucleotides, which would take longer. Also, the diagnosis of chromosomal abnormalities by karyotype analysis requires cells to be arrested in metaphase generally by overnight incubation in spindle inhibitors (8). Similarly, the use of nonisotopic or conventional fluorescent methods for detecting in situ hybridization has reduced the time for analysis in comparison with the use of radiolabeled probes, but they still require 24 hours for completion (9). This time limitation could be overcome if biopsy was performed at early stages on day 2 after insemination. However, several authors have suggested that mouse (10) and bovine (Loskutoff et aI., unpublished data, 1991) embryos at early stages may be more sensitive than later stages to manipulation in vitro. In addition, Somers et al. (11) have shown in the mouse a reduction in the inner cell mass: trophectoderm ratio at the blastocyst stage after removal of one cell at the four-cell stage (3/4 embryos). This reduction may result in an insufficient total Vol. 58, No.5, November 1992
number of inner cell mass cells and a reduced capacity to further development of embryos after implantation because fetuses are derived from the inner cell mass (12). Therefore, before attempting diagnosis and transfer of human embryos biopsied on day 2 after insemination, a study assessing any damage on embryo viability and development in vitro is needed. In the present study, several parameters were evaluated in control and biopsied embryos on day 2 after insemination as follows: [1] the percentage of embryos reaching the blastocyst stage to assess any block to embryo development; [2] daily pyruvate uptake as an indirect measure of embryo viability (13); [3] the total number of cells at the blastocyst stage to evaluate any effect on cleavage rate; and [4] the allocation of cells to the trophectoderm and inner cell mass of blastocysts because of the importance of number of inner cell mass cells for fetal development (12). MATERIALS AND METHODS Patients and Protocols
This study is composed of 28 infertile women who, between March 25 and May 23, 1991, underwent IVF -treatment at Hammersmith Hospital. After approval of the project by the Voluntary Licensing Authority for Human In-Vitro Fertilization and Embryology and the Ethics Committee of the Royal Postgraduate Medical School and after obtaining patients' consent, normally fertilized surplus day 2 embryos of good morphology were studied. The IVF protocol has been described by Rutherford et al. (14). Briefly, pituitary desensitization with D-Ser(TBU)6ethylamide-Iuteinizing hormone-releasing hormone (buserelin acetate, Suprefact; Hoechst United Kingdom Ltd, London, United Kingdom) was achieved and maintained during superovulation with human menopausal gonadotropin (Pergonal; Serono Laboratories Ltd, Welwyn Garden City, United Kingdom). A dose of 10,000 IU human chorionic gonadotropin (hCG, Pregnyl; Organon Laboratories Ltd, Cambridge, United Kingdom) was administered when at least three follicles had attained a minimum mean diameter of 17 mm with consistent serum estradiol levels. Oocyte collection was performed 34 hours later. Oocytes and embryos were cultured individually in 1 mL of Earle's balanced salt solution (EBSS; GIBCO, Paisley, United Kingdom), supplemented with 0.47 mM sodium pyruvate, antibiotics, and 10% (vol/vol) heat-inactivated maternal serum in a gas Tarin et al.
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phase of 5% CO 2 , 5% O2 , and 90% N 2 • Oocytes were inseminated at 40 hours after hCG administration and checked for the presence of pronuclei and polar bodies 16 to 18 hours later. After a further 24 hours in culture, two, or exceptionally, three normally fertilized embryos were selected for transfer on the basis of morphological grade and stage of development.
differentially labeled but simply fixed and labeled in bisbenzimide (Hoechst 33342; Sigma Chemical Co., St. Louis, MO) in absolute alcohol at 4°C for 30 minutes. After this time, the labeled blastocysts were washed in absolute alcohol at 4°C for another 30 minutes. The methods used for counting nuclei by fluoresence microscopy are described by Hardy et al. (15).
Embryo Biopsy
One hundred twenty-nine human embryos with two to eight cells approximately 48 hours after insemination were studied. Only normally fertilized embryos of good morphology, graded I and II according to Hardy et al. (15), were suitable for inclusion in the study. Each patient had one half of her spare embryos with three to eight cells randomally allocated for biopsy (n = 65), and the remaining embryos served as controls (n = 57). However, all of two-cell embryos (n = 7) were allocated to the control group because the aim of the present work was to biopsy a quarter of the embryo cells. Further culture of biopsied and control embryos as well as the biopsy procedure were as described by Hardy et al. (5), although some modifications were introduced. Embryos were biopsied in 30 ILL droplets of Hepes-buffered medium M2 supplemented with 4 mg/mL bovine serum albumin (BSA; ICN ImmunoBiologicals, Bucks, United Kingdom) in a Petri dish under silicone oil (BDH, Poole, United Kingdom). Control embryos were exposed to identical conditions to the biopsied group in adjacent droplets. The internal diameter of the holding, acid Tyrode's and sampling micropipettes was 20, 5 and 35 ILm, respectively. One (1/4) or two (2/8) blastomeres were removed according to the embryo stage and/or ease of biopsy. Biopsied and unmanipulated control embryos were cultured until day 6 after insemination in 30-ILL droplets of EBSS (n = 97) or in 5 ILL of a modified T6 medium (HLT6) (n = 32), containing 0.47 mM pyruvate, 1 mM glucose, 5 mM lactate, and 4 mg/mL BSA (16) under silicone oil. Both media were kept at 4°C so that the embryos could be transferred to a new relatively fresh droplet every 24 hours. Labeling of Blastocyst Nuclei
Differential labeling of trophectoderm and inner cell mass nuclei of 36 blastocysts on day 6 after insemination was attempted with polynucleotide-specific fluorochromes using the modification of the method of Handyside and Hunter (17) described by Hardy et al. (15). In 21 blastocysts, after removing the zona pellucida (ZP), the blastocysts were not 972
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Measurement of Pyruvate Uptake
After biopsy, 16 control and 16 biopsied embryos from six patients were washed individually through three droplets of HL T6 before culture in 5 ILL-droplets under silicone oil. Droplets of medium alone incubated adjacent to the embryo containing droplets acted as controls. After daily culture, spent, as well as control droplets from the embryos that reached the blastocyst stage, were diluted 200 times with bidistilled water and frozen immediately at -70°C. The pyruvate assay was based on the conversion of pyruvate to lactate, catalyzed by lactate dehydrogenase (LDH), coupled to the oxidation of the reduced form of nicotinamide-adenine dinucleotide (NADH). The decrease in florescence of NADH, as the reaction goes to completion, was measured using a Technicon AutoAnylizer (New York, NY) with fluorometric attachment and was directly proportional to the concentration of pyruvate in the assay mixture. All of the samples were assayed for pyruvate levels in a single run on the analyzer, and pyruvate depletion by each embryo was calculated by comparing levels in spent medium to that of the control droplets. Statistical Analysis
For comparison ofthe means, Student's t-test was applied. Chi-squared test, with continuity correction when needed, was used for frequency comparisons. Significance was considered as P ::; 0.05. Cleavage index was defined as follows: [(no. of blastomeres on day X + 1 - no. of blastomeres on day X)/no. of blastomeres on day X] X 100. RESULTS
Normally fertilized surplus embryos approximately 48 hours after insemination ranged between the two- and eight-cell stages. The percentage of embryos in each stage was 5.4% (7/129),14.7% (19/ 129), 55% (71/129), 14% (18/129), 7.8% (10/129), 0.8% (1/129), and 0.8% (1/129), respectively. Because embryos from each IVF patient were randomFertility and Sterility
ally allocated to the control or biopsied group, the number of embryos at each stage was not significantly different between these groups.
i
60
:I0
50
. . CXJflRl.
.A
. . BIOPSED
E E
.&
~
Development In Vitro
The numbers ofbiopsied and control human embryos on day 2 that reached the blastocyst stage by day 5 or 6 in different culture media are presented in Table 1. Because embryo development did not vary significantly with the kind (HLT6 or EBSS) and/or amount (5 or 30 JLL) of medium used, the observations are presented together. Thirty-two of 64 control (50%) and 31 of 65 biopsied embryos (47.7%) reached the blastocyst stage. Embryo stage at the time of biopsy did not affect their development capability in vitro either, although among control embryos, the proportion developing to blastocyst tended to be higher in those at more advanced stages at the time of biopsy. Overall, the proportion of blastocysts that hatched from the ZP by day 6 in the biopsied group (10 of 31,32.3%) was significantly (P < 0.01) higher than the control group (1 of 32, 3.1%). Pyruvate Uptake In Vitro
The pyruvate uptake by six control and seven biopsied embryos at four- and five-cell stage that developed to the blastocyst stage is shown in Figure 1. In the control group, pyruvate utilization increased from 21.2 ± 1.9 to 52.3 ± 6.3 pmoljembryo per hour by day 4.5 before dropping to 20.5 ± 4.5 pmoljembryo per hour on day 5.5. The biopsied embryos showed a similar pattern, increasing from 19 ± 1.92 to 42 ± 2.20 pmoljembryo per hour from day 2.5 to day 4.5 before declining to 27.3 ± 6.9 pmolj embryo per hour on day 5.5. The reduction in the biopsied group on days 2.5, 3.5, and 4.5 was 10.3%, 10.4%, and 18.4%, respectively. No significant dif-
Table 1 Development of Human Embryos Biopsied on Day 2 After Insemination in Different Culture Media No. of blastocysts
Control HLT6 EBSS Total Biopsied HLT6 EBSS Total
40
C
No. of embryos
Day 5
Day 6
Total
16 48 64
4 (25.0)* 14 (29.2) 18 (28.1)
2 (12.5) 12 (25.0) 14 (21.9)
6 (37.5) 26 (54.2) 32 (50.0)
16 49 65
5 (31.3) 15 (30.6) 20 (30.8)
2 (12.5) 9 (18.4) 11 (16.9)
7 (43.8) 24 (49.0) 31 (47.7)
* Values in parentheses are percents. Vol. 58, No.5, November 1992
Ii::::) w
~
II:
30
20
:0IL
10 2
3
4
5
6
DAY POST-INSEMINATION
Figure 1 Pyruvate uptake by six control and seven biopsied embryos that developed to the blastocyst stage.
ferences were found either between control and biopsied embryos or with the 75% expected in the biopsied group. Although pyruvate dropped for both biopsied and control embryos over the last 24 hours in culture, the most significant drop was by those embryos that had reached the blastocyst stage by day 5 in culture. The uptakes for these embryos were markedly lower for biopsied and control embryos (20.9 and 16.7 pmoljembryo per hour) than for embryos that reached blastocyst on day 6 (37.8 and 24.2 pmolj embryo per hour, respectively). Total Number of Cells and Numbers of Trophectoderm and Inner Cell Mass Cells
Accurate counts of the numbers of trophectoderm and inner cell mass cells both in control and biopsied embryos were obtained in blastocysts from embryos originally at four- (n = 37) or five-cell (n = 11) stages. Among the other cleavage stages (2- [n = 2], 3- [n = 8], 6- [n = 4], 7- [n = 1], and 8-cell [n = 1] stage), only control or biopsied embryos could be successfully labeled, respectively. Total number of cells, numbers of trophectoderm and inner cell mass cells, and inner cell mass:trophectoderm ratios did not vary significantly between four- and five-cell embryos and are presented together in Table 2. Biopsied embryos had significantly fewer total cells and trophectoderm and inner cell mass cells than the control group. The numbers were not uniformly reduced (52.6%, 46.7%, and 68.6%, respectively) and were disproportionately reduced with respect to the values expected on the basis of a 25% reduction in cell mass. The ratio of inner cell mass:trophectoderm cells was strikingly decreased in the biopsied embryos compared with the controls. Two populations of four- or five-cell embryos were evident in terms of their rate of development and Tarin et aI.
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Table 2
Numbers of Trophectoderm and Inner Cell Mass Cells in Human Blastocysts After Biopsy at the Four or Five-Cell Stage Trophectoderm
Inner cell mass
Inner cell mass: trophectoderm ratio
Total
%
Control' Morula stage Morula stage Biopsied:j: Morula stage Morula stage Expected:j::j: Morula stage Morula stage
45.0 ± 6.4 (28 to 55) t 50.7 ± 4.3 (42 to 55) 28 24.0 ± 5.2 (11 to 45) § 37.0 ± 6.1 (25 to 45) 14.3 ± 1.8 (11 to 19) 33.8 ± 4.8 (21.0 to 41.3) 38.0 ± 3.3 (31.5 to 41.3) 21.0
on day 4 after day 4 on day 4 after day 4 on day 4 after day 4
24.5 ± 1.4 (21 to 28) 24.3 ± 2.0 (21 to 28) 25 7.7 ± 2.2 (3 to 18) II'~ 11.7 ± 4.1 (4 to 18) § 4.8 ± 1.2 (3 to 8) 18.4 ± 1.1 (15.8 to 21.0) 18.3 ± 1.5 (15.8 to 21.0) 18.8
59.5 ± 11.7 (38.2 to 89.3) 49.5 ± 8.7 (38.2 to 66.7) 89.3 34.7 ± 7.9 (9.8 to 72.0) 36.9 ± 18.4 (9.8 to 72.0) 33.0 ± 6.8 (25.0 to 53.3) 59.5 ± 11.7 (38.2 to 89.3) 49.5 ± 8.7 (38.2 to 66.7) 89.3
62.4 ± 4.7 (26 to 85) 63.3 ± 5.1 (26 to 85) 53 29.6 ± 3.1 (14 to 58) ··,tt 40.8 ± 4.3 (30 to 58) § 22.1 ± 1.5 (14 to 27) 47.7 ± 3.8 (19.5 to 63.8) 47.5 ± 3.8 (19.5 to 63.8) 39.8
• The number of trophectoderm and inner cell mass cells are based on 3 and 1 embryos, whereas the total number of cells in 11 and 1 embryos for the normal and retarded embryos, respectively. The total number of cells was determined in four differentially and eight uniformly labled blastocysts). t Values are means ± SE with ranges in parentheses. :j: The number of trophectoderm and inner cell mass cells are based on three and four embryos, whereas the total number of cells in six and nine embryos for the normal and retarded embryos, respectively. The total number of cells was determined in seven differentially and eight uniformly labeled blastocysts). § P < 0.05, significantly different from control group. II P < 0.0005, significantly different from control group. ~ P < 0.01, significantly different from expected group . •• P < 0.0001, significantly different from control group. tt P < 0.001, significantly different from expected group. :j::j: Values are calculated as 75% of the control embryos.
whether the morula stage was reached on or after day 4 after insemination (Table 3). Ninety-one percent of control embryos that developed to blastocyst by day 5 or 6 reached the morula stage on day 4 after insemination, whereas the rest did not reached this stage until later. A similar dichotomy in developmental rate was observed in the biopsied embryos. However, the number of retarded embryos was significantly (P < 0.05) greater. The retardation in embryo development was mainly because of a significant (P < 0.001) decrease in the cleavage index over the period between days 3 and 4. The percentage of blastocysts on day 5 or 6 did not show significant differences either between embryos that reached the morula stage on or after day 4 after insemination or between control and biopsied groups. The total number of cells, numbers of trophectoderm and inner cell mass cells, and inner cell mass:
trophectoderm ratio of both populations of embryos is shown in Table 2. Biopsied embryos that reached the morula stage by day 4 had a significantly (P < 0.05) smaller total number of cells and numbers of inner cell mass cells than the control group. Furthermore, the total and inner cell mass numbers were disproportionately reduced (35.5% and 51.9%, respectively), whereas the number of trophectoderm cells was close to the value expected on the basis of a 25% reduction in cell mass. Therefore, the inner cell mass:trophectoderm ratio was lower in the biopsied than in control group and lower than predicted. Similarly, the retarded population ofbiopsied embryos had a relatively smaller total number of cells and numbers of trophectoderm and inner cell mass cells, and a lower inner cell mass:trophectoderm ratio than control or expected values, although the differences with respect to the expected values
Table 3 Cleavage Index of Human Embryos Whereas Developed to Blastocyst Stage by Day 5 or 6 After Biopsy at the Four- or Five-Cell Stage Cleavage index No. of embryos Control Morula Morula Biopsied Morula Morula
stage on day 4 stage after day 4 stage on day 4 stage after day 4
23 21 (91.3)' 2 (8.7) 25 14 (56.0) 11 (44.0) §
Day 2 to 3
Day 3 to 4
Day 5
Day 6
64.1 ± 7.9t 25.0 ± 0.0
:j: 0.0 ± 0.0
12 (57.1) 2 (100.0)
9 (42.9) 0(0.0)
:j: 10.5 ± 5.711
10 (71.4) 5 (45.5)
4 (28.6) 6 (54.5)
67.8 ± 11.9 53.7 ± 9.5
• Values in parentheses are percents. t Values are means ± SE. :j: Unknown value because of the impossibility of counting the number of blastomeres at the morula stage.
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No. of blastocysts
§ Value significantly different from control group (P < 0.05). II Value significantly different from day 2 to 3 group (P < 0.001).
Fertility and Sterility
were higher than embryos that reached the morula stage on day 4. The reduction in the total number of cells and numbers of trophectoderm and inner cell mass cells was 58.3%, 48.9%, and 80.8%, respectively. DISCUSSION
The present study shows that biopsy of a quarter of human embryo on the 2nd day after insemination has no effect on the proportion developing to the blastocyst stage by day 5 or 6. Similar results have been found after biopsy of one or two blastomeres from eight-cell human embryos on day 3 (5). However, in the mouse, conflicting results have been published. Wilton and Trounson (18) reported that single-cell biopsy at the four-cell stage had no effect on the proportion of embryos reaching the blastocyst stage, whereas Krzyminska et al. (10) found a lower proportion of blastocysts in 3/4 embryos than in intact-controls or 6/8 embryos, suggesting that the developmental potential of embryos is affected by the cleavage stage at the time of biopsy. The overall pattern of pyruvate uptake for both control and biopsied embryos was similar to that reported previously (16), and pyruvate consumption dropped sharply as expected on day 5.5, when the embryos preference is expected to switch from pyruvate to glucose. No clear relationship W/lS demonstrated between pyruvate uptake and cellular mass. Although the pyruvate uptake by manipulated embryos was lower than control embryos, it did not reflect the expected 25% reduction. Similar results have been obtained after biopsy of human embryos at eight-cell stage (5). Furthermore, although pyruvate dropped for both biopsied and control embryos over the last 24 hours in culture, the most significant drop was by those embryos that had reached the blastocyst stage by day 5 in culture. These results suggest that the culture medium is unable to sustain the metabolic requirements of the embryo once the blastocyst stage is reached. Two populations of four- or five-cell human embryos were evident according to their developmental rate and whether they reached the morula stage on or after day 4 after insemination. In both populations, the cleavage rate was retarded after biopsy, although the retardation was more manifest in embryos that reached the morula stage after day 4. This effect was evidenced by a smaller total number of cells at the blastocyst stage in comparison with the 75% expected values. These results contrast with the proportional reduction in the total number of cells at the blastocyst stage found in seven of eight Vol. 58, No.5, November 1992
and six of eight human biopsied embryos (5) and suggest that as in the mouse (10) and cow (Loskutoff et aI., unpublished data, 1991), human embryos at early stages may be more sensitive than later stages to manipulation in vitro. This effect may be caused by embryo exposure to acid Tyrode's solution because exposure of human oocytes is detrimental to later pre implantation development after fertilization (19). The reduction in cell numbers at the blastocyst stage in biopsied embryos (whether or not they reached the morula stage on day 4 after insemination) was disproportionately distributed between the trophectoderm and inner cell mass. In particular, a reduced inner cell mass to trophectoderm ratio was observed in both populations. This result contrasts with the proportional reduction in the number of trophectoderm and inner cell mass cells and unaltered inner cell mass:trophectoderm ratio found in 7/8 and 6/8 human embryos biopsied on day 3 after insemination (5), but it is in agreement with previous studies that analyzed these parameters in 3/4 (11) and 1/2 mouse embryos (20). In the mouse, the allocation of embryonic cells to the trophectoderm and inner cell mass occurs during the fourth (21) and fifth cleavage divisions (22). However, little is known about allocation to the trophectoderm and inner cell mass in human embryos. Hardy et al. (15) estimated by extrapolating from trophectoderm and inner cell mass numbers in day 5 to 7 human blastocysts that there would be approximately 6 and 12 cells allocated to the inner cell mass at the 16- and 32-cell stages, respectively. In the present study, it is possible that after biopsy either less inner cell mass cells were allocated at the morula stage and cleavage overall was retarded, or the same proportion of inner cell mass cells was allocated but division of only within the inner cell mass was retarded. Although it is not possible from the present data to discriminate between these alternatives, the fact that in the more retarded biopsied embryos the inner cell mass cells:trophectoderm ratio was reduced with respect to embryos that reached the morula stage on day 4 supports the first hypothesis. In conclusion, the present paper shows that the biopsy of a quarter of embryo on day 2 following the aspiration (after zona drilling with acid Tyrode's solution) method does not have an adverse effect either on the proportion developing to the blastocyst stage or on embryo viability, measured indirectly through pyruvate uptake. In addition, it appears that zona drilling with acid Tyrode's assists hatching and could therefore facilitate implantation (23). However, Tarin et al.
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there is clear evidence of retardation of cleavage and an altered inner cell mass:trophectoderm ratio. In the human, it is not known whether retardation of cleavage and a reduction in the inner cell mass: trophectoderm ratio will affect implantation or development in vivo. However, studies using the mouse as a model suggest that the proportion of biochemical pregnancies may be increased. In this species, a decrease in the total number of cells at the blastocyst stage does not affect implantation and initiation of a decidual reaction in the uterine stroma (12) but is positively correlated with a decrease in the proportion of inner cell mass (11, 20) and reduced development in vivo (12, 24). Unfortunately, it is not possible to determine from the present data whether the retardation of cleavage and reduction in the inner cell mass:trophectoderm ratio were because of the aspiration (after zona drilling with acid Tyrode's solution) method used or to a high sensitivity of human embryos at early stages to manipulation in vitro. Further work is needed to discriminate between both alternatives.
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9. Griffin DK, Handyside AH, Penketh RJA, Winston RML, Delhanty JDA. Fluorescent in situ hybridization to interphase nuclei of human preimplantation embryos with X and Y chromosome specific probes. Hum Reprod 1991;6:101-5. 10. Krzyminska DB, Lutjen J, O'Neill C. Assessment of the viability and pregnancy potential of mouse embryos biopsied at different preimplantation stages of development. Hum Reprod 1990;5:203-8. 11. Somers GR, Trounson AO, Wilton LJ. Allocation of cells to the inner cell mass and trophectoderm of 3/4 mouse embryos. Reprod Fertil Dev 1990;2:51-9. 12. Zhouji W, Trounson AO, Dziadek M. Developmental capacity of mechanically bisected mouse morulae and blastocysts. Reprod Fertil Dev 1990;2:683-91. 13. Gardner DK, Leese HJ. Assessment of embryo viability prior to transfer by the noninvasive measurement of glucose uptake. J Exp Zool 1987;242:103-5. 14. Rutherford AJ, Subak-Sharpe R, Dawson K, Margara RA, Franks S, Winston RML. Dramatic improvement in IVF success following treatment with LHRH agonist. Br Med J 1988;296:1765-8. 15. Hardy K, Handyside AH, Winston RML. The human blastocyst: cell number, death and allocation during late preimplantation development in vitro. Development 1989;107:597604. 16. Hardy K, Hooper MAK, Handyside AH, Rutherford AJ, Winston RML, Leese HJ. Noninvasive measurement of glucose and pyruvate uptake by individual human oocytes and preimplantation embryos. Hum Reprod 1989;4:188-91. 17. Handyside AH, Hunter S. A rapid procedure for visualising the inner mass and trophectoderm nuclei of mouse blastocysts in situ using polynucleotide-specific fluorochromes. J Exp Zool 1984;231:429-34. 18. Wilton LJ, Trounson AO. Biopsy of pre implantation mouse embryos: development of micromanipulated embryos and proliferation of single blastomeres in vitro. Bioi Reprod 1989;40:145-52. 19. Malter HE, Cohen J. Partial zona dissection of the human oocyte: a non traumatic method using micromanipulation to assist zona pellucida penetration. Fertil Steril 1989;51:13948. 20. Rands GF. Cell allocation in half- and quadruple-sized preimplantation mouse embryos. J Exp ZooI1985;236:67 -70. 21. Handyside AH. Immunofluorescence techniques for determining the number of inner and outer blastomeres in mouse morulae. J Reprod Immunol 1981;2:339-50. 22. Balakier H, Pedersen RA. Allocation of cells to inner cell mass and trophectoderm linages in preimplantation mouse embryos. Dev Bioi 1982;90:352-62. 23. Cohen J, Elsner C, Kort H, Malter H, Massey J, Mayer MP, et al. Impairment of the hatching process following IVF in the human and improvement of implantation by assisting hatching using micromanipulation. Hum Reprod 1990;5:713. 24. Van Steirteghem A, Liu J, Van den Abbeel E, Liebaers I, Devroey P. In vitro fertilization and pre implantation diagnosis. In: Verlinsky Y, Kuliev A, editors. Preimplantation genetics. New York and London: Plenum Press, 1991:15564.
Fertility and Sterility
