ORIGEN Centro de Tecnologia em Genetica e Reproducião Humana, R. Otoni 881/15, Belo Horizonte, Minas Gerais, CEP 30270150, Brazil
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Abstract |
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Key words: blastocyst/embryo biopsy/embryo selection/implantation/IVF
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Introduction |
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In order to reduce the number of embryos transferred, it is vital to have very efficient selection criteria to identify those embryos that are more likely to implant and develop into pregnancy. Moreover, it is also important to select spare embryos with the ability to implant for freezing and future transfer. A great number of methods have been suggested to evaluate and choose embryos suitable for transfer. Routinely, morphological and development assessment (Edwards et al., 1984; Cummins et al., 1986
; Steer et al., 1992
) has been used to eliminate arrested or degenerate embryos with minimal potential for implantation. In addition, other methods have been proposed to measure several metabolic parameters of the embryos, for example: pyruvate uptake (Conaghan et al., 1993
), O2 consumption (Magnusson et al, 1986
), secretion of platelet activation factor (O'Neill and Saunders, 1984
), and the production of interleukin-1
(Sheth et al., 1991
). These methods however, have not led to increased implantation and pregnancy rates, especially when more than three similar `good' (whatever the criteria used) embryos are present. So the question still remains: which of the identical embryos of good morphology, good cleavage rate and with adequate metabolic milieu should be selected for transfer?
We therefore propose a new method to evaluate embryo viability, its capacity to develop to blastocyst stage, and to implant and develop normally. We analysed the in-vitro development of isolated blastomeres, biopsied on day 3 after fertilization, and compared it to the development of the original embryo within a 3 day co-culture, in order to find a relationship that could show the embryo's potential future development and so increase implantation rates. This is the first known invasive method proposed to select embryos for transfer and presents a very close relationship with embryo development.
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Materials and methods |
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Ovulation induction
Treatment started on day 2 of the cycle with s.c. administration of 3.6 mg of gonadotrophin releasing hormone analogue (GnRHa, Goserelin, Zoladex; Zeneca, Brazil) for suppression of the pituitary function. To confirm pituitary suppression, serum oestradiol concentrations and vaginal ultrasound were performed 1014 days later. If the oestradiol concentration was <30 pg/ml and the ultrasound showed an endometrial thickness of <3 mm, patients were considered ready to start ovulation induction.
After confirmation of suppression, patients were superovulated with daily human menopausal gonadotrophin (HMG) i.m. injections (Humegon; Organon, Brazil). The dose of HMG was tailored according to the oestradiol concentrations and follicular growth was monitored by vaginal ultrasound (Tosbee; Toshiba, Japan). Human chorionic gonadotrophin (HCG, Pregnyl; Organon, Brazil) was given when at least three follicles reached a mean size of 17 mm with concordant oestradiol concentrations.
IVF procedure
Oocyte retrieval was performed ~34 h after HCG injection by vaginal ultrasound-guided aspiration. Oocytes were inseminated 5 h after retrieval (day 0) either by classical IVF or by intracytoplasmic sperm injection (ICSI). On the following day, i.e. 1719 h later (day 1) the oocytes were checked for normal fertilization by the presence of two pronuclei. The embryos were cultured in 20 µl of Earle's balanced salt solution (Sigma, USA) with 10% heat-inactivated maternal serum at 37°C in a Petri dish (Falcon; BD, USA) under mineral oil (Sigma), under a gas phase of 5% CO2. On day 2 or 3 after oocyte retrieval, the embryos were examined and a maximum of four were selected for embryo transfer.
Cleavage stage biopsy
Normally fertilized embryos which had reached the 6- to 10-cell stages on day 3 irrespective of grade were transferred into drops of HEPES-buffered medium (Earle's) in a Petri dish under mineral oil. Biopsy was performed using a pair of micromanipulators (Leitz, Germany) in conjunction with an inverted microscope (Leitz). Each embryo was immobilized by suction with a flame-polished holding pipette held in one micromanipulator. The second micromanipulator with a double holder controlled a drilling pipette (internal diameter 10 µm) containing acid Tyrode's solution (pH 2.2) and a sampling pipette (internal diameter 30 µm) containing buffered medium. The drilling pipette was placed in close contact with the zona pellucida and a hole made with a controlled stream of acid Tyrode's solution. Immediately the zona was penetrated this pipette was removed, and the sampling pipette was pushed through the hole. One or two cells judged to be at the equivalent of the 8-cell stage were then removed by gentle suction. In all cases, an interphase nucleus was observed in the isolated blastomeres.
Embryo and blastomere co-culture
The biopsied embryos and blastomeres were co-cultured in 20 µl drops of Earle's medium supplemented with 10% heat-inactivated maternal serum under mineral oil in a Petri dish at 37°C under a gas phase of 5% CO2 in air. The embryos and blastomeres were assessed daily for morphological development until day 6.
Cavity formation was considered when fluid began to accumulate either intracellularly or in intercellular cavities between 2 or more cells. Cell number at the blastocyst stage was counted by Giemsa (BDH, USA) staining and the nuclei of biopsied blastomeres were labelled either by Giemsa or by vital labelling (Hoechst 33342) as described by Geber et al. (1995b). Cell numbers were estimated on the assumption that they were equivalent to the number of nuclei counted.
Statistical analysis
Statistical analysis of embryo and blastomere development was performed using the 2-test and Fisher's exact test. Moreover a model for logistic regression was adjusted in order to identify whether the blastomere development variables influence embryo development to blastocyst stage, and with this model it was possible to evaluate the probability of blastocyst formation. A difference was considered significant when P < 0.05. The odds ratio was calculated in order to quantify the degree of association between two groups.
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Results |
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Embryo development
A total of 33 (50%) biopsied embryos reached the blastocyst stages on day 5 or day 6. Twenty-one blastocysts developed from the 44 embryos which had one cell removed (47.7%) and 12 developed from 22 embryos which had two cells removed (54.5%). The remainder arrested at earlier stages. Sixteen out of these 33 blastocysts (48.5%) hatched out from the zona pellucida on day 5 (n = 3) or day 6 (n = 13).
Blastomere development
Approximately 64% of the isolated blastomeres divided over 3 days in culture, i.e. 42 out of the 66. Among the rest (24 out of 66), isolated blastomeres failed to divide over the same period. In 33 cases out of 66 biopsies (50%) the isolated blastomere cavitated within the 3 days of culture. Cavitation and division were observed in 30 out of the 66 biopsied blastomeres (45.5%) as described in detail in Table I.
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We found that the proportion of blastomeres, biopsied from embryos that developed to blastocyst stage, that cavitated was significantly higher than that biopsied from embryos that arrested (P < 0.001). When we considered blastomere division, we also found a statistically significant difference between those biopsied from embryos that reached the blastocyst stage, and from arrested embryos (P < 0.001). A statistically significant difference was also found when we compared the blastomeres that either cavitated and divided, between the groups of blastomeres originated from embryos that reached blastocyst stage and from embryos that arrested (P < 0.001). When analysing the blastomeres that neither divided nor cavitated we also found a statistically significant difference (P < 0.001). As can be seen in Table II, more blastomeres divided, cavitated and divided/cavitated when biopsied from embryos that reached blastocyst stage than those biopsied from embryos that arrested. Moreover, more blastomeres neither divided nor cavitated when biopsied from embryos that arrested.
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The potential future development of the day 3 biopsied human embryos was calculated using the odds ratio (Table II). Embryos whose biopsied blastomeres showed cavitation after 3 days in culture were 13.8 times more likely to develop to the blastocyst stage. Embryos whose biopsied blastomeres presented cell division were 11.1 times more likely to develop to the blastocyst stage. Embryos whose biopsied blastomeres showed either cavitation or division were 17.5 times more likely to develop to blastocyst stage. Finally, embryos whose biopsied blastomeres did not cavitate or divide were 12.0 times more likely to arrest.
The data were also analysed with logistic regression for multivariate analysis. The results demonstrated that cell division and cavitation are significant factors for predicting the probability of embryos to develop to the blastocyst stage (Table III). Using this model we were able to estimate the probability of an embryo to develop to blastocyst stage (Table IV
). Embryos that had biopsied blastomeres showing cell division and cavitation had an 81.6% probability of developing to the blastocyst stage. If cavitation was the only observed phenomenon, the probability was 50%; if cell division was the only phenomenon, 37.5%; and if none of them occurred, 11.9%.
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Discussion |
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With regard to cleavage stage it has been shown that at day 2 after insemination, pregnancy rates were significantly higher if the embryos were at the 4-cell stage (Trounson et al., 1982; Cummins et al., 1986
; Claman et al., 1987
; Giorgetti et al., 1995
). These results imply that there is an optimal cleavage speed after IVF. Concerning morphology, embryos with fewer anuclear fragments and without irregular cells have a higher probability of implanting. Moreover, when analysing all of these parameters together (Puissant et al., 1987
; Steer et al., 1992
; Giorgetti et al., 1995
) there was a very high relationship to implantation rates. Other authors have suggested that early cleaving embryos have an improved chance of achieving a pregnancy (Shoukir et al., 1997
).
Some biochemical methods have been used to try to improve the methods of embryo selection. Pyruvate uptake (Conaghan et al., 1993) was significantly lower in embryos that implanted after day 2 or day 3 embryo transfer; however, due to the wide variation in individual results the authors concluded that this method alone could not predict the most suitable embryos to implant and that morphological grading is the most consistent indicator. Another study performed in natural cycles (Turner et al., 1994
) has shown that pyruvate uptake alone must not be used as a definitive test to determine which embryos should be transferred.
Other metabolic and non-invasive methods have been proposed: glucose uptake and lactate production (Hardy et al., 1989; Lane and Gardner, 1996
), O2 consumption (Magnusson et al., 1986
) and platelet activation factor (O'Neill and Saunders, 1984
). These methods, however, did not prove to be more effective than the morphological criteria.
A very important point that must be addressed is how to avoid multiple pregnancies without reducing pregnancy rates. Some authors have pointed out the necessity of increasing the number of embryos to be transferred in order to increase pregnancy rates (Azem et al., 1995). On the other hand, several authors have demonstrated that transfer of two embryos did not reduce the pregnancy rate (Waterstone et al., 1991
; Englert et al., 1993
; Nijs et al., 1993
). The transfer of two embryos should be considered only for patients with good prognosis, i.e. younger patients and with embryos of good morphology, in order not to reduce the chance of pregnancy in patients with a poor prognosis. For the latter, it is not very difficult to select three to six embryos for transfer (Shulman et al., 1993
) using morphological criteria. For the patients with a good prognosis, however, it is not very easy to select two or three embryos among a large number of good morphology embryos, as they can be very similar.
Performing embryo transfer on day 3 after insemination instead of day 2 was proposed by Dawson et al. (1995) in order to allow good morphology embryos to develop for one more day and, consequently, avoid transferring embryos with the potential to arrest. The results showed significantly higher implantation rates following transfer on day 3, and a lower miscarriage rate.
There is a current consensus to limit the number of good morphology embryos to be transferred in order to reduce the rates of multiple pregnancies. This number differs from one country to another, but ideally two embryos should be the limit. In situations where many embryos of the same morphology and cleavage rate are suitable for transfer, a further diagnostic technique must be performed to select which embryos are most likely to implant after transfer. Our study shows that embryos with the same cleavage rates at day 3 after insemination have 50% probability of arresting, confirming the need for a new selection method. Our results demonstrate a very important relationship between the development of the embryos and the biopsied blastomeres. We found that embryos whose biopsied blastomeres presented cavitation and cell division were 17.5 times more likely to develop to the blastocyst stage. Moreover, we were also able to calculate the probability of an embryo developing to the blastocyst stage, i.e. when blastomeres presented cavitation and cell division, the embryo had 81.6% probability of developing to the blastocyst stage.
Freezing the embryos that were not transferred to the uterus is a well-established method of improving pregnancy rates without the need to repeat ovulation induction (Trounson and Mohr, 1983). Pregnancies have been established even using biopsied embryos (Carson et al., 1997
). The results, however, are not very good and not all embryos cleave after freezingthawing (Kondo et al., 1996
; Karlström et al., 1997
). The possibility of predicting embryo development could avoid unnecessary transfer after thawing of embryos with a low probability of cleavage.
The development of preimplantation genetic diagnosis (PGD) techniques (Handyside et al., 1990) has given many couples with a high risk of transmitting genetic pathology the chance to have children without the disease (Griffin et al., 1994
). Allowing biopsied blastomeres to multiply in vitro will increase the number of cells available for analysis and thus improve the results of the genetic study (Geber et al., 1995b
); moreover, PGD might be offered to a greater number of patients, increasing the range of indications. Also important is the fact that creating a hole in the zona pellucida (assisted hatching) might improve the implantation rates as previously demonstrated (Cohen et al., 1990
).
One criticism of this technique is the need to micromanipulate all embryos. This point, however, is not very important nowadays as many IVF centres worldwide are performing ICSI, PGD or assisted hatching, methods that are based on micromanipulation techniques.
To our knowledge, this is the first published study that shows an invasive method to predict embryo development and to select the most suitable embryos to implant after IVFembryo transfer. In this study we show a very close relationship between embryo development and its biopsied blastomere. This new technique can be used to select the embryos for transfer in the cases where many good morphology embryos are available and also for PGD. Moreover, we can evaluate whether it is worth keeping the remaining embryos frozen. In conclusion, we believe that embryo biopsy for selection of the embryos for transfer can improve implantation rates and decrease multiple pregnancy rates, after fresh or freezingthawing good prognosis cycles.
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Notes |
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References |
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Submitted on June 23, 1998; accepted on November 30, 1998.