1 Laboratory for Biology and Psychology of Human Fertility, the Faculty of Medicine, Free University of Brussels, 2 Clinic of Fertility, Department of Obstetrics and Gynaecology, Hospital Erasme, and 3 IRIBHN Statistical Unit, Free University of Brussels, Route de Lennik 808, 1070 Brussels, Belgium
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Abstract |
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Key words: embryo cohort size/embryo quality/maternal age/oocyte maturity
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Introduction |
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Numerous studies have analysed the factors that influence the pregnancy rates in human IVF and embryo transfer; however, only a few have analysed the impact that female factors or parameters of the follicular phase could have on embryo development and morphology prior to transfer. Maternal age (Janny and Ménézo, 1996), causes and duration of infertility, ovarian stimulation protocol (Pellicer et al., 1989
; Fluker et al., 1993
; Testart et al., 1993
) or the hormonal profile during the follicular phase (Burns et al., 1994
) as well as culture conditions are some of the factors that could affect the embryo viability. On the other hand, variation in embryo quality has been seen in human embryos produced in vivo (Sauer et al., 1987
), suggesting that poor quality embryos can be an inherent feature in human fertility.
Furthermore, facing the highly criticized increasing incidence of multiple births in IVF and embryo transfer, several programmes limit the number of transferred embryos up to two, with a maximum of three. If the number of triplets has dramatically decreased with this transfer policy, patients still experience a risk of ~25% of a twin gestation (Kodama et al., 1995; Devreker et al., 1999
). To reduce further the risk of twin gestations, the replacement of only one embryo could be an adequate alternative for patients with good prognosis. However, this means that choosing the right embryo becomes a crucial issue in order to maintain the chances of pregnancy. To understand how embryo viability can be affected by maternal or paternal factors, ovarian stimulation or culture conditions can help to improve embryo selection. This retrospective study, including 949 IVF and embryo transfer cycles, analysed different parameters with reference to the embryo quality. A stepwise multiple linear regression analysis taking into account the maternal and paternal ages, causes and duration of infertility, ovarian stimulation parameters, number of oocytes collected, fertilization rate and number of embryos obtained was performed. Furthermore, 176 couples who underwent three successive cycles were analysed to determine if embryo quality remained similar from cycle to cycle.
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Materials and methods |
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Another group of 176 couples taken from the original 949 couples, who underwent three successive attempts, were selected to evaluate the embryo quality cohort from cycle to cycle. Couples that performed more than three cycles were excluded from the analysis because of the small number of patients available.
Overall, the main indications of infertility for IVF and embryo transfer were tubal (31%), endometriosis (9%), pure male infertility (5%), unknown (12%) and mixed male and female (43%). For intracytoplasmic sperm injection (ICSI) and embryo transfer, the main causes were pure male infertility (42%), mixed male and tubal (18%), endometriosis (9%) or other combinations (31%). The mean maternal age and duration of infertility was 32.5 ± 4.7 years and 5.2 ± 3.4 years respectively. The overall pregnancy rate was 40%, including 33.5% deliveries and 6.5% miscarriages. The implantation rate per embryo reached 19%. Thirty-five percent of the deliveries were multiple, with 4.2% of triplets. The delivery rates were similar between IVF and ICSI cycles, 31.6 and 36.2% respectively.
Ovarian stimulation and IVF protocol
Patients were treated with human menopausal gonadotrophin (HMG; Pergonal®; Serono, Geneva, Switzerland; Humegon®; Organon, Oss, The Netherlands, Netherlands) in combination with a luteinizing hormone-releasing hormone agonist (Buserelin, Suprefact®; Hoechst, Brussels, Belgium) (see Devreker et al., 1996).
Preincubation, insemination or ICSI of oocytes and embryo culture before transfer were carried out in Earle's Balanced Salt solution containing 5.56 mmol/l glucose and supplemented with 25 mmol/l sodium bicarbonate (Sigma, Bornem, Belgium), 0.33 mol/l pyruvic acid (Sigma) and 0.5% human serum albumin (Red Cross, Brussels, Belgium) in a gas phase of 5% CO2, 5% O2 and 90% N2 (Van den Bergh et al., 1995). Normal fertilization was confirmed 1416 h after insemination by the presence of two pronuclei (day 1). On the morning of embryo transfer, 4244 h after insemination, embryos were examined under an inverted microscope to determine the evenness and number of blastomeres as well as the extent of extracellular fragmentation. A numerical score was calculated on the basis of embryo morphology and cleavage rate (Puissant et al., 1987
). Four points were given for an embryo with regular blastomeres and no anucleate fragments, three points for an embryo with uneven blastomeres and one or two anucleate fragments, two or one points for an embryo with uneven blastomeres and anucleate fragments of the embryonic surface
1/3 or
1/3 respectively. Two more points were added if the embryo had reached the four-cell stage. The transfer policy applied during the period analysed was based on the morphology of the embryos available on the day of transfer. When the sum of the three best embryos reached a total of
15 only two embryos were replaced (a protocol suggested to reduce the risk of multiple pregnancies; Puissant et al., 1987; Staessen et al., 1992). For all the other couples, three embryos were replaced if available except for patients who specifically requested a double transfer or a triple transfer. Since then, the transfer policy has been modified (Devreker et al., 1999
) in order to reduce the high multiple pregnancy rate. Embryos were transferred vaginally through a Frydman® catheter (CCDT, Paris, France) and luteal support was maintained by daily injection of 100 mg i.m. of oily progesterone or with intravaginal pessaries (three times 200 mg/day of micronized progesterone). The remaining good quality embryos were frozen.
The mean score of the embryo cohort is defined by the sum of scores for all the normally fertilized embryos divided by the number of embryos. The mean score of the transferred embryos is the ratio between the sum of the scores and the number of transferred embryos. The delivery rate is the number of deliveries divided by the number of transfers. The implantation rate is the ratio between the number of fetuses with cardiac activity visualized by ultrasound during the first trimester of gestation and the number of embryos transferred. Each cause of infertility was given a numerical score according to the severity of the disease. Tubal pathologies were scored as one if absent, two if tubes were still patent, three if non-patent tubes or four if not defined. Anomalies of spermatozoa were scored: one if normal, two if two of the following parameters were abnormal including sperm count, percentage of motility or abnormal forms, or three if the three parameters were abnormal. Endometriosis was given a score of one if absent, two if moderate or three if severe. The absence or presence of polycystic ovary syndrome (PCOS) was given a score of one or two respectively. Maternal age, causes of infertility, number of ampoules of HMG injected, number of days of stimulation, oestradiol concentrations, fertilization rate, number of collected oocytes and number of embryos available were analysed to determine what effects, if any, these variables might have on the quality of the transferred embryos.
Further analysis was carried out by subdividing the patients according to the number of embryos available at the time of transfer; group A: fewer than five embryos, group B: between five and 15 embryos, group C: >15 embryos.
Statistical analysis
Quantitative variables were summed up by their means ± SD. Statistical comparisons of means were carried out with analysis of variance and Scheffé test for multiple comparisons; bivariate correlation (Pearson or Spearman for qualitative data) were tested between maternal age, means of score of the embryo cohort, means of score of the transferred embryos, duration of infertility and the different parameters of the ovarian response. A stepwise multiple linear regression was performed between the quality of the transferred embryos, the quality and size of the embryo cohort and the maternal age, the causes and the duration of infertility, the duration of stimulation, the number of ampoules of HMG, the number of oocytes collected, and the fertilization rate. Chi-square test or Fisher's exact probability test was used to look for possible relationships between qualitative variables. Analysis was completed using Statistics Package for Social Sciences (SPSS) 6.1 for Windows 95.
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Results |
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The probability of good quality embryos for transfer was dependent on the ovarian response. The mean scores of the transferred embryos correlated positively with the maximum oestradiol concentrations reached after the injection of human chorionic gonadotrophin (HCG) (r = 0.20, P < 0.001), with the mean number of follicles punctured (r = 0.21, P < 0.01), with the mean number of oocytes collected (r = 0.30, P < 0.001) and with the size of the embryo cohort (r = 0.42, P < 0.001). Furthermore, a negative correlation was observed between the duration of stimulation, the number of ampoules of HMG injected and the means of scores of the transferred embryos (r = 0.10 and r = 0.16 respectively, P < 0.001). The mean score of the transferred embryos was analysed according to the number of collected oocytes for the transfers of two or three embryos separately (Tables II and III). The mean score of the transferred embryos was significantly higher when the cohort contained more than six oocytes (P < 0.001) for both groups of transfer (Tables II and III
). Furthermore, implantation and delivery rates significantly increased with the size of the cohort (Tables II and III
).
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Patients had a tendency to produce a similar embryo quality from cycle to cycle. For the group of patients who underwent three successive IVF or ICSI cycles (n = 176), a strong correlation was observed between the means of embryo scores from cycle to cycle. The Pearson correlation coefficients for the means of embryo scores were r = 0.25 (P < 0.01) between the first and second cycles and r = 0.24 (P < 0.01) between the first and third cycles. These coefficients were higher for the means of scores of the transferred embryos, r = 0.33 (P < 0.01) between the first and second cycles, r = 0.31 (P < 0.01) between the first and third cycle and r = 0.47 (P < 0.01) between the second and third cycles.
A stepwise linear regression demonstrated that the quality of the embryos (partial coefficient r = 0.81, P < 0.001) and the size of the cohort (partial coefficient r = 0.65) influenced the quality of the transferred embryos (multiple r square = 0.66, P < 0.001).
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Discussion |
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In human IVF, the selection of a good embryo, i.e. one that should implant and give rise to a baby, remains a major issue. Consequently, the general practice is to transfer several embryos to boost the chances of pregnancy with the associated risk of multiple pregnancy. In fact, most IVF programmes select the transferred embryos on their morphological appearance on the day of transfer. For cleavage stage embryos, several criteria have been shown to correlate with embryo viability including the evenness and number of blastomeres and the presence or absence of cytoplasmic fragmentation. The presence of fragments was shown to reduce implantation rates (Staessen et al., 1992; Giorgetti et al., 1995
; Alikani et al., 1999
). Furthermore, severely fragmented embryos had a lower ability to develop to the blastocyst stage in vitro and the resulting blastocysts had lower cell counts (Hardy et al., 1989
). Cleavage rate is another important factor reflecting the embryo viability (Edwards et al., 1984
; Edwards and Beard, 1999
). Slower cleaving embryos were demonstrated to have lower implantation rates (Bavister et al., 1995). However, the determinant factor seems to be the time frame necessary for the embryo to accomplish the different cell division. The score used in this study takes into account both the percentage of fragmentation and the cleavage stage reached by the embryo at the time of transfer. It has been correlated to implantation and delivery rates in previous reports (Puissant et al., 1987
; Devreker et al., 1999
). Furthermore, a comparison between homogeneous transfer of 2-cell versus 4-cell stage embryos showed that 4-cell stage yielded higher implantation rate for a similar degree of fragmentation compared to 2-cell stage embryos (unpublished data).
Maternal age is a critical parameter in human fertility. With increasing maternal age, pregnancy rates decrease while spontaneous abortion rates increase, reducing the likelihood of a delivery. Compromised oocyte or embryo viability, impaired endometrial receptivity or rather a combination of these factors can explain this decline in fertility. Oocyte donor programmes demonstrated that the more important factor is embryo viability (Navot et al., 1991; Sultan et al., 1994
). In this study, the main factor that influenced the probability of a good morphological embryo was the size of the cohort irrespective of maternal age. Patients >38 years had a similar embryo quality compared to their younger counterparts when taking into account the number of collected oocytes or the size of the embryo cohort. Rather, maternal age influences the ovarian response. With ageing, progressive depletion of the ovarian follicle store occurs with a decrease in the number of oocytes that can be harvested (Janny and Ménézo, 1996
). With a decreasing number of oocytes, fewer embryos are available, reducing the possibility of choice for transfer.
However, embryo morphology imperfectly reflects the embryo viability. It has been shown that embryos that appear perfect can contain chromosomal abnormalities and that the frequency of these abnormalities increased with the age of the women (Munné et al., 1995). This could explain the lower implantation rate observed in this study for the older women when compared with a similar embryo quality for each embryo cohort size.
Having taken into account the embryo cohort size and the maternal age, the distribution of embryo quality was equivalent whatever the underlying causes of infertility. This suggests that infertility problems affect IVF outcome by interfering with other parameters than embryo quality (Flucker et al., 1993). Endometriosis could interfere in the interactions between the embryo and the endometrium explaining the lower implantation rates observed (Damewood et al., 1990; Dmowski et al., 1995; Arici et al., 1996
). Furthermore, in this study patients were treated with gonadotrophin-releasing hormone (GnRH) analogue in a long protocol, probably reducing the impact endometriosis could have on embryo quality. PCOS patients were reported to have impaired embryo viability due to recruitment of immature follicles producing immature oocytes with reduced potential even if it fertilized normally (Pellicer et al., 1989
; Dor et al., 1990
). However, blastocyst development and embryo metabolism were not impaired in PCOS patients compared to patients suffering from tubal infertility (Hardy et al., 1995
). Although male infertility has been related to poor embryonic development (Ron-El et al., 1991
; Parinaud et al., 1993
; Janny and Ménézo, 1994
), a lower embryo quality was not observed in couples with abnormal semen compared with other causes of infertility. Although culture to the blastocyst stage would be more appropriate to observe a paternal effect on embryo development.
Patients tended to produce a similar embryo quality from cycle to cycle. This was independent of maternal age. Despite their young age, several women produced embryo cohorts of poor quality. This reflects that embryo quality is an inherent feature of the woman and probably depends, among other factors, on oocyte maturity. Patients with consistently poor quality embryos could produce a majority of incompetent oocytes, an independent feature that could be responsible for the infertility. This favours the supporters of a policy of low dose stimulation protocol, as whatever the number of embryos collected the embryo quality will remain the same. However, the relationship observed between the size of the cohort and the embryo quality suggests that for these patients a mild form of ovarian stimulation could compromise their chances.
The main factor, then, which determines the probability of good quality embryos was the embryo cohort size. The quality of the transferred embryos was higher for patients that had more than five embryos compared to patients with fewer than five embryos available (P < 0.01).
This confirms a previous observation (Templeton and Morris, 1998) in a large retrospective study where the number of embryos available was more important in determining outcome than the actual number of transferred embryos.
The transfer of embryos with a high viability will allow reduction of the number of embryos transferred, leading to a reduction in the risk of multiple pregnancy. This is a major issue in assisted reproduction programmes. To avoid multiple pregnancies as much as possible, including twins, the transfer policy should be modified to replace only one embryo in a selected group of patients. This policy favours authors recommending the use of milder forms of ovarian stimulation (Edwards et al., 1996). This transfer policy indirectly implies that the collection of a maximum of oocytes and embryos is unnecessary. Why is it necessary to collect
10 embryos if only one or two embryos are going to be transferred? There is no doubt that mild ovarian stimulation will reduce the inherent risk of this treatment, including hyperstimulation syndrome, excessive exposure to high oestradiol levels or the controversial risk of ovarian cancers (Edwards et al., 1996
). Several patients could benefit from a milder form of stimulation (Olivennes et al., 1996
), although no randomized studies comparing those low dose protocols to standard protocols have been performed. In the current study, the embryo cohort size was the major factor determining the quality of the transferred embryos. A low dose protocol could result in a small embryo cohort that in turn will limit the choice if there is going to be a choice at all. On the other hand, supernumerary embryos of good morphology can be frozen. Freezing programmes increase the chance of pregnancy per cycle without patients undergoing the complete IVF treatment.
However, the evaluation of embryos on day 2 post-insemination is probably inaccurate, because during the first 48 h post-fertilization the embryo relies on maternal transcripts and has not already activated its own genome. Moreover, patients produced heterogeneous embryo cohorts, as reflected by differences in their development and implantation potential (Gregory, 1998). It becomes necessary to define new criteria to help in the selection of embryos with high viability. These criteria could include closer observations of oocytes (Scott and Smith, 1998
), culture in vitro to the blastocyst stage or identification of new markers of follicular maturity. The embryo's, viability directly depends on the oocytes from which it comes and oocyte viability depends on events occurring during the oocyte maturation (Jones et al., 1983
). A better understanding of follicular maturation could help to understand failure in embryo development or implantation (Gregory, 1998
). It has been observed that cell division followed a polarity that specifically distributed cell constituents between blastomeres which could be important for further development (Payne et al., 1997
; Edwards and Beard, 1999
). Human blastocyst culture in vitro with the new sequential media should help in the selection of the most viable embryo. To reach the blastocyst stage, embryos experienced different important steps including the activation of the embryonic genome, formation of the blastocoele cavity and the first differentiation between the trophectoderm and inner cell mass. Authors reported encouraging results for a selected group of patients (Gardner et al., 1998
; Menezo et al., 1998
). However, that the blastocyst transfer to all patients is the best solution still remains to be proven in well conducted randomized studies. A scoring system using a combination of oocyte and embryo morphology parameters together with markers of follicular maturity would probably help in the understanding of factors influencing the embryo viability.
In conclusion, a large embryo cohort increased the frequency of embryos of good morphology in the cohort independently of the maternal age or the causes of infertility. The possibility of choosing embryos for transfer remains the main determinant of outcome. Prospective and randomized studies should determine the best ovarian stimulation protocol and improve embryo selection for each patient, resulting in the highest chances of pregnancy associated with a minimum risk related to the procedure.
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Acknowledgments |
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Notes |
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References |
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Submitted on January 15, 1999; accepted on August 27, 1999.