Comparison of implantation and pregnancy rate using two methods of embryo selection: Pronuclear morphology and embryo quality and embryo morphology alone
Robab Davar, M.D.,Shamsi Beigi, M.D.,Mehrdad Soleimani, Bs.C.
Code Number: mf07038
Keywords: implantation, pronuclear morphology, pregnancy
Despite many advances in the field of reproductive medicine, success rate of IVF and embryo transfer in the human is low, with <30% of embryos that are transferred ever resulting in clinical pregnancies (1). Because of this implantation rates relatively low, the practice of transferring multiple embryos (n = 2-6) in order to increase the likelihood of pregnancy, is a common practice, which has led to an unacceptable level of multiple pregnancies.
Selection of the best embryo for transferring was the focus of multiple studies by scientists. Current techniques for this purpose are on the embryo morphology, its metabolic activity and culture of them on the advance media to achieve blastocystic phase. Researchers try to find the reliable markers for susceptibility of embryo life. One of these markers is distance between pronucleus and their size, number and situation of nucleolus precursor bodies at the one-cell stage diagram no. 1 (5,12). This technique is a noninvasive easy and suitable method for embryo selection (2, 3) overall the occurrence of pregnancy is predictable only by the embryological factors therefore clinical parameters is important as well in some cases. (4)
This study compared implantation and pregnancy rate in two methods of embryo selection by "pronuclear morphology and embryo quality" and by "embryo morphology alone.
MATERIALS AND METHODS
In this randomized double blind clinical trial, 95 patients(45 in A group and 50 in B group) referring to the Yazd Research and Clinical Center for Infertility for IVF (in vitro fertilization) or ICSI (Intra cytoplasmic sperm injection) treatment were included from March to December 2004. These patients randomly allocated in two groups. Different questioners (A & B) for each group were prepared and randomly were distributed to the patients.
In group A, embryo selection was done by pronuclear morphology(Scott PN scoring) (2 , 5) and embryo quality while in group B, this process was done by embryo morphology methods alone(3). ≤4 of the best selected embryo was transfer to the uterus.
All women with age of less than 40 years old and any causes of infertility were included in our study. Variables such as age of patients, infertility duration, reason of infertility, chemical and clinical pregnancy, and implantation rate were evaluated in this study. Oocyte quality (3) was documented as immature (GV), immature (M I), mature (M II) and very mature (M II) and post mature. Pronuclear morphology was recorded Z1 Z2 Z3 and Z4 pattern. Embryo morphology was assessed according to 1: poor, 2: fair, 3: good, and 4: excellent.
In case group Embryo selection was done by pronuclear morphology 16-18 h after ICSI or insemination and embryo quality in third day while in control group this process was done only by embryo quality in third day. All data were collected via questioner, interview and examination during study with 6 weeks follow-up time.
In present study, at first, 0.5 cc GnRH-a was injected at mid Luteal phase of previous cycle (21st day) and its dosage were reduced to 0.25 cc on first day of cycle. Then HMG 2-3 Amp/day was started from second day of cycle. Monitoring of follicular growth was done by transvaginal sonography started from 9th day of the cycle and stradiol measurement. When one follicle ≥ 18 mm and 3 - 4 follicle ≥ 14 mm was developed and estradiol level was 600 pg/ml and 36 hours after hCG injection, ovum retrieval was done. Oocytes were classified according to their quality. According to cause of infertility ICSI or IVF was done.
In case group pronuclear morphology was assessed 16-18 hours after ICSI or insemination. In both groups embryo morphology was evaluated in third day and 72 hours after ovum retrieval. Finally ≤4 embryos with best quality were selected and transferred to uterus. The best embryos were selected from Z1 or Z2 quality or and if there was no Z1 or Z2 embryo, from Z3, Embryo with Z4 quality wasnt transferred. Luteal phase support was done by 100 mg progesterone. Diagnosis of chemical pregnancy was done by b-hCG measurement 14 days after transfer.
Diagnosis of clinical pregnancy was performed by detection of fetal heart beat in sonography 5 - 6 weeks after transfer and implantation rate was measured by number of pregnancy sac per number of embryo transferred.
Data was entered in SPSS statistical software and were analyzed by chi-square, Fisher Exact and ANOVA tests and probabilities less that 0.05 were assigned significant.
In total, 95 IVF and ICSI cycle were analyzed in 10 months period. In case group, 419 (9.3 ± 6.4 in each cycle) and in control group, 444 oocytes (8.8 ± 4.8 in each cycle) were collected. Overall, 275 oocytes in case and 283 oocytes in control group were metaphase II mature oocytes.
In case group 129 embryos and in control group 151 embryos were transferred.
Table 1. Situation of variables of samples in two groups of present study
Mean age of women were 28.97 ± 4.5 and 28.97 ± 4.3 years in case and control groups respectively. Mean duration of infertility period were 7.95 ± 5.2 years in case and 7.92 ± 5 years in control group. There werent significant differences between two groups according to age, duration of infertility and number of oocytes. (Table 1)
Regarding the causes of infertility, there werent significance differences between two groups. ART procedures (IVF or ICSI) were the same in two groups. (P-value = 0.945) the result indicated that 15 chemical pregnancy occurred in case group and 10 in control group. The chemical pregnancy rates were 33.3% and 20%. In cases and control group respectively. Numbers of clinical pregnancies were 14 and 9 in case and control groups and theses rates were 31.1% and 18% respectively. Significant differences regarding the clinical and chemical pregnancy werent seen between groups. Implantation rate was 14% in case and 7.9% in control group. Implantation rate was significantly higher in case group than control group. (P-value = 0.05) . (Table 2)
There was 4 twin pregnancy in case and 3 in control group. In addition, there were 3 abortions in case and 2 abortions in control group. There was no abortion in twin pregnancy.
Regarding the quality of embryo comparison between two groups, there was no differences in quality 4 embryo (P-value=0.15), but in quality 3 embryo significant difference was seen between two groups. (P-value = 0.048) (Figure 1).
The number of transferred embryos in control group was more than that in case group. (Figure 2)
In total, Embryos from Z2 morphology pattern of pronuclear were more transferred than other patterns. (P-value = 0.013) while Embryos from Z1 morphology pattern of pronuclear were more than Z2 or Z3 patterns in positive pregnancy cases. (P-value = 0.0056) (Figure 3).
According to pronuclear morphology classification, if there was at least one embryo from Z1 pattern it was classified as X type and if there was no embryo from Z1 pattern as nX type. Clinical pregnancy rate in cases that had at least one transferred embryo from Z1 pattern was significantly higher than other groups. (P-value = 0.024) in addition implantation rate was more in type X than nX group, but this difference wasnt significant (Table 3).
According to other type of classification, embryos that only derived from Z1 or Z2 pattern were named type A and if at least had one embryo derived from Z1 or Z2 were named type B and in cases that hadnt any embryo derived from Z1 or Z2 morphology pattern were named type C. There wasnt significant difference between type A and B in clinical pregnancy rate. (P-value=0.363) (Table 4).
Table 2. Comparison of chemical and clinical pregnancy and implantation rate in two study groups Implantation
In case group, when implantation and pregnancy rate were more than cases that 2 or 4 embryos were transferred. While in control group similar results were seen if 4 embryos were transferred. (Table 5)
In case group, from 419 derived oocytes, 275 oocytes were mature (M II). And fertilization was occurred in 239 oocytes (86%). while in control group, from 444 derived oocytes, 283 oocytes were mature (M II), and fertilization occurred in 234 oocytes (82%).
In case and control groups, 60% of embryos with quality 4 were derived from mature oocytes (MII) while no embryo with quality 4 was derived from very mature oocytes. (Figure 4a, b)
18 (6.8%) and 20 (7.2%) embryos respectively had arrested from Z3 or Z4 pattern in case and control groups, while 118 (72%) embryos with quality 4 were derived from Z1 or Z2 pattern.
Table 3. Comparison of clinical pregnancy and implantation rates according to transferred embryos pattern in case group.
Thus embryos with 4 level of quality were more derived in Z1 or Z2 pattern than other pattern and risk of arrest increases in embryos from other patterns (Figure 5).
Z1 and Z2 embryos were derived from mature situation (M II) in 61% of cases were derived and wasnt derived no Z1 and Z2 embryos. In immature situation (M I) only Z3 or Z4 embryos. (Figure 6)
in classification of transferred embryos according to quality of them, cases that only quality 4 embryo were derived from them were named type D, while cases that at least had one quality 4 embryo was named type E and cases that hadnt any quality 4 embryo was named type F.
Clinical pregnancy and implantation rates were higher if quality 4 embryos that were selected with consecutive morphologic and pronuclear evaluation than embryos that were selected only with embryo morphology in third day but this difference wasnt significant. (Table 6)
Table 4. Comparison of clinical pregnancy and implantation rates according to transferred embryos pattern in case group.
The present study showed that there is a relationship between the zygote morphology 16 18 hours after insemination or ICSI and their maturation potential in vitro or in vivo. The results indicated that Implantation rate was significantly higher in case group than this rate in. control group, while chemical and clinical pregnancy rate weren't significantly higher in case group. Performing another study with more sample size could possibly show significance differences in these rates. According to our results, implantation and pregnancy rates were increased with embryo selection using pronuclear morphology and embryo morphology in third day.
C. Wittemer had the same results with our study, study which showed the pregnancy and implantation rates of 34.4% and 19.8% respectively (4). In case group, Clinical pregnancy and implantation rates were 38% and 21% in Zsolt P study (2).
Table 5. Comparison of pregnancy and implantation rate according to number of transferred embryos in our study Groups
In Scott study, implantation and pregnancy rate in cycles that were transferred in third day with pronuclear scoring system were significantly higher than other groups. (5) Our results were same to Lukaszuk study that was done on 62 cases of ICSI. He reported significant results for pregnancy rate in embryos that were transferred with 0, 1 (equal Z1 and Z2 ) or 2 pattern of zygote morphology (6).
Frequencies distributions of our pronuclear pattern (Z1, Z2, Z3, and Z4) were 14.8%, 41.8% 34.6% and 8.7% consequently. In Wittemer study, 58% of cases were Z1 and Z2 and 42% of that were Z3 and Z4 this results were same to our study. Lynette and Gardner reported the same frequencies for Z1, Z2, Z3 and 6% for Z4 (4, 5).
Table 6. Comparison of pregnancy and Implantation rates according to transferred embryos quality patterns in two study groups
Pregnancy rate in cases that had Z1 and then Z2 and Z3 morphology pattern and in X type were higher than other pattern or types, but difference wasn't significant. Montag had written same result. (7) In present study, pregnancy rate in A and B morphology groups weren't significantly different. But in Wittemer study, this difference was significant (4).
Production of 4 embryos quality was increased with elevation in oocytes quality and maturation (M II). These results were confirmed in other studies (8). We must consider to selection of better ovulation induction protocol for improving oocyte quality and achievement to better embryos.
According to number of transferred embryo, pregnancy rate was higher in case group than control group with transferring 2 or 3 embryos. With transferring 4 embryos pregnancy rate in control group was similar to case group by transferring 3 embryos. Implantation rate was higher in case group with 3 embryos transferring than control group with 3 or 4 embryos transferred. Thus with improving scoring method in embryo selection, number of embryos that were needed for transferring were decreased without decreasing PR and we can limited multiple pregnancies.
According to results of one study that was done on the 956 embryos, pregnancy rate per each time of transferring of 1, 2, 3 or 4 embryos were 12%, 40%, 53%, 43% consequently. In poor response cycles, stimulation cycles must be forbidden and prevent form selecting less than 3 oocytes. (9) We must consider some factors such as age of mother, embryo quality, and history of unsuccessful IVF cycles for embryo selection. In most of the therapeutic programs 3 or 4 embryo were transferred to uterus and transferring of less than 3 embryo in under 35 years old women was not recommended. Affinity to embryo transferring in blastocystic phases (5 days after oocyte retrieval) was increased due to new improvement in embryo implantation techniques. (10) Because of unknown causes, homozygotic multiple pregnancies was increased with embryo transferring in blastocytic phases and delivery complications were increased (11).
Training for improving duration of embryo growth was done in some laboratories. And they use morphologic parameters for embryo selection. Pregnancy and implantation rates were higher with selection of suitable therapeutic method and better embryos of Z1 or Z2 levels, than selection only on the embryo quality in third day. According that we can decrease number of embryos and decrease chance of multiple pregnancies. Until to achievement of better methods and confirmation of blastocytic transferring method, we can use this method in infertility centers.
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