Comparison of Implantation and Pregnancy Rate Using Two Methods of Embryo Selection: “Pronuclear Morphology and Embryo Quality” and “Embryo Morphology Alone”

Middle East Fertility Society Journal Vol. 12, No. 3, 2007 © Copyright Middle East Fertility Society

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. Mehrdad Soleimani, Bs.C. Shamsi Beigi, M.D.

Research and clinical center for infertility, Shahid Sadoughi University of medical sciences and health services, Yazd, Iran

ABSTRACT

Objective: Despite many advances in the field of reproductive medicine, with <30% of embryos that are transferred ever resulting in clinical pregnancies. This study compared implantation and pregnancy rate in two methods of embryo selection. Design: randomized double blind clinical trial Materials and Methods: Research and clinical center for infertility, Yazd, Iran Subject: In this randomized double blind clinical trial, 95 patients referring to the Yazd Research and Clinical Center for Infertility for IVF or ICSI treatment were included from March to December 2004. These patients randomly allocated in two groups. (A & B) In group A (Case), embryo selection was done by pronuclear morphology and embryo quality while in group B (Control), this process was done by embryo morphology methods alone. Oocytes were classified according to their quality. According to cause of infertility ICSI or IVF was done. Finally ≤ 4 embryos with best quality were selected and transferred to uterus. Main outcome measures: Implantation rate, Pregnancy Rate Results: In case group, 419 and in control group, 444 oocytes were collected. In case group 129 embryos and in control group 151 embryos were transferred. Number and percent of clinical pregnancies was 14 (31.11%) and 9 (18%) and implantation rate was 18 (13.95%) and 12 (7.94%) in case and control groups. Conclusion: Until to achievement of better methods and confirmation of blastocystic transferring, we can use from this method in Infertility center. Keywords: implantation, pronuclear morphology, pregnancy

Despite many advances in the field of transferring multiple embryos (n = 2-6) in order to reproductive medicine, success rate of IVF and increase the likelihood of pregnancy, is a common embryo transfer in the human is low, with <30% of practice, which has led to an unacceptable level of embryos that are transferred ever resulting in multiple pregnancies. clinical pregnancies (1). Because of this Selection of the best embryo for transferring implantation rates relatively low, the practice of was the focus of multiple studies by scientists. Current techniques for this purpose are on the Corresponding Author: Robab Davar, M.D. ; Email: embryo morphology, its metabolic activity and [email protected], Research and clinical center for culture of them on the advance media to achieve infertility, Shahid Sadoughi University of medical sciences blastocystic phase. Researchers try to find the and health services, Bouali Avenue, Safayeh reliable markers for susceptibility of embryo life. P.O. Box: 89195-999, Postal Code: 8916877391, Yazd, Iran, Telephone: +98 351 8247085, Fax: +98 351 8247087 One of these markers is distance between pronucleus and their size, number and situation of

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Diagram 1. The Z score describes the number, size and position of the nucleoli and the equality between the nuclei for these characteristics. Z-1 zygotes have equal numbers of nucleoli between 3-7 that are aligned at the pronuclear junctions. Z-2 zygotes have equality in size and number between the nuclei, but the nucleoli have not yet aligned at the pronuclear junction. Z-3 zygotes are characterized by inequality between the nuclei; unequal sized nucleoli, unequal numbers of nucleoli, or unequal alignment at the pronuclear junction. Z-4 zygotes are grossly abnormal and present with unequal sized nuclei, nuclei that have not aligned, small, and misplaced nuclei.

nucleolus precursor bodies at the one-cell stage included from March to December 2004. These diagram no. 1 (5,12). This technique is a patients randomly allocated in two groups. noninvasive easy and suitable method for embryo Different questioners (A & B) for each group were selection (2, 3) overall the occurrence of pregnancy prepared and randomly were distributed to the is predictable only by the embryological factors patients. therefore clinical parameters is important as well in In group A, embryo selection was done by some cases. (4) pronuclear morphology(Scott PN scoring) (2 , 5) This study compared implantation and and embryo quality while in group B, this process pregnancy rate in two methods of embryo selection was done by embryo morphology methods by "pronuclear morphology and embryo quality" alone(3). ≤4 of the best selected embryo was and by "embryo morphology alone. transfer to the uterus. All women with age of less than 40 years old and any causes of infertility were included in our MATERIALS AND METHODS study. Variables such as age of patients, infertility duration, reason of infertility, chemical and clinical Patients pregnancy, and implantation rate were evaluated in this study. Oocyte quality (3) was documented as In this randomized double blind clinical trial, 95 immature (GV), immature (M I), mature (M II) and patients(45 in A group and 50 in B group) referring very mature (M II) and post mature. Pronuclear to the Yazd Research and Clinical Center for morphology was recorded Z1 – Z2 – Z3 and Z4 Infertility for IVF (in vitro fertilization) or ICSI pattern. Embryo morphology was assessed (Intra cytoplasmic sperm injection) treatment were according to 1: poor, 2: fair, 3: good, and 4: excellent.

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Figure 1. Distribution of transferred embryos regarding the quality of embryo. (129 & 151 embryos respectively in case & control group). Z-test , * p = 0.15 , ** p = 0.048.

In case group Embryo selection was done by both groups embryo morphology was evaluated in pronuclear morphology 16-18 h after ICSI or third day and 72 hours after ovum retrieval. Finally insemination and embryo quality in third day while ≤4 embryos with best quality were selected and in control group this process was done only by transferred to uterus. The best embryos were embryo quality in third day. All data were selected from Z1 or Z2 quality or and if there was collected via questioner, interview and no Z1 or Z2 embryo, from Z3, Embryo with Z4 examination during study with 6 weeks follow-up quality wasn’t transferred. Luteal phase support time. was done by 100 mg progesterone. Diagnosis of chemical pregnancy was done by β-hCG Methods measurement 14 days after transfer. Diagnosis of clinical pregnancy was performed In present study, at first, 0.5 cc GnRH-a was by detection of fetal heart beat in sonography 5 - 6 injected at mid Luteal phase of previous cycle weeks after transfer and implantation rate was (21st day) and its dosage were reduced to 0.25 cc measured by number of pregnancy sac per number on first day of cycle. Then HMG 2-3 Amp/day was of embryo transferred. started from second day of cycle. Monitoring of Data was entered in SPSS statistical software follicular growth was done by transvaginal and were analyzed by chi-square, Fisher Exact and sonography started from 9th day of the cycle and ANOVA tests and probabilities less that 0.05 were stradiol measurement. When one follicle ≥ 18 mm assigned significant. 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 RESULTS were classified according to their quality. According to cause of infertility ICSI or IVF was In total, 95 IVF and ICSI cycle were analyzed done. in 10 months period. In case group, 419 (9.3 ± 6.4 In case group pronuclear morphology was in each cycle) and in control group, 444 oocytes assessed 16-18 hours after ICSI or insemination. In (8.8 ± 4.8 in each cycle) were collected. Overall, 275

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Distribution of transferred embryos

Figure 2. Distribution of 129 transferred embryos in case group according to PN morphology.

oocytes in case and 283 oocytes in control group Mean age of women were 28.97 ± 4.5 and 28.97 ± were metaphase II mature oocytes. 4.3 years in case and control groups respectively. In case group 129 embryos and in control group Mean duration of infertility period were 7.95 ± 5.2 151 embryos were transferred. years in case and 7.92 ± 5 years in control group. There weren’t significant differences between two groups according to age, duration of infertility and Table 1. Situation of variables of samples in two groups of number of oocytes. (Table 1) present study Regarding the causes of infertility, there

Case Control p-value weren’t significance differences between two groups. ART procedures (IVF or ICSI) were the Range of Infertility 1-20 1-19 - same in two groups. (P-value = 0.945) the result duration indicated that 15 chemical pregnancy occurred in Mean of Infertility 7.95 ± 5. 2 7.92 ± 5 0.973 duration ± SD (year) case group and 10 in control group. The chemical Range of ages 20-38 20-39 - pregnancy rates were 33.3% and 20%. In cases and Mean of ages ± SD 28.97 ± 4.5 28.92 ± 4.3 0.949 control group respectively. Numbers of clinical (year) pregnancies were 14 and 9 in case and control Range of oocyte were 3-30 3-22 - groups and theses rates were 31.1% and 18% gotten per each cycle Mean of oocyte per 9.31 ± 6.4 8.88 ± 4.8 0.710 respectively. Significant differences regarding the cycle ± SD clinical and chemical pregnancy weren’t seen Range of transferred 2-4 2-4 - between groups. Implantation rate was 14% in case embryos per each cycle and 7.9% in control group. Implantation rate was Mean of transferred 2.87 ± 0.55 3.04 ± 0.7 0.185 embryos per cycle ± SD significantly higher in case group than control group. (P-value = 0.05) . (Table 2)

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Transferred embryos Transferred

Pronuclear morphology

Figure 3. Distributing of 43 transferred embryos in positive pregnancy case according to PN morphology from 129 transferred embryos. Z-Test , * p =0.013 , **p =0.0056

There was 4 twin pregnancy in case and 3 in type X than nX group, but this difference wasn’t control group. In addition, there were 3 abortions significant (Table 3). in case and 2 abortions in control group. There was According to other type of classification, no abortion in twin pregnancy. embryos that only derived from Z1 or Z2 pattern Regarding the quality of embryo comparison were named type A and if at least had one embryo between two groups, there was no differences in derived from Z1 or Z2 were named type B and in quality 4 embryo (P-value=0.15), but in quality 3 cases that hadn’t any embryo derived from Z1 or embryo significant difference was seen between Z2 morphology pattern were named type C. There two groups. (P-value = 0.048) (Figure 1). wasn’t significant difference between type A and B The number of transferred embryos in control in clinical pregnancy rate. (P-value=0.363) (Table group was more than that in case group. (Figure 2) 4). In total, Embryos from Z2 morphology pattern of pronuclear were more transferred than other Table 2. Comparison of chemical and clinical pregnancy and patterns. (P-value = 0.013) while Embryos from Z1 implantation rate in two study groups Implantation morphology pattern of pronuclear were more than Z2 or Z3 patterns in positive pregnancy cases. (P- value = 0.0056) (Figure 3). Study groups Case Control P-value

According to pronuclear morphology Cycles (N) 45 50 classification, if there was at least one embryo Transferred embryos (N) 129 151 from Z1 pattern it was classified as X type and if Chemical Pregnancy (N) 15 10 there was no embryo from Z1 pattern as nX type. Clinical Pregnancy (N) 14 9 Clinical pregnancy rate in cases that had at least Gestational Sac (N) 18 12 Chemical pregnancy Rate (PR%) 33.33 20 0.14 one transferred embryo from Z1 pattern was Clinical pregnancy Rate (PR%) 31.11 18 0.136 significantly higher than other groups. (P-value = Implantation Rate (IR %) 13.95 7.94 0.05 0.024) in addition implantation rate was more in

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Figure 4. Distribution of quality of embryos arrest of growth 3 PN & no fertilization in case (4-a) and control groups (4-b) according to quality .of oocyte.

In case group, when implantation and pregnancy Thus embryos with 4 level of quality were more rate were more than cases that 2 or 4 embryos were derived in Z1 or Z2 pattern than other pattern and risk transferred. While in control group similar results of arrest increases in embryos from other patterns were seen if 4 embryos were transferred. (Table 5) (Figure 5). In case group, from 419 derived oocytes, 275 Z1 and Z2 embryos were derived from mature oocytes were mature (M II). And fertilization was situation (M II) in 61% of cases were derived and occurred in 239 oocytes (86%). while in control wasn’t derived no Z1 and Z2 embryos. In immature group, from 444 derived oocytes, 283 oocytes were situation (M I) only Z3 or Z4 embryos. (Figure 6) mature (M II), and fertilization occurred in 234 in classification of transferred embryos according oocytes (82%). to quality of them, cases that only quality 4 embryo In case and control groups, 60% of embryos were derived from them were named type D, while with quality 4 were derived from mature oocytes cases that at least had one quality 4 embryo was (MII) while no embryo with quality 4 was derived named type E and cases that hadn’t any quality 4 from very mature oocytes. (Figures 4-a , 4-b) embryo was named type F. 18 (6.8%) and 20 (7.2%) embryos respectively Clinical pregnancy and implantation rates were had arrested from Z3 or Z4 pattern in case and higher if quality 4 embryos that were selected with control groups, while 118 (72%) embryos with quality 4 were derived from Z1 or Z2 pattern. Table 4. Comparison of clinical pregnancy and implantation rates according to transferred embryos pattern in case group.

Transferred embryos A B C Total P-value Table 3. Comparison of clinical pregnancy and implantation pattern rates according to transferred embryos pattern in case group. Number of cycles 16 25 4 45 Transferred embryos pattern X nX Total P-value Transferred embryos 44 76 9 129 number Number of cycles 18 27 45 Mean of transferred 2.75 3.04 2.25 2.87 Transferred embryos number 53 76 129 embryos number Mean of transferred embryos 2.94 2.81 2.87 Clinical pregnancy (N) 4 10 0 14 Clinical pregnancy (N) 9 5 14 Gestational sac (N) 5 13 0 18 Gestational sac (N) 11 7 18 Clinical pregnancy rate 25 40 0 31.11 P=0.363 Clinical pregnancy rate (PR%) 50 18.51 31.11 P=0.024 (PR%) implantation rate(IR%) 20.75 9.21 13.95 P=0.063 implantation rate(IR%) 11.36 17.10 0 13.95 P=0.396

Pearson chi-square Pearson chi-square

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Figure 5. Distribution of 263 zygotes according to pronuclear morphology

consecutive morphologic and pronuclear evaluation group, while chemical and clinical pregnancy rate than embryos that were selected only with embryo weren't significantly higher in case group. morphology in third day but this difference wasn’t Performing another study with more sample size significant. (Table 6) could possibly show significance differences in these rates. According to our results, implantation and pregnancy rates were increased with embryo DISCUSSION selection using pronuclear morphology and embryo morphology in third day. The present study showed that there is a C. Wittemer had the same results with our study, relationship between the zygote morphology 16 – study which showed the pregnancy and implantation 18 hours after insemination or ICSI and their rates of 34.4% and 19.8% respectively (4). In case maturation potential in vitro or in vivo. The results group, Clinical pregnancy and implantation rates indicated that Implantation rate was significantly were 38% and 21% in Zsolt P study (2). higher in case group than this rate in. control

Table 5. Comparison of pregnancy and implantation rate according to number of transferred embryos in our study Groups

Embryo transferred per cycle 2 3 4

case control Case Control Case Control Number of cycles 10 12 31 25 4 13 Number of embryos transferred 20 24 93 75 16 52 Number of clinical pregnancies 2 0 12 5 0 4 Number of gestational sac 2 0 16 5 0 7 Clinical Pregnancy rate (PR %)* 20 0 38.70 20 0 30.76 Implantation rate (IR %) 10 0 17.20 6.66 0 13.46

Pearson chi – square * P-value = 0.08 (Clinical pregnancy rate compared in transferring three embryos but this difference was non-significant. Probably with increasing the number of samples would be significant.)

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Mature (M-II) Very Mature (M-II) Immature (M-I) n= 239 n= 22 n= 2 Quality of fertilized oocyte

Figure 6. Distribution of PN morphology according to quality of oocyte from 263 zygote in case group.

In Scott study, implantation and pregnancy rate in Frequencies distributions of our pronuclear pattern cycles that were transferred in third day with (Z1, Z2, Z3, and Z4) were 14.8%, 41.8% 34.6% pronuclear scoring system were significantly higher and 8.7% consequently. In Wittemer study, 58% of than other groups. (5) Our results were same to cases were Z1 and Z2 and 42% of that were Z3 and Lukaszuk study that was done on 62 cases of ICSI. Z4 this results were same to our study. Lynette and He reported significant results for pregnancy rate in Gardner reported the same frequencies for Z1, Z2, embryos that were transferred with 0, 1 (equal Z1 and Z3 and 6% for Z4 (4, 5). Z2 ) or 2 pattern of zygote morphology (6).

Table 6. Comparison of pregnancy and Implantation rates according to transferred embryos quality patterns in two study groups

Transferred embryos pattern in D E F each cycle Case control Case Control Case Control Number of cycles 30 26 9 20 6 4 Transferred embryos number (N) 89 78 28 65 12 8 Number of clinical pregnancies 10 5 3 4 1 0 Number of gestational sac 14 6 3 6 1 0 Pregnancy rate (PR %)* 33.33 19.23 33.33 20 16.66 0 Implantation rate (IR %) 15.73 7.69 10.71 9.23 8.33 0 P value for PR% 0.397 Chi-square 0.209 Fisher ~1 fisher

* Pattern D: If only quality 4 embryo were transferred. * Pattern E: If at least one quality 4 embryo were transferred. * Pattern F: If no quality 4 embryo were transferred. (Pregnancy rates were compared in two groups. Differences were not significant statistically but pregnancy rate was more in case group. Probably with increasing the number of sample would be significant.)

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Pregnancy rate in cases that had Z1 and then CONCLUSION Z2 and Z3 morphology pattern and in X type were higher than other pattern or types, but difference Training for improving duration of embryo wasn't significant. Montag had written same growth was done in some laboratories. And they result. (7) In present study, pregnancy rate in A use morphologic parameters for embryo selection. and B morphology groups weren't significantly Pregnancy and implantation rates were higher different. But in Wittemer study, this difference with selection of suitable therapeutic method and was significant (4). better embryos of Z1 or Z2 levels, than selection Production of 4 embryos quality was increased only on the embryo quality in third day. with elevation in oocytes quality and maturation According that we can decrease number of (M II). These results were confirmed in other embryos and decrease chance of multiple studies (8). We must consider to selection of pregnancies. Until to achievement of better better ovulation induction protocol for improving methods and confirmation of blastocytic oocyte quality and achievement to better transferring method, we can use this method in embryos. infertility centers. According to number of transferred embryo, pregnancy rate was higher in case group than control group with transferring 2 or 3 embryos. REFERENCES With transferring 4 embryos pregnancy rate in control group was similar to case group by 1. Edwards RG and Beard HK. Blastocyst stage transfer: transferring 3 embryos. Implantation rate was pitfalls and benefits. Hum. Reprod. 1999; 14: 1–6 2. Zsolt P Nagy MD. Pronuclear morphology evaluation higher in case group with 3 embryos transferring with subsequent evaluation of embryo morphology than control group with 3 or 4 embryos significantly increases implantation rates. Fertil Steril. transferred. Thus with improving scoring method 2003: 80; 67-74 in embryo selection, number of embryos that were 3. Alan o. Trounson. Handbook of IVF 2nd ed. CRC 1999: 188-195 needed for transferring were decreased without 4. Wittemer C. Zygote evaluation: an efficient tool for decreasing PR and we can limited multiple embryo selection. Hum Reprod 2000; 15: 2591 – 97. pregnancies. 5. Lynette Scott, The morphology of human pronuclear According to results of one study that was embryo is positively related to Blastocyst development done on the 956 embryos, pregnancy rate per each and implantation. Hum Reprod . 2000; 15: 2394 – 2403. 6. Lukaszuk, liss j, Prognostic value of the pronuclear time of transferring of 1, 2, 3 or 4 embryos were morphology pattern of zygotes for implantation rate. 12%, 40%, 53%, 43% consequently. In poor Ginekol pol 2003; 74: 508-13. response cycles, stimulation cycles must be 7. Montag M, van der H. Evaluation of pronuclear forbidden and prevent form selecting less than 3 morphology as the only selection criterion for further embryo culture and transfer: results of a prospective oocytes. (9) We must consider some factors such multicentre study. Hum Reprod 2001; 16: 2384-9 as age of mother, embryo quality, and history of 8. Pasquale patrizio. Micheel J. Tucker .A color Atlas for unsuccessful IVF cycles for embryo selection. In Human Assisted Reproduction Laboratory & clinical most of the therapeutic programs 3 or 4 embryo insights: Lippincot Williams & Wilkins. 2003: 71-88. were transferred to uterus and transferring of less 9. Tarlatzis B. Ovulation induction. ELSEVIER. 2003, 228. 10. Kenneth J RYAN .Kistner’s Gynecology & women’s than 3 embryo in under 35 years old women was health 7th edition . Mosby .1999 .346-350. not recommended. Affinity to embryo transferring 11. Janathan S. Berek. Novak's Gynecology 13th edition. in blastocystic phases (5 days after oocyte Lippincot Williams & Wilkins.2002 .1029 – 1033. retrieval) was increased due to new improvement 12. David K Gardner, Ariel Weissman, Colin M Howles, Zeev Shoham. “Hand book of assisted reproductive in embryo implantation techniques. (10) Because techniques” Martin Dunitz. 2001, 196 of unknown causes, homozygotic multiple pregnancies was increased with embryo transferring in blastocytic phases and delivery complications were increased (11).

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