{"paper_id":"73f0c534-90ab-4b3b-87af-799de5a7a532","body_text":"An important issue in assisted reproductive techniques\n(ART) is how many embryos could be transferred\nfor each couple. A number of variables considered\nfor a high success rate in  in vitro  fertilization\n(IVF) treatment may be followed by a high rate of\nmultiple pregnancies. Over 30% of IVF pregnancies\nare multiples which are associated with increasing\nmaternal and infant morbidity and mortality such as\n( 1 ,  2 ) hypertension, polyhydramnios, premature labor,\n( 1 ) low birth weight, higher perinatal mortality\nand congenital anomalies ( 1 ). Therefore, it is crucial\nto find proper methods to reduce multiple pregnancies\nwithout reducing the overall pregnancy rate.\nDespite recent recommendations for achieving acceptable\npregnancy rate with few multiple pregnancies,\none or two good quality embryos needs to be\nconsidered for transfer ( 3 - 6 ), but still in some countries\nincluding Iran, patients have an impression that\nincreasing the number of embryos transferred is associated\nwith increased pregnancy rate.\nSome studies have reported elective singleembryo\ntransfer (eSET) in IVF-intracytoplasmic\nsperm injection (ICSI) cycles prevents multiple\npregnancies without reduction of overall pregnancy\nrate ( 3 ,  4 ,  6 ), while some other studies believe\nthat eSET could be associated with a lower pregnancy\nrate per cycle, especially in an unselected\npopulation ( 5 ,  7 ,  8 ). Despite the efforts have been\nmade in order to limit the incidence of multiple\npregnancies after ART (e.g. by SET), the average\nIVF treatment includes transfer of two, three\nor sometimes even more embryos into the uterus,\nwhile use of eSET in clinical practice has not yet\nbeen performed. This may be due to the factors\ninfluencing the number of embryos transferred\nsuch as cost-effectiveness of eSET technique, professional\nattitudes and the financial situation of\ncouples. In a recent systematic review, Pandian\net al. ( 8 ) reported that insufficient data are available\non the outcome of two versus three and four\nETs policies. In Iran and some other parts of the\nworld where no legal restrictions exist, this is the\nresponsibility of infertility specialists and patients\nto make decision about the number of embryos\ntransferred with respect to the risks associated\nwith multiple gestations and acceptable pregnancy\nrates. A recent guideline stating the suitable number\nof embryos to transfer following an IVF cycle\nsuggests that a maximum of three or four embryos\ncan be transferred in women over the age of 39 ( 9 ).\nThe primary purpose of the present study was\nto investigate whether increasing the number of\nembryos transferred from two to three leads to an\nincrease in the overall pregnancy and live birth\nrates in women undergoing ICSI cycles, then the\nsecondary objective was to evaluate the impact of\nmaternal age on the outcome of IVF/ICSI according\nto the number of embryos transferred.\n\nThis retrospective study was performed at Reproductive\nBiomedicine Research Center, Royan\nInstitute, Tehran, Iran, from September 2006 to\nJune 2010. The Institutional Review Board of\nRoyan Institute was approved the study. All patients\nsigned a consent form in their initial visit\ngiving permission to use their results without using\ntheir names in the future studies. The study\npopulation consisted of 387 women with primary\ninfertility and with at least one fresh ET in good\nquality. Exclusion criteria were as following: use\nof clomiphene citrate; use of human menopausal\ngonadotropin (hMG) only in antagonist protocols;\nwomen with advanced age (≥40), women with\nbody mass index (BMI) ≥30, as well as history of\novarian hyperstimulation syndrome, uterine factor\ninfertility, severe endometriosis, hydrosalpinges\nand repeated implantation failure. All patients according\nto number of ETs were categorized into\ntwo groups: ET2 and ET3 groups, indicating two\nand three embryos were transferred.\nIn this study, all of the patients underwent a\nstandard long protocol using 500 μg gonadotropinreleasing\nhormone (GnRH)-a (Buserelin, Superfact,\nAventis Pharma Deutshlan, Frankfurt, Germany).\nOnce down-regulation was confirmed by\nlinear endometrium thickness in ultrasonography\nand serum estradiol concentration <50 pg/ml, the\nSuperfact dose was reduced by one-half (250 μg),\nand gonadotrophin stimulation with recombinant\nfollicular stimulating hormone (rFSH, Gonal-F,\nSerono Laboratories Ltd., Geneva, Switzerland)\nwas applied and continued until the day of human\nchorionic gonadotropin (hCG, IBSA, Switzerland)\nadministration. The first ultrasound scan was performed\non day 6 and the dose of rFSH was adjusted\naccording to the ovarian response. When at\nleast two follicles >18 mm were seen, 10000 IU\nurinary hCG (uhCG, Choriomon, IBSA, Switzerland) was injected intramuscularly and oocyte\nretrieval was performed 34-36 hours later. The\npresence of two pronuclei and two polar bodies\nwas assessed 16-18 hours after ICSI. Approximately,\n48 hours after injection, embryos were\nclassified based on morphological criteria ( 10 ).\nEmbryos with the best morphology and with the\nmost advanced stage of development were selected\nfor transfer. In our institute, the grading\nof embryos was performed by two embryologists\nwith same background. Two expert clinicians\nperformed the ETs and the difficult ETs\nwere excluded from study.\nIn our institute, a number of factors, including\nthe patient’s age, cause and duration of infertility,\nthe number and grade of the available embryos\nand requests of the couples, were taken into consideration\nin order to decide how many embryos to\ntransfer. ET performed on day 2 or 3 after oocyte\nretrieval and two or three embryos per patient were\ntransferred. Luteal-phase support was provided\nwith 400 mg vaginal progesterone (Aburaihan co.,\nTehran, Iran) twice a day until the day of beta-hCG\n(β-hCG) assay. If the result of β-hCG assay was\npositive, the same dosage of progesterone was\ncontinued up to 10 weeks of gestation. Clinical\npregnancy was defined as a positive pregnancy test\nfollowed by the presence of fetal sac on transvaginal\nultrasound 4 weeks later.\nData were analyzed using the Statistical Package\nfor the Social Sciences 16.0.0 (SPSS, SPSS\nInc. Chicago, IL, USA). Demographic factors,\nreproductive history, ART cycle-specific parameters,\nand pregnancy outcomes were compared\nbetween two groups using the chi-square\ntest for categorical variables and Student t test\nfor continuous variables when data was normally\ndistributed, whereas Mann-Whitney test\nwas used for abnormal cases. All tests were\ndone two tailed. Descriptive statistics are presented\nas mean ± standard deviation (SD) and\npercentage. Multiple logistic regression analysis\nwas used to evaluate the association between\nthe number of ET and live birth rate, adjusting\nfor potential confounding variable (age). We\nused Receiver Operating Curve (ROC) analysis\nto find the best cut point of age for prediction\nof live birth by regression equation. A value of\nP<0.05 was considered to be statistically significant.\n\nA total of 387 patients were included in this\nstudy, among whom 193 patients with two ETs and\n194 patients with three ETs. Pregnancy outcomes\nwere compared between ET2 and ET3 groups.\nThe demographic characteristics are demonstrated\nin  table 1 . Two groups had no difference in\nterms of infertility diagnosis, women’s BMI, infertility\nduration and the number of previous ART cycles.\nThe mean of maternal age in ET2 group was\nsignificantly higher than ET3 group (P<0.001).\nDemographic characteristics of women according to\nthe number of embryos transferred (ET)\nSD; Standard deviation and BMI; Body mass index.\nThe mean total dose of rFSH, duration of gonadotropin\nadministration, and the number of MII\noocyes were similar between two groups (P=0.7,\nP=0.6 and P=0.3, respectively). Furthermore, the\nmean number of oocytes retrieved and total embryos\nwere significantly higher in ET3 group compared\nto the ET2 group (P=0.001,  Table 2 ). Chisquare\ntest showed that the percent of patients with\nGrade C ET in ET3 group was higher significantly\n(P=0.02).\nOur results showed that pregnancy rates in patients\nwith two and three ETs were similar (P=0.7).\nThere was also no significant differences in terms\nof live birth (P=0.4), miscarriage and intrauterine\ndeath rate between two groups. Multiple pregnancy\nrate was significantly higher in the ET3 group compared to ET2 group ( Table 3 ).\nThe cut point for maternal age obtained by\nROC analysis for clinical pregnancy rate was 33\nyears. Logistic regression analysis revealed that\nonly age was predictable for clinical pregnancy\nrate in stimulating ICSI cycles [Odds ratio (OR):\n1.6, Confidence interval (CI):1.05-2.8] ( Table 4 ).\nFurthermore, logistic regression analysis for predictive\nfactors of live birth rate showed that the\nquality of transferred embryos and number of ET\nwere significantly predictable, so that the quality\nof ET was directly related to the live birth, but the\nnumber of ET had an inverse relationship with this\nvariable ( Table 5 ).\nAs shown in  table 6 , live birth rates were similar\nbetween ET2 and ET3 in women younger and\nolder than 33 years old. Therefore, a reduction in\nnumber of embryos transferred did not decrease\nthe clinical pregnancy and live birth rates in both\nage levels.\nCharacteristics of ICSI cycles of study population according to the number of embryos transferred (ET)\nData are presented as mean ± SD. ICSI; Intracytoplsamic sperm injection, SD; Standard deviation and M2; Metaphase II.\nICSI outcomes according to the number of embryos transferred (ET)\n*NS; Not significant, ICSI; Intracytoplsamic sperm injection and SD; Standard deviation.\nLogistic regression analysis for predicting the clinical pregnancy rate in ICSI cycles\nICSI; Intracytoplsamic sperm injection, OR; Odds ratio and CI; Confidence interval.\nLogistic regression analysis for predicting the live birth rate in ICSI cycles\nICSI; Intracytoplsamic sperm injection, OR; Odds ratio and CI; Confidence interval.\nAge related results according to number of embryos transferred (ET)\n*NS; Not significant and SD; Standard deviation.\n\nOur study indicated that in good prognosis patients\naged 39 years or younger, two and three ETs\nhave same pregnancy and live birth rates, while the\nmultiple pregnancy rate was significantly higher in\nET3 group; therefore, it is recommended to transfer\ntwo instead of three embryos.\nBased on a recent guideline, individual IVF-ET\ncenters should evaluate their own data to identify\npatient-specific, embryo-specific, and cycle-specific\nto determine factors of implantation and live\nbirth in order to develop ET protocols minimizing\nthe occurrence of multi-fetal gestation, while\npreserving acceptable overall pregnancy and live\nbirth rates ( 11 ).\nIn a recent study, Min et al. ( 9 ) presented a guideline\nfor the number of embryos transferred considering\nthe maternal age; however, this numbers can\nbe different in various infertility centers according\nto the laws of those countries. In a number of\ncountries, including Norway, Sweden, Denmark,\nBelgium, England, Italy, Germany, and Australia,\nthe complications associated with multiple pregnancies\nare reduced through use of SET by legal\nrestrictions, while many other European countries\nhave bordered to a maximum of two ETs.\nIn other parts of the world like Iran, there is no\nlegal restriction in this regard, and it is the responsibility\nof infertility specialists and patients\nto make decision about the number of embryos\ntransferred. Various strategies for eSET depend on\ndifferent funding methods of infertility treatments.\nThere are countries where the public sector covers\nthe majority of costs, whereas in some other courtiers,\npatients have to undertake the costs directly\nor indirectly through private insurance systems.\nDespite the recent emphasis and supports on eSET\n( 3 - 6 ,  12 ,  13 ), this technique in not generally used\nin Iran because of heavy treatment costs. SET in\nour institute is necessarily for some patients with\nspecial conditions such as poor ovarian response\nand male factor cases.\nIn agreement with the previous studies ( 14 -\n 17 ), we found similar clinical pregnancy and live\nbirth rates in patients with two versus three ETs,\nbut the multiple pregnancy rate in our study was\nsignificantly greater in group with three embryos\nthan two embryos transferred. Despite the fact\nthat women in ET3 group were younger than ET2\ngroup, but due to higher number of couples with\nmale infertility in the ET3 group, the number of\npatients with grade C embryos transferred were\nsignificantly higher than ET2 group. Therefore, no\nsignificant difference was observed between the\ntwo groups in terms of implantation, clinical pregnancy\nand live birth rates.\nWe evaluated the influence of maternal age on\nIVF/ICSI outcome for the number of ET and the\nobtained data indicated lower rate of multiple pregnancies\nin group with two embryos transferred as\ncompared to ET3 group with individuals of different\nages (younger or older than 33 years), while\nthe pregnancy and live birth rates were similar. In\ncontrast to our results, Giannini et al. ( 18 ) showed\nthat in older women (≥35 years), a reduction in the\nnumber of embryos transferred significantly decreased\nthe chances of pregnancy.\nLogistic regression analysis showed that one\nsignificant prognostic variable for clinical pregnancy:\nmaternal age (as categorical variable); it\nmeans that in women younger than 33 years old,\nthe chance of clinical pregnancy increased 1.6-\nfold. Our results were in line with Chuang et al.\n( 19 ) that age is a good predictor for pregnancy\npotential. Implantation, pregnancy and live birth\nrates in women younger than 33 years old in both\ngroups (ET2 and ET3) were higher than older\nwomen.\nOn the other hand, logistic regression analysis\nindicated two significant prognostic variables for\nlive birth that were quality and number of transferred\nembryos; it means that the chance of live\nbirth following ICSI treatment increased 3.2-fold\nwhen the embryo with top quality (grade A) was\ntransferred. Our result is in agreement to Dennis\net al. ( 20 ) study, in which they suggested that embryo\ngrade is a significant predictor for live birth\nrate. But the number of ET had an inverse relationship;\nit means that probability of live birth in\nwomen with transfer of two embryos was three\ntimes greater than those who had three embryos\ntransferred.\nOne of the limitations of this study comes from\nits retrospective nature; however, we tried with regression\ntest to control confounding factors.\nPresent study was performed in fresh ET cycles,\nand because the probability of no synchronization between the time of fresh ET and the window of\nimplantation in the endometrium is existed ( 21 ),\nwe suggest future study to evaluate the association\nbetween number of embryos transferred and live\nbirth rate in cycles with frozen and/or blastocyst\nstage ETs.\nAccording to a recent guideline, women aged\n35 years or younger, in first or second IVF attempt,\nwith at least 2 good quality embryos\ntransferred should be considered as good-prognosis\npatients, and the eSET strategy should\nbe used in order to avoid multiple pregnancies\n( 9 ). In our study, the rate of twin pregnancy in\nwomen with double ET was 27%, which is not\nacceptable; therefore, it is recommended that the\neSET strategy be considered, although there are\nno executive arrangements to enable our institution\nto enforce the eSET strategy. Moreover it is\nimportant to remind the patients about the high\nrisk nature of multiple pregnancies that requires\nequipped labor which may not be available in\nsome rural or smaller areas. Concerns in relation\nto multiple pregnancies and even twin pregnancy\nresulting from ART specify the importance of\ncounseling before ET for IVF patients ( 22 ).\n\nDue to the difficulty of implementing the eSET\nstrategy in some infertility centers in the world\nincluding Iran, we suggest transferring of a maximum\nof two embryos with at least one good quality\nembryo for good prognosis women aged 39\nyears or younger. However, further clinical trials\nare required to evaluate the effect of the number\nof embryos transferred (single versus double ET)\nfor women in different age groups using fresh or\nfrozen ET technique.","source_license":"CC-BY-4.0","license_restricted":false}