|Year : 2020 | Volume
| Issue : 1 | Page : 18-25
Clinical outcomes of fresh versus frozen embryo transfers and its influence on mid-trimester miscarriage and fetal birth weight: A retrospective study
Priya Selvaraj, Kamala Selvaraj, S Valarmathi, Mahalakshmi Sivakumar, HP Vasundra
GG Hospital, Fertility Research and Women's Speciality Centre, Chennai, Tamil Nadu, India
|Date of Submission||07-Jul-2020|
|Date of Acceptance||31-Oct-2020|
|Date of Web Publication||30-Jan-2021|
Dr. Priya Selvaraj
GG Hospital, Fertility Research and Women's Speciality Centre, Chennai, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Aim: The aim of the study was to compare the clinical outcomes of fresh and frozen embryo transfer (FET) in women undergoing assisted reproductive technology and its influence on mid-trimester miscarriage and fetal birth weight.
Materials and Methods: The study was conducted between June 2016 and June 2019. The study group comprised 778 cycles, of women, who were undertaken in this retrospective analysis of in vitro fertilization cycles who underwent fresh and FET. These women were randomly divided into two groups, fresh embryo transfers (n = 410) and FETs (n = 368). Both groups were compared with their respective outcome measures such as the clinical pregnancy rates (CPRs), miscarriage rates, multiple pregnancy rates as well as live births and birth weights.
Results: The mean age of the participants in fresh and FET groups was 31.13 ± 4.39 and 31.45 ± 4.25, respectively. There were statistically significant differences with respect to clinical pregnancy (P = 0.002) between the groups. Fetal miscarriage rates (30.14% vs. 23.53%) as well as birth weights were not statistically significant (P = 0.164) between fresh versus FET. The results clinically signified that the FET gives a lower miscarriage rate and higher CPRs while not affecting the average fetal birth weight.
Conclusion: Adjusting the baseline characteristics and exclusion criteria of participants, women undergoing FET were associated with better clinical outcomes than women undergoing fresh ET cycles Contrary to existing literature, our incidence of neonatal morbidities, or large birth weights were not increased and fetal macrosomia did not occur. Gaining debates on the advantage of frozen over fresh transfers and its effects on obstetric and neonatal outcomes may require a large multicenter pooling of data and analyses.
Keywords: Fresh embryo transfer, frozen embryo transfer, sequential, stimulation protocols
|How to cite this article:|
Selvaraj P, Selvaraj K, Valarmathi S, Sivakumar M, Vasundra H P. Clinical outcomes of fresh versus frozen embryo transfers and its influence on mid-trimester miscarriage and fetal birth weight: A retrospective study. Onco Fertil J 2020;3:18-25
|How to cite this URL:|
Selvaraj P, Selvaraj K, Valarmathi S, Sivakumar M, Vasundra H P. Clinical outcomes of fresh versus frozen embryo transfers and its influence on mid-trimester miscarriage and fetal birth weight: A retrospective study. Onco Fertil J [serial online] 2020 [cited 2021 Feb 28];3:18-25. Available from: https://www.tofjonline.org/text.asp?2020/3/1/18/308406
| Introduction|| |
Assisted reproductive technology (ART) is the treatment of choice for couples with prolonged and unresolved infertility. As the field of assisted reproduction evolves rapidly, there have been increasing numbers of data reflecting on several factors that influence the outcomes in in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) cycles. Despite recent advances in assisted reproduction, the implantation rate is still relatively low. To increase pregnancy rates and reduce multiple pregnancy rates, there is a need for reliable embryo selection method for determining embryo potential. For instance, in the past few years, frozen embryo transfer (FET) and single embryo transfers (SET) have taken precedence over fresh and multiple (sequential) embryo transfers (ET). Another debatable issue is the rates of miscarriages in fresh versus FETs and if there are truly any contributing factors causing an increase of the same in either of these two methods. A surge of higher birth weight has been implicated following FETs. Our aim was to analyze our available data and with comparison to already published data and establish a cause-effect relationship for any increased risks of miscarriages or increased fetal birth weight (4 kg) with respect to FETs.
It has been published that, among 148,494 ART pregnancies, between the years 1999 until 2002, in the United States, had resulted in a pregnancy loss rate of 29%. Similarly, in the UK, a total of 52,288 women underwent 67,708 cycles of IVF or ICSI in the year 2014 comprising 1.5% of all births from ART procedures. The live birth rates in the UK are about 26.5% of IVF treatments (human fertilization and embryology authority, 2016), while 22.3% of IVF pregnancies ended in miscarriage. An early miscarriage is defined as pregnancy losses<12 weeks of gestation, while anything after that time period after that time period till the period of viability which is 26 weeks, is regarded as second-trimester loss. Some of the risk factors that have been identified that contribute to early and late miscarriages are increased maternal age, history of previous miscarriages pointing to implantation and genetic factors, polycystic ovarian syndrome, male factor infertility, uterine anomalies, and endometriosis. Specifically, IVF-related losses may even implicate FETs, cleavage stage embryos, and poor responders with poor embryo quality.,
| Materials and Methods|| |
A total of 778 women who underwent embryo transfer (ET) procedures in our center between the time period June 2016 and June 2019 were undertaken in this retrospective analysis. The mean age group of the women was 31 years, and they were demographically similar. The women were divided into two groups. Those who underwent fresh ETs (n = 410, designated as Group A) and those who underwent FETs (n = 368, designated as Group B). The clinical pregnancy rates (CPRs), miscarriage rates (first and mid-trimester), and live birth rates were analyzed and compared. In addition, live birth weights in both groups were also compared. Any identifiable or contributory factors that increased risks (miscarriages, twinning rates, and large for gestational age) were identified. This also meant that the number of embryos transferred may be regarded as a contributory factor to the above possible outcomes.
All data collection, entry, management, and analysis were coordinated and performed at our center with outsourced statistician. Patient's data included maternal age, etiology, and pregnancy-related outcomes as enlisted above were recorded. The primary outcome measures were miscarriage rates in second trimester and the live birth weights in both groups. The secondary outcome data were preclinical pregnancies, implantation rates, overall miscarriage rates including first trimester, ongoing pregnancies, and live birth rate. In secondary outcomes, we have divided the data to compare the number of ETs in both fresh and frozen transfer that is a transfer of three embryos was termed as sequential transfer, whereby two cleavage embryos and one blastocyst were transferred. The other group comprised those cases where only ≤2 embryos were transferred. The two groups with its respective outcomes were compared, giving importance to multiple pregnancy rates and outcomes.
Ovarian stimulation protocols
The study group was subjected to three different stimulation protocols – A long protocol using gonadotrophin-releasing hormone (GnRH) agonist, at a dose of 3.6 mg on day 21 of an Oral Contraceptive Pill (OCP)-induced cycle otherwise known as the long protocol. The second protocol used GnRH antagonist which was administered as 0.25mg after follicular diameter is diameter is 1.4 x 1.4 cm following ovarian stimulation. A third minimal stimulation protocol using clomiphene citrate during the early menstrual phase along with human menopausal gonadotrophins (HMG). While the protocols differed, the evaluation of patients remained the same. The stimulation regimens for the agonist and antagonist cycles were also the same with respect to recombinant follicle-stimulating hormone (FSH) and gonadotropins. The criteria for selecting stimulation according to the type of response, that is whether they were poor responders (POSEIDON Criteria) or hyper-responders (polycystic ovary syndrome [PCOS]). These two groups were largely subjected to antagonist protocol with dual trigger using GnRHagoinst plus recombinant human chorionic gonadotropin (hCG). The normo-responders were largely subjected to long agonsit protocol. The choices of fresh and frozen were based on several criteria. Antagonist and long agonist protocol with no risk of hyper-stimulation, modified luteal phase support when GnRH agonist was used for trigger were eligible for fresh embryo transfer, Poor endometrial thickness, serum progesterone level more than 1.2 nmol/L, and previous failures were suitable for frozen transfers.
A basic evaluation was conducted by an ultrasound examination on day 2/3 of the menstrual phase to rule out functional/follicular cysts and assess antral follicle count, followed by blood test for hormone levels (FSH, luteinizing hormone, E2, and prolactin). The stimulation injections were commenced on day 3 of the cycle and comprised recombinant FSH (rec-FSH) in a dose range of 200 IU-300 IU for the first 4 days followed by a combination of rec-FSH (at half dose) and HMG with starting doses at 150 IU and incremental up to 300 IU for a maximum of 9–12 days of total stimulation duration. In the antagonist cycle, 0.25 mg of cetrotide (Merck, Germany) was administered subcutaneously when the dominant follicles reached a mean diameter of 1.4 cm × 1.4 cm in size. The minimal stimulation protocol involved clomiphene citrate at a dose of 200 mg for 7 days with two doses of HMG given on 5th and 7th day, respectively, followed by a first follicular study ultrasound on day 9 to asses for further doses of HMG at 150 IU. In all three protocols, the trigger was given when the dominant follicles reached a mean diameter of 1.8 cm × 1.8 cm, using either a recombinant HCG (Injection Ovitrelle 250 μg, Merck Specialities Private Limited, Mumbai, India) for the long protocol, or a GnRH agonist (Gonapeptyl/Decapeptyl, Ferring Pharmaceuticals Pvt Ltd., Thane, India) given in doses of either 0.2 or 0.1 mg as subcutaneous injections, or urinary HCG given as a dose of 10,000 IU in minimal stimulation.
Lab procedure, cryopreservation technique, thaw, and transfer protocol
Post trigger the transvaginal oocyte, pick-up was performed at 36 h and the resultant oocytes were fertilized by ICSI ±3 h postretrieval. In all the cases either, fresh or frozen semen samples were used, after employing sperm washing techniques such as double density gradient centrifugation or microfluidic sperm sorter. The culture media used were cleavage media (Quinn's, Sage IVF, Trumbull, CT, USA), and V-One step media (Vitromed, Germany). A PN check was done at 16–18 h with further evaluation of subsequent embryo formations using strict morphological criteria. The resultant embryos were transferred fresh or frozen, depending on individualized treatment protocols. In women who were assigned to the FET, the embryos and blastocysts were frozen by Vitrification using Irvine Scientific Media and loaded in a closed system by high-security vitrification straws.
In fresh cases, transfer was performed on 2nd or 3rd day of fertilization as day 2 or 3 cleavage stage embryos (2 nos) with a single day 5/6 blastocyst in the sequential transfer group. In ≤2 embryos transfer group, either day 2/3 embryos were transferred (2 nos) or two blastocysts or single blastocyst on day 5/6.
The ETs were performed using the Labotect catheter (Labotect GmBH, Gottinggen, Germany) for majority of cases, and for few others, Cook transfer catheter (Cook Ireland Ltd, Limerick, Ireland) plain or USG guided were used. We found that this had no influence on the outcomes in our study.
For FETs, the endometrial preparation for the cycle, comprised incremental doses of estradiol valerate (2 mg) tablets followed by endometrial thickness assessment, once in 2 days from day 13 of cycle. When the endometrial thickness was ≥8 mm and serum progesterone was<1.1 ng/ml, the luteal phase progesterone support (P-day 0) was initiated. The embryos were then thawed using the same media and transferred as either sequential (day P-19 and P-day 21) or just cleavage day 2/3 embryos (P-day 19) or blastocysts (P-day 21) as mentioned.
The one-sample K-S test was used to check for normality (age, infertile years, etiology, and neonatal birth weight). The independent t-test was used to assess between-group differences in continuous variables, and these variables were expressed as the mean ± standard deviation categorical variables were represented as the number of cases (n), and the percentage (%) z-test for proportions were used to compare the factors. Statistical significance was set at P < 0.05.
Pregnancy outcomes were recorded at completion, and they were in regular checkup to follow-up the miscarriage, delivery rate, and fetal birth weight of each individual while all of live births occurred between 27 and 38 weeks of gestation.
Epi Info, Free domain suite, United States of America, Centers for Disease Control (CDC) was the statistical tool used for the analysis.
| Results|| |
A total of 922 women were undertaken in this retrospective analysis of IVF cycles and their outcomes with respective to fresh versus frozen transfer cycles were analyzed. Four hundred and ten cycles were in the fresh ET group and 368 cycles in the FET group. One hundred and forty-four cycles were excluded of women who had >3 embryos transferred (n = 22), those who had biopsied embryo or blastocyst transfer (n = 109), and women with anomalous fetuses (n = 13) [Figure 1].
Characteristics of the study groups
Maternal age and duration of infertility years were equally distributed (P > 0.05) in both groups which is depicted in [Table 1]. Furthermore, the endometrial thickness in both the groups (0.99 ± 0.16 vs. 0.95 ± 0.15) was similar. Duration of infertility years, the etiology of infertility, and the number of ET were comparable between the two groups. [Table 1] indicates the baseline maternal characteristics.
|Table 1: Summarized demographics and clinical characteristics of fresh and frozen embryos|
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In [Table 1], the proportion of male factor infertility was higher in both groups, equally followed by PCOS and combined factor infertility. Probabilities of transferring two or more embryos than SETs are influenced by etiology, previous ART failures, endometrial factor, or morphology of the embryos and/or blastocysts.
Pregnancy outcomes between fresh ET and Frozen ET (FET) were compared.
In fresh transfer as well as frozen transfer, there were no statistically significant differences in CPRs with regard to different stimulation protocol employed [Table 2] and [Table 3]. Since the number of patients with minimal stimulation were few it showed a higher pregnancy rate compared to agonist or antagonist protocol. The live birth rates were also comparable.
From [Table 4], it is clear that there is no significant statistical difference in mean neonatal birth weight of singletons and twins between FET and Fresh ET (singleton – 2.46 ± 0.41 vs. 2.47 ± 0.44; Twins –1.97 ± 0.41 vs. 1.97 ± 0.44). The proportion of miscarriage in the first trimester was higher in the fresh ET group in comparison to the FET group (26.71% vs. 20.32%), but it was not found to be statistically significant.
|Table 4: Pregnancy outcomes between fresh embryo transfer and frozen embryo transfer|
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The CPRs were significantly higher in frozen transfers with an increase in live birth rates when compared to fresh transfers. The mean birth weight was similar in both the groups as evidenced by nonsignificant P value. The overall miscarriage rates were lower in frozen transfer cycles as compared to the fresh ET.
The second-trimester pregnancy loss in both groups was similar. In fresh transfer, five women and in frozen transfer six women had second-trimester miscarriages. Out of five women in fresh transfer, one had intrauterine death at 20–21 weeks, and four had inevitable spontaneous miscarriages between 17 and 23 weeks of gestation. In FET transfer, three women had intrauterine deaths between 14 and 19 weeks and three women had inevitable spontaneous miscarriages between 14 and 23 weeks of gestation.
In [Table 5], the CPR rates are higher in frozen cycles when the number of transferred embryos was three embryos. Overall, CPRs were higher in both fresh and frozen, when number of embryos were equivalent to three embryos. The miscarriage rates in sequential transfer of three embryos (two embryos and one blastocyst) were significantly lower in frozen transfers as compared to fresh (fresh ET = 22.22% vs. FET = 16.30%). Overall transferring two embryos or less yielded a much lower CPR.
|Table 5: Pregnancy outcomes between fresh embryo transfer and frozen embryo transfer|
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The incidence of multiple pregnancy rate is also higher in frozen cycle compared to fresh ET rather than the number of embryos transferred (three ETs [fresh ET = 23.23% vs. FET = 30.37%] vs. < [<2 ETs fresh ET = 17.02% vs. FET = 15.38%]).
In [Table 6], the proportion of fresh and FET with gestational age was equally distributed as depicted in the table. The mean birth weight along with the gestation age of FET in singleton pregnancies was better when compared to fresh transfer cycle, as shown in [Table 7].
| Discussion|| |
The increasing number of FETs and the implications of outcomes such as large for gestational birth weights, an increased incidence of pregnancy-induced hypertension, or even postpartum hemorrhage or that the miscarriage rates have been lower with FETs prompted us to put together our data and highlight some of the key issues. We decided to compare few variables such as etiology, stimulation protocols, and number of ETs, multiple pregnancy rate, live birth weights, and miscarriage rates to provide further evidence of current clinical practice.
Chen et al., 2016, reported that the frequency of live births after FET was significantly higher in comparison to that of fresh transfers (49.3% vs. 42.0%; P = 0.004). FETs also resulted a higher frequency of singleton live births than fresh-ET (33.5% vs. 27.8%), and a lower frequency of pregnancy loss (22.0% vs. 32.7%). Similarly, our study revealed in a higher frequency of live births in FET (69.86 vs. 76.47%) and a higher singleton birth rate (45.89% vs. 49.20%) than fresh ET.
Lambalk et al., 2017, had found that using GnRH agonist protocols yielded higher pregnancy rates in comparison to antagonist with increased risk of ovarian hyperstimulation syndrome (OHSS). He postulated that the use of agonist in normo-responders was beneficial, while the use of antagonist in PCOS and hyperresponders would reduce risks of OHSS and improve outcomes. In concordance, our study found higher CPRs with the agonist protocol in both fresh as well as frozen groups, though it did not reach statistically significant (39.10% vs. 55.93%). However, the first-trimester miscarriage rates were higher with the agonist in comparison to antagonist in the FET group, while in the fresh transfer groups, they were comparable as quoted in [Table 2] and [Table 3].
In their study of a retrospective analysis of full-term singleton births from fresh versus frozen transfers, Zhang et al. concluded that the children born after FETs were associated with higher birth weights than those born from fresh transfers (3386.7 ± 448.1 vs. 3468.7 ± 475.3, P = 0.000). The reasons have been several including effects of stimulation protocols in fresh cycles affecting endometrial and subendometrial blood flow as well as effects on gene transcriptions and likelihood of defective implantation and placentation. However, in our study, the average birth weights in both fresh and frozen were 2.46 ± 0.41 and 2.47 ± 0.44 kg, respectively. For twins, it was 1.97 ± 0.41 overall. Even with respect to the number of embryos transferred, there did not seem to be a statistically significant difference in birth weights as cited in [Table 2]. As the statistics show, the birth weights have all conformed to standard average Indian birth weights.
Wikland et al., 2010, have claimed that the risks to offspring resulting from Vitrification as compared with the slow-freezing technique may be higher owing to the high concentrations of potentially toxic cryoprotectants. Furthermore, in his study, the birth weight was significantly higher in singletons born after transfer of vitrified blastocysts, as compared with, transfer of fresh blastocysts. More singletons born after transfer of fresh blastocysts were small for gestational age compared with singletons born after transfer of vitrified blastocysts (12.1% vs. 3.0%, P = 0.0085). No adverse neonatal outcomes were observed in children born after transfer of vitrified, as compared with fresh blastocysts or after transfer of slow-frozen early cleavage stage embryos. In comparison to our study, we did not see any significant difference in birth weights in both groups as well as no increased risks to neonates with respect to birth weights or development.
Wael et al. reported that there was no difference in multiple pregnancy rates (25% vs. 25%, P = NS) when compared with the sequential group and conventional day 3 transfers. On the contrary, our study revealed a higher incidence of multiple pregnancy rate in sequential transfer (fresh ET = 23.23% vs. frozen ET = 30.37%) compared to transfer of ≤2 embryos cycle (fresh ET = 17.02% vs. frozen ET = 15.38%) in fresh and FETs rather than the number of embryos transferred.
Zhao et al. found that singleton pregnancy after FET seems to have a better perinatal outcome compared with that after IVF/ICSI. Other two comparative studies also found that births from FET have a better perinatal outcome and a similar neonatal birth outcome compared with fresh ET., In our study, there was no incidence of perinatal complications as reported by Zhao et al. There were no neonatal deaths observed, and all the babies projected in these data have been discharged without adverse outcomes.
Since these key factors may influence miscarriages, we did not delve deep into the obstetric history or comorbidities. On an average, the overall mode of delivery was either elective or emergency C-sections. The average gestational age of presentation was 27–38 weeks. The incidence of chronic or pregnancy-induced hypertension or gestational diabetes complicating pregnancies was not taken into account as we had adequately managed them, and the only variable it affected in uncontrolled cases or emergencies were gestational age at delivery. We report no neonatal death or intrauterine demise in these study groups.
| Conclusion|| |
There are not many published Indian data on the outcomes of fresh versus FETs, especially with respect to miscarriage rates and neonatal morbidity or birth weights. Moreover, it becomes imperative to also know the incidence of multiple pregnancies in the compared groups with respect to number of embryos transferred. In conclusion, this study has highlighted the safety and efficacy of FETs versus fresh transfers. Contrary to western literature and publications, our incidence of neonatal morbidities, or large birth weights were not increased and fetal macrosomia did not occur. The miscarriage rates were also lower in FET cycles compared to fresh cycles owing to already known fact of discordancy in hormonal milieu and an endometrial advancement in fresh transfers. However, pooled multicenter data among the Indian population would add more value to this study as well as toward safer practice guidelines.
We thank Ms. Suguna Balakrishnan and Ms. Srimathi.S from HR department for their valuable inputs in constructing the manuscript.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]