Elective Single Embryo Transfer: Is Frozen Better Than Fresh?

Hala Gomaa; Rania Baydoun; Sakina Sachak; Ilyn Lapana; Samuel Soliman

JBRA Assist. Reprod. 2016;20 (1):3-7
ORIGINAL ARTICLE
doi: 10.5935/1518-0557.20160002

Elective single embryo transfer: Is frozen better than fresh?

Hala Gomaa¹, Rania Baydoun¹, Sakina Sachak¹, Ilyn Lapana¹, Samuel Soliman¹

¹NewLife Fertility Center, Mississauga, Ontario, Canada

Received July 09, 2015
Accepted October 20, 2015

Corresponding author:
Hala Gomaa
New Life Fertility Centre
Ontario, Canada
E-mail: halaobgyn99@yahoo.com

CONFLICT OF INTERESTS
No conflict of interest have been declared.

ABSTRACT
Objective: Single embryo transfer (SET) has been recommended to avoid multiple births following assisted reproductive technology (ART) procedures. Many studies have shown that frozen embryo transfer may yield better pregnancy rates than fresh embryo transfer. This study looked into pregnancy rates following fresh versus frozen single embryo transfer procedures in age-matched patients.
Methods: This retrospective case control study was carried out at a private clinic [NewLife Fertility Clinic, ON, Canada]. Patient groups included infertile women treated with IVF/ICSI and elective single embryo transfer (eSET) given either fresh or frozen embryos. Cycle outcomes were compared between patient groups matched by age. The primary endpoints were positive testing for ß-hCG and viable ongoing pregnancy. The secondary endpoints were live birth and miscarriage rates.
Results: A total of 583 eSET cycles (212 fresh transfer cycles and 371 frozen transfer cycles) were performed. Significantly higher pregnancy and live birth rates were observed among patients aged ≤ 39 years given frozen embryos.
Conclusion: Frozen single embryo transfer was associated with higher pregnancy and live birth rates when compared to fresh single embryo transfer.

Keywords: IVF, eSET, Fresh embryo transfer, Frozen embryo transfer, Pregnancy rate

INTRODUCTION
Infertility has been estimated to affect 10–15% of couples in developed nations (Evers, 2002). Assisted reproductive technology (ART) has been increasingly used to address this relevant societal issue. Over the past ten years, Canada has seen a significant increase in the use of ART procedures (Gunby et al., 2011). The most common side effect of ART is multiple births, an event associated with higher levels of maternal morbidity, perinatal morbidity, and mortality of twins (The ESHRE Capri Workshop Group, 2000). Limiting the occurrence of multiple births while maintaining high pregnancy rates in patients given IVF is a significant challenge. The high rates of twin births after IVF stem from the current standard practice of implanting more than one embryo in each patient. Elective single embryo transfers have been shown to decrease the occurrence of multiple births without affecting overall pregnancy rates (Tiitinen et al., 2003). In Canada, the mean rate of multiple births caused by IVF/ICSI of 33% in 2001 (Gunby et al., 2005) dropped to 18.8% in 2012 (Canadian Fertility and Andrology Society 2013). In Quebec, the government has sponsored eSET and found that the procedure decreased the number of births from IVF by approximately a third (Gleicher, 2011). In addition to better morbidity outcomes, a prospective study revealed that single embryo transfers yield additional economic benefits. The transfer of a single good quality embryo is equally effective and less expensive than a double embryo transfer in women <38 years of age in their first IVF/ICSI cycle when the cost of maternal and neonatal care given until the newborn is three months old is considered (Gerris et al., 2004).
The use of cryopreservation programs may help maximize pregnancy rates while minimizing multiple birth rates. Successful cryopreservation of human blastocysts allows blastocyst transfers to be performed routinely in IVF treatments, thus reducing the number of implanted embryos.
Additionally, frozen embryo transfers (FET) may increase both the cumulative pregnancy rates of IVF and ICSI procedures (Lurie et al., 2001) and take-home baby rates (Bergh et al., 1995). Embryos have been successfully cryopreserved in the blastocyst stage using various freezing protocols (Fehilly et al., 1985). Vitrification performed better than slow freezing in terms of post-thawing survival rates (Loutradi et al., 2008).
This study aimed to find the best way to decrease overall multiple birth rates while maintaining live birth success rates. To this end, the outcomes of elective single embryo transfers (eSET) using fresh and frozen embryos of patients matched by age were compared.

MATERIALS AND METHODS
This retrospective study looked into the outcomes of 583 eSET cycles performed at the New Life Fertility Clinic from 2009 to 2013. Fresh embryos were used in 145 and frozen embryos in 310 eSET procedures performed in patients aged 35 years and under, whereas 67 fresh eSET and 61 frozen eSET procedures were carried out in patients aged 36-39 years.
The data collected from patients at baseline included demographic information, number of embryos, embryo quality, and pregnancy rates.
All eSET (fresh and frozen) cycles were performed using non-donor oocytes.

Enrollment criteria:
1. Female individuals under 35 years of age in their first IVF/ICSI cycle with at least two good quality embryos.
2. Female individuals of all ages with at least four good quality embryos (NICE guidelines).

Exclusion criteria:
1. Female individuals with more than one failed treatment cycle.
2. Abnormal uterine cavity findings on sonohysterogram or presence of hydrosalpinx.

The elected primary endpoints were clinical pregnancy and live birth rates.
The patients enrolled in the study gave informed consent to their participation and to the collection of data concerning IVF/ ICSI, cryopreservation, number of embryos transferred, and pregnancy outcomes.

Ovarian Stimulation and Fresh Embryo Transfer
Ovarian stimulation was done using a recombinant or urinary FSH or HMG combination with GnRH agonist or antagonist. Follicular development was followed with ultrasound scanning and serial serum hormonal level of FSH, LH, E2 and progesterone. When more than four follicles above 18 mm in diameter were identified by transvaginal ultrasound, 10 000 IU of urinary human chorionic gonadotrophin was given S.C to trigger ovulation. Guided by transvaginal ultrasound, egg retrieval was done 34 to 36 hours after hCG injection. All mature eggs retrieved were fertilized either by IVF or ICSI based on the semen quality and the number of mature eggs retrieved. In fresh transfer cycles, for luteal-phase support, 600 mg of progesterone was used vaginally started on the day of oocyte retrieval. Fresh ET,was performed on day 5 or day 6 after oocyte retrieval.

Protocol for Vitrification and Warming
Blastocysts deemed suitable for vitrification on Day 5 were selected. Artificial shrinkage of fluid volume in blastocysts was performed to enhance survival rates. Expanded blastocysts with a cavity encompassing > ½ of the embryo were mechanically collapsed prior to vitrification using an ICSI needle. The collapsed blastocysts were vitrified using Cryotop straws and commercially available vitrification solutions (Kitazato BioPharma Co). Collapsed blastocysts were dipped in equilibration solution (15% ethylene glycol – EG – plus dimethyl sulfoxide – DMSO) for seven minutes; then they were placed in a vitrification solution with 15% EG, 15% DMSO, and 0.5 mmol/L sucrose for 30 seconds; Cryotop straws were then filled with 1 μl of vitrification solution. Vitrified embryos were warmed in subsequent cycles. The Cryotop straws holding the vitrified embryos were removed from liquid nitrogen and dipped for one second in a thawing solution containing 1 mol/L sucrose for one minute at 37°C. The embryos were first equilibrated in Kitazato diluent solution for three minutes at room temperature and then flushed with washing solution for five minutes and kept at 37°C for one minute. Culture time for retrieval was two hours before transfer.

Endometrial Preparation and Frozen Embryo Transfer
In frozen embryo transfer cycles, vitrified-warmed embryo was transferred either during a natural cycle after confirmed ovulation or after preparation of the endometrium; using estradiol valerate [Estrace; Shire Canada Inc, Saint-Laurent, QC] which was administered orally at a starting dose of 4 mg daily, beginning from day 3 of the menstrual cycle. After ultrasonography confirmed an endometrial thickness reaching 8 mm or more with triple line pattern , progesterone 600 mg is initiated. Transfer of day-5 vitrified-warmed top quality embryo [2AA, 3AA or 4 AA] blastocysts. The quality was based on Gardner classification system(Gardner and Schoolcraft 1999, Gardner et al. 2000). In which the components of the blastocysts are graded, and the score depends on expansion of the embryo’s cavity and goes from 2-6, inner cell mass grade and trophoectoderm quality [ Table 1]. Assisted hatching is not a common practice at our clinic especially first ones and If assisted hatching was done on the group of patients that was included in our study, it would be on a very insignificant number of patients which will not affect our data analysis. The transfer was performed under ultrasound guidance using a cook catheter (COOK IRELAND LTD, Ireland). Luteal-phase support was achieved with vaginal progesterone 600 mg/day, which was continued daily until positive serum βhCG concentrations were measured 7 days after transfer, and the clinical pregnancy rates per embryo transfer procedure and implantation rates per transferred blastocyst were based on the detection of fetal heartbeats by ultrasound at 5 weeks after embryo transfer.
Patients were considered to have had a miscarriage when they lost their fetuses before 20 weeks of gestation (Regan & Rai, 2000).

Table 1: Embryo Morphology (Gardner et al., 2000; Gardner & Schoolcraft, 1999)

Statistical Method
Individual data points were collected from the IVF database and entered into Microsoft Excel 2007 (Microsoft Corporation). Data sets were described in terms of their mean values and standard deviations. Comparisons were carried out using the chi-square test. Statistical significance was attributed to differences with P-values <0.05.

RESULTS
There was a total of 583 eSET cycles, 455 were patients ≤ 35 y. There was no significant difference in age or the quality of blastocyst transferred in both groups (2AA, 3AA or 4AA). In the group ≤ 35 y, our results show a significantly higher pregnancy rate in the frozen group (73.22%) versus the fresh group (59.31%) (P= 0.003). A higher but non-significant viable pregnancy rate was observed in the frozen group (55.80%) versus the fresh group (48.96%) (P=0.2). When live birth was compared between the frozen group and the fresh cycle group a significant difference was observed (51.93% vs 42.06%) (P=0.05) (Table 2).
In the older age groups [36-39 years] (Table 3), 61 frozen eSET and 67 fresh eSET, there was a significant higher positive β hCG in the frozen eSET cycles when compared to fresh eSET (72.13%vs35.82%) (P<0.0001), the clinical pregnancy rate was (52.45% versus 20.89%) (P= 0.0002). Also, there was a significant higher live birth rate in the frozen eSET cycles (52.45% versus 20.89%) (P=0.0002).

Table 2: Summary of the results found in patients aged 35 years and under [total = 455 cycles]

Table 3: Summary of the results found in the group aged 36-39 years [total=128 cycles]

DISCUSSION
This study aimed to assess the pregnancy rates derived from fresh and frozen single embryo transfer cycles. Elective SET has been defined by the Society for Assisted Reproductive Technology (SART) as the transfer of one embryo despite the availability of multiple high quality embryos (Practice Committee of the Society for Assisted Reproductive Technology & Practice Committee of the American Society for Reproductive Medicine, 2012; Regan & Rai, 2000; Gardner et al., 2000; Gardner & Schoolcraft, 1999). (Gerris et al., 1999) carried out the first randomized trial in which elective single-embryo transfer (SET) and double-embryo transfer procedures were compared. Another study (Lundin & Bergh, 2007) compared the cumulative pregnancy rates of eSET versus double embryo transfers and reported similar results. Elective SET combined with subsequent frozen-thawed embryo transfer was shown to produce high pregnancy rates and a decrease in multiple births.
Pregnancies resulting from the use of IVF eSET had reportedly decreased risk of low birth weight and prematurity when compared to DET, but higher risk of prematurity, placenta previa, gestational diabetes, and ectopic pregnancy than spontaneous pregnancies (Grady et al., 2012).
Successful implantation, pregnancy, and embryo development require synchronization between a receptive endometrium and the embryo (Perrier d’Hauterive et al., 2007; Dal Prato et al., 2002). Despite the advancements seen in IVF, implantation failure remains an unsolved problem in reproductive medicine and is considered a major cause of recurrent IVF failure in otherwise healthy women. The mean IVF cycle implantation rate is around 25% (de los Santos et al., 2003). Many authors have shown that endometrial receptivity can be adversely affected by controlled ovarian stimulation (Haouzi et al., 2009). Others have found that the endometrium is usually more receptive in natural non-stimulated cycles (Fatemi et al., 2010) and that avoiding fresh embryo transfers and freezing all embryos destined for transfer might improve the safety and effectiveness of IVF and ICSI cycles (Maheshwari & Bhattacharya, 2013). The hypothesis, therefore, was that frozen embryo transfers could yield better success rates than fresh embryo transfers.
Based on these studies, starting in 2012 our patients were offered a protocol comprised of cryopreservation of their good quality embryos followed by single-embryo transfers through frozen-thawed embryo transfer cycles.
This study compared the outcomes reported for the protocol based on fresh embryo transfer cycles previously in effect at our clinic and the outcomes of the new protocol in effect since 2012.
Top quality frozen embryos (2AA, 3AA or 4 AA) in terms of morphology at freezing (Hartshorne et al., 1990) and lack of damage after thawing (Salumets et al., 2006) were used in this program. Embryo morphological traits were preserved after thawing when high-quality characteristics existed before FET (Veleva et al., 2013).
Blastocyst transfer (BT) is effective at improving implantation and clinical pregnancy rates and yields better clinical outcomes (Cruz et al., 1999). In our study, the pregnancy rates of vitrified-warmed blastocysts were significantly higher than the rates seen with fresh blastocyst transfer (BT) cycles in both ≤ 35 year-old and 36-39 year-old patient groups (73.22% vs. 59.31%, P=0.003; and 72.13% vs. 32.82%, P=<0.0001).
The ongoing pregnancy rate in the vitrified-warmed blastocysts significantly higher only in the 36-39 year-old group (55.80 % vs. 48.96%; P= 0.2), in patients aged 35 years and under (52.45% vs. 20.89%; P= 0.0002), and in the 36-39 year-old group (Zhu et al., 2011; Veleva et al., 2006; Niinimaki et al., 2013).
SET using top quality embryos could serve as a predictor of early pregnancy outcome (De Neubourg et al., 2004). In the present study, LBR in the group with top quality frozen embryos was significantly higher when compared to the group given fresh embryo transfers group in patients aged 35 years and under [51.93% vs. 42.06%; P=0.05) and in the group aged 36-39 years [52.45% vs. 20.89%; P= 0.0002).
SET using good quality frozen blastocysts reduces the incidence of ectopic pregnancies and twin births while maintaining comparable pregnancy rates (Yanaihara et al., 2008).
No cases of ectopic pregnancy and only three twin births were observed in the population included in our study.
This descriptive study faces methodological limitations and calls for the organization of randomized prospective trials to corroborate the results reported herein.
In conclusion, the results of this study demonstrate the superiority of the frozen eSET cycle over a fresh eSET and it can be performed with good pregnancy outcome in all age groups, which will result in a further decline in the twin pregnancy rate.