The Impact of Age on Gestation and Implantation Rates and on Blastocyst Scoring, after Single and Double Embryo Transfers

Research Article

Austin J Reprod Med Infertil. 2015;2(2): 1010.

The Impact of Age on Gestation and Implantation Rates and on Blastocyst Scoring, after Single and Double Embryo Transfers

Bos-Mikich A¹*, Michels MS¹, Dutra CG², Oliveira NP², Ferreira MO², Aquino DC¹ and Frantz N²

¹Department of Morphological Sciences, Federal University of Rio Grande do Sul, Brasil

²Nilo Frantz Human Fertility Center, Brasil

*Corresponding author: Adriana Bos-Mikich, Department of Morphological Sciences, ICBS/UFRGS, Federal University of Rio Grande do Sul, Porto Alegre,RS, Brasil

Received: March 27, 2015; Accepted: May 10, 2015; Published: May 29, 2015


Successful single embryo transfer (SET) cycles depend primarily on the choice of the best embryo to be transferred. Recent study on single blastocyst transfers has shown that trophectoderm (TE) quality is the most important parameter for successful pregnancy and live birth. Patient´s age however, is a key component of a woman´s reproductive potential. The present retrospective study aimed to analyze first the effect of maternal age on clinical gestation and implantation rates after single and double blastocyst transfers. Second, patients were divided into two age groups (<35 and >35 years old) and their blastocyst scores were recorded after single or double (DET) transfers resulting in single or twin pregnancy or non-pregnancy.

Our data clearly shows that for young women (<35 years of age) the transfer of a single blastocyst results in similar gestational rates as DETs, without the risk of twin pregnancies. In addition, our data show that for both young and older women TE score is the most important parameter to be assessed for embryo selection. In addition, inner cell mass (ICM) plays an important role in blastocyst selection in older (>35 years of age) patients. We suggest that blastocyst grading for patients aged 35 years or above shall be performed using a strict grading policy, possibly not of a single parameter, but TE, ICM and expansion grades together to choose the “best combined-score blastocyst” and DETs should be considered, particularly after previous cycles with pregnancy failures.

Keywords: Blastocyst score; SET; DET; Maternal age


Despite the risks of multiple implantation and gestation, most IVF clinics around the world do not perform SET for fear of dropping their implantation and pregnancy rates. Successful cycles of SET represent the gold standard of an ART Institution. Success on a SET cycle depends primarily on the choice of the best embryo to be transferred. Since the advent of embryo culture to the blastocyst stage, “natural” in vitro selection takes place, as not all cleavage stage embryos reach the blastocyst stage and are naturally eliminated from the cohort of putative candidates for transfer. Although there is the possibility that a patient may have no embryo for transfer, embryos that reach the blastocyst stage on day-5 or -6, have higher chances of implantation and pregnancy after transfer [1]. On the other hand, it is not uncommon that patients have more than one or two blastocysts on day-5 or -6 for transfer or cryostorage. Again, a selection must take place to choose the embryo with the highest chances of implantation and gestation, most of the times based on morphological parameters. Possibly, the most widely used blastocyst scoring system is the that one proposed by Gardner & Schoolcraft [2], which takes into account three scores for each embryo: its inner cell mass quality, its trophectoderm quality and finally the blastocyst expansion / hatching (EH) status. Not surprisingly, high implantation and live birth rates were obtained when transferred blastocysts presented top grading for all three scores. Considering that the developing embryo, gastrula and eventually fetus will develop from the inner cell mass, it was reasonable to consider that the inner cell mass score was the major criterion to be taken into account, when choosing one embryo from a group of blastocysts with similar grading for the three scores. A retrospective study on single blastocyst transfers, however have shown that trophectoderm quality is the single most important parameter for a successful pregnancy and live birth [3]. Subsequently, the same group of authors reported that similar blastocyst quality criterion should be used in frozen embryo transfers [4], where, in addition to the trophectoderm quality, the degree of blastocoel reexpansion post-cryopreservation also played an important role in the prediction of live birth.

One point that was not taken into account in those previous studies was the age of the patients, when blastocyst scores were evaluated [3,4]. In a previous work on early embryo development [5] we observed that the early cleavage (EC) phenomenon is dependent on maternal age. Early cleavage embryos, which presented the highest implantation and gestation rates, occurred more frequently in younger women. Other studies have already shown the effects of maternal age on hormonal treatment to induce follicular growth, ovulation induction, zygote and embryo quality, implantation and gestation rates [6-10]. The original work from Gardner et al. [1] did not find any statistical difference in age between the three groups of patients that presented one, two or none top grading blastocysts. However, the overall mean age of the patients was quite low, around 33 years old. The number of patients older than 35 years of age grows continuously in Assisted Reproduction (AR) programs. Thus, it is important to assess the impact of age on blastocyst quality in this population of women, in order to best counsel them about the treatment and their pregnancy probabilities with extended embryo culture.

The aims of the present retrospective study were first to evaluate the effect of maternal age on chemical and clinical gestation and implantation rates after single or double blastocyst transfers. Second, the impact of the three blastocyst scores on gestation [2,8] was assessed on single and double embryo transfers of fresh and vitrified/ rewarmed blastocysts taking into account maternal age.

Material and Methods

In this retrospective study, fresh and cryopreserved cycles of Assisted Reproduction using intracytoplasmic sperm injection (ICSI) were analyzed, in which patients received a single or two fresh or vitrified/rewarmed blastocyst(s), between 2012 and 2014. Only cycles in which blastocyst grading scores were clearly registered were included. Clinical gestation and implantation rates were analyzed for SET and DET, where patients were divided into four age groups (<35, 35-37, 38-39, 40-42 years old; [11]). To analyze the impact of ICM, TE and EH scores on clinical gestation rates from SET or DET cycles that resulted in single (SETs) or twin (DETs) pregnancies, or nongestations, patients were divide in two groups: < 35 and >35 years of age.

Stimulation protocol and embryo transfer

Pituitary suppression was achieved using GnRh antagonist and ovarian stimulation was achieved using recombinant FSH or hMG. When at least one follicle reached 18mm in diameter, patients received a single dose of hCG. Oocyte collection was performed 36 hours after hCG administration and insemination was performed by ICSI. Embryo transfer was performed on day-5 or -6 if one or two good quality blastocysts were available. One or two embryos with the best score were transferred either day-5 or day-6 post-insemination. The study outcomes were positive serum βhCG test and the presence of gestational sac(s) by ultrasound (US), two to three weeks after a positive βhCG test.

Embryo culture and blastocyst scoring

Embryos were cultured from the pronuclear to the blastocyst stage in Global® medium supplemented with 20% SSS. On the morning of day-5 or -6 of culture, the percentage of blastocysts was recorded. Blastocysts from each patient were photographed prior to transfer. For the vitrified/rewarmed embryos, pictures were taken on the day of rewarming and transfer. For grading, blastocysts were classified using Gardner & Schoolcraft [2,8] scoring system. By using this scoring method, embryos received a score from 1 to 6 according to their EH status, being that grade “6” related to hatching blastocysts. ICM and TE were given scores A, B or C, being that scores A and B corresponded to the best organized ICMs and trophectoderm cells forming a continuous epithelium. Grade C related to very small or scattered ICM cells and few and large trophectodermal cells. In order to make a correlation between maternal age, clinical gestation and blastocyst scoring, only SETs and DETs in which 2 or 0 gestational sacs were detected by US were considered for analysis.


After transfer, surplus embryos that reached the blastocyst stage at day-5 or -6 were vitrified with a cryoloop [12] using Cryotech Vitrification Kit, and rewarmed according to the “Cryo top” technique.

Outcome measures and Statistical Analysis

Chemical (β-hCG) and clinical pregnancy (CPR) and implantation (IR) rates were tabulated and compared for fresh and cryopreserved transfers for patients in SET and DET groups. Blastocyst scores were assessed on SET and DET in pregnant and non-pregnant women, divided in 2 age groups: <35 and >35 years old. Clinical pregnancy was defined as the presence of one (or two) gestational sac in the uterus.

Differences between groups were assessed by two-tailed Fisher exact-test. A difference of p<0.05 was considered statistically significant.


A total of 35 fresh and 129 cryopreserved single and double blastocyst transfers were retrospectively analyzed.

For two cycles of cryopreserved embryo transfers, no gestational sac US data were registered, so they were excluded from the implantation rate and blastocyst score analysis.

Chemical and clinical gestation and implantation rates

Results show that patients aged <35 years old presented similar chemical, clinical and implantation rates for fresh or cryopreserved, single or double blastocyst transfers (Tables 1 and 2).

No fresh SET was performed for patients older than 35 years of age.

For patients aged 35-37 years, the transfer of a single cryopreserved embryo resulted in significantly lower chemical, clinical and implantation rates compared with the transfer of two cryopreserved embryos (P<0.001).

Patients aged 38-39 and 40-42 years old, cryopreserved SETs did not result in any gestation.

Double blastocyst transfers resulted in no significant differences in chemical, clinical and implantation rates for fresh and cryopreserved cycles, between patients aged <35 compared with 35-37, 38-39 and 40-42 years old (Table 2).