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Predictive value of serum HCG concentrations in pregnancies achieved after single fresh or vitrified-warmed blastocyst transfer

Reproductive BioMedicine Online, September 2017, Volume 35, Issue 3, Pages 272-278


Possible differences between serum HCG levels in pregnancies achieved after transfer of a single fresh or a vitrified-warmed blastocyst were evaluated. Out of 1130 single blastocyst transfers resulting in positive HCG results, 789 were single fresh blastocyst transfers and 341 single vitrified-warmed blastocyst transfers. The initial serum HCG levels of 869 clinical intrauterine pregnancies were evaluated, 638 after the transfer of a single fresh blastocysts and 231 after the transfer of a single vitrified-warmed blastocysts. The HCG levels from cycles resulting in a clinical intrauterine pregnancy were significantly higher after the transfer of a single vitrified-warmed blastocyst (383 ± 230 IU/l) versus a fresh transfer (334 ± 192 IU/l; P = 0.01). Threshold values for predicting a clinical pregnancy for a fresh blastocyst were 111 IU/l and for a vitrified-warmed blastocyst 137 IU/l. Our study shows that the overall beta-HCG levels are comparable after the transfer of a fresh or vitrified-warmed blastocyst, suggesting that vitrification most probably does not affect the ability of the embryos to produce beta-HCG. This study further shows that when clinicians counsel patients, they should take into account that higher HCG levels are needed after a vitrified-warmed blastocyst transfer to predict a clinical intrauterine pregnancy.

Keywords: Clinical pregnancy, Fresh blastocyst, HCG, Single embryo transfer, Vitrified-warmed blastocyst.


Transfer of a single embryo is important in reducing the occurrence of multiple pregnancies. This is made possible by the increasing use of single embryo transfer and improving embryo cryopreservation for supernumerary embryos. It is imperative that the technique of cryopreservation minimally affects the viability and potency of the embryos. Transfer of vitrified embryos has been shown to be associated with a similar pregnancy outcome compared with that of fresh embryo ( Feng et al., 2012; Takahashi et al., 2005 ), even when a single embryo is transferred ( Roy et al., 2014 ).

HCG dimer is secreted from the syncytiotrophoblast as early as 6–8 days after fertilization, and may serve as a marker for embryo viability ( Butler et al., 2013; Rull and Laan, 2005 ). It is known that HCG levels are correlated with pregnancy outcome ( McCoy et al., 2009; Shamonki et al., 2009 ). Therefore, it is important to evaluate whether embryo vitrification followed by warming has an effect on the developmental potential of the embryo and its ability to produce and secrete HCG. In one study, serum HCG levels in pregnancies after transfer of a cryopreserved embryo was lower than those after fresh embryo transfer ( Sites et al., 2015 ). Yet, the study numbers were small, cleavage and blastocyst embryos were included and various methods of cryopreservation were applied, including slow freezing and vitrification.

Transfer of more than one embryo makes it impossible to evaluate the true contribution of the embryo in the very early stages of development. It is possible that, in cases in which multiple embryos are transferred (more than one implant), only one embryo survives, resulting in a singleton clinical pregnancy and live birth. As expected, multiple implantations are associated with higher initial levels of HCG ( Urbancsek et al., 2002; Singh et al., 2013 ). The vanishing twin phenomenon is reported to be as high as 10%, undoubtedly affecting the initial level of HCG measured in the maternal serum ( Poikkeus et al., 2007 ).

The aim of the present study was to evaluate initial serum HCG levels after transfer of a single fresh blastocyst embryo or of a single vitrified-warmed blastocyst embryo.

Materials and methods

All fresh and vitrified-warmed single blastocyst transfers (SBT) carried out between January 2010 and December 2014 at the reproductive unit of the McGill University Health Center (MUHC) in Montreal, Canada were analysed. Only SBT of day 5 fresh and vitrified-warmed embryos from non-donor oocytes were included. For standardization purposes, only serum HCG levels drawn 11 days after the transfer of either fresh or vitrified-warmed blastocyst embryos were included. Day 11 after transfer was chosen as it is equivalent to 16 days after oocyte retrieval in fresh cycles and applicable to both fresh and vitrified-warmed cycles. The Research and Ethics Board of MUHC approved the study (MUHC Study code 12-283-SDR).

Standard stimulation protocols were used, including either microdose flare protocol, a fixed antagonist protocol or a mid-luteal long agonist protocol ( Oron et al., 2014 ). All embryos were cultured in COOK medium (Medical, Sydney, Australia) during all stages of embryo development. Extended culture to blastocyst was conducted according to the sequential media system. When needed, assisted hatching with laser zona drilling (20 µm) was first carried out under an inverted microscope in a microdrop (40 µl) using a ZILOS-tk Zona Infrared Laser Optical System (version 3.29, Hamilton Thorne Instruments Biosciences, Beverly, MA). On day 5, embryos were scored for blastocyst formation. Blastocysts were graded according to the level of expansion, the presence and the quality of inner cell mass and quality of trophectoderm. Good-quality blastocysts were defined as those that scored 3BB or over based on the Gardner and Schoolcraft scoring system ( Gardner et al., 2004 ). Vitrified-warmed blastocysts were graded twice: before vitrification and before transfer. Only grading before transfer was included. Surplus embryos not selected for transfer were cryopreserved.

Since 2010, vitrification was carried out using ethylene glycol and dimethyl sulphoxide using Cryotop (Kitazato, BioPharma). Vitrification of expanded blastocysts was carried out after induced collapse of the blastocoele with laser ( Son et al., 2003 ). After complete shrinkage of the blastocoele, the blastocysts were equilibrated in equilibration medium containing 7.5% (v/v) ethylene glycol +7.5% (v/v) 1,2-propanediol for 3 min at room temperature and then transferred to the vitrification medium containing 15% (v/v) ethylene glycol + 15% (v/v) 1,2-propanediol + 0.5 M sucrose at room temperature for 45–60 s. Warming was carried out by transferring the vitrification device containing embryos to a 100 µl drop of 0.5 mol/l sucrose. After 1 min, the blastocysts were transferred sequentially to 100 µl drops of 0.35M mol/l of sucrose with 2.0 min and 0.2 M mol/l of sucrose with 3.0 min and 0 mol/l of sucrose with 5 min twice at room temperature.

Luteal support after fresh and vitrified-warmed embryo transfer was provided using oral oestradiol (Estrace 2 mg X2/d) and vaginal progesterone (either Crinone 8% once daily, Endometrin 100 mg X2/d or Prometrium 200 mgX3/d). Luteal support was administered until positive pregnancy test and was continued until 10 weeks of gestation when applicable.

For standardization purpose, only serum HCG test measured 11 days after embryo transfer was included in the analysis. Blood sampling was between 7 and 9 am. Serum HCG measurement was carried out using immunometric sandwich assay with Immulite 2000 system (Siemens Medical Solutions Diagnostics, Flanders, NJ). The sensitivity of the assay was 0.4 mIU/ml. The same standard assay was used for all cycles analysed.

Pregnancy was indicated by serum HCG levels of 5 mIU/l or more, a second measurement was taken 2 days later when the initial HCG levels were lower than 100 mIU/ml. Transvaginal ultrasound was carried out 2 weeks after the initial beta-HCG measurement to evaluate the location of the pregnancy.

Cycle outcome was analysed as biochemical pregnancy, ectopic pregnancy or clinical intrauterine pregnancies (intrauterine gestational sac on transvaginal ultrasound). Clinical pregnancy outcome was analysed as miscarriage (fetal loss before 20 weeks of gestation), stillbirth (fetal loss after 20 weeks of gestation), an ongoing pregnancy (a pregnancy ongoing beyond 20 weeks of gestation at the time of the study) or a live birth. Multiple pregnancies were excluded from the analysis.

Statistical analysis

Statistical analysis was carried out using SAS 9.2 (SAS Institute Inc., USA). Patient characteristics and clinical outcomes were tabulated by day of fresh or vitrified-warmed transfer. Chi-squared or Fisher's exact test was used for categorical variables and Wilcoxon Rank Sum tests for continuous variables. To account for the fact that a patient might contribute to multiple cycles, and hence observations from such cycles might be correlated, the generalized estimating equation approach was used to obtain consistent parameter inferences.

A multivariable logistic regression model was created separately to determine the association of potential factors with clinical intrauterine pregnancy resulting from fresh and vitrified-warmed blastocyst transfers. A receiver operating characteristic (ROC) was created for fresh and vitrified-warmed blastocyst embryos using a non-parametric distribution method to assess the predictive value of HCG for a clinical intrauterine pregnancy and to assess optimal sensitivity and specificity. The percentage for the area under the curve (AUC) and the 95% confidence interval were generated for each ROC curve. The AUC measures the diagnostic accuracy of HCG on pregnancy outcome; an AUC closer to 1.0 denotes a perfect test, whereas AUC closer to 0.5 means no better than chance.


Of a total of 4821 non-donor blastocyst transfers, 2015 resulted in a positive serum HCG. After including only HCG results drawn 11 days after embryo transfer for fresh and vitrified-warmed cycles, 1144 cycle transfers were available for analysis. Two multiple gestations resulting from a single vitrified-warmed blastocyst embryo and 12 multiple gestations from a single fresh blastocyst embryo transfer were excluded from the analysis.

A total of 789 pregnancies were achieved after the transfer of a single fresh blastocyst and 341 pregnancies were achieved after the transfer of a single vitrified-warmed blastocyst; 256 transfers (148 from fresh and 108 from vitrified-warmed blastocyst transfers) resulted in a biochemical pregnancy and five (three from fresh and two from vitrified-warmed blastocyst transfers) were ectopic pregnancies. A total of 638 clinical intrauterine pregnancies resulted from the transfer of a single fresh blastocyst and 231 resulted from the transfer of a single vitrified-warmed blastocyst ( Figure 1 ).


Figure 1
Cycles included in the study.


Demographic characteristics of both groups are presented in Table 1 . The number of good- quality embryos was higher than the number of assisted hatching in the fresh blastocyst transfer group compared with the vitrified-warmed blastocyst transfer group. Mean HCG levels resulting from single fresh blastocyst transfers (298 ± 204 IU/l) and from single vitrified-warmed blastocyst transfers (306 ± 242 IU/L) were comparable ( Table 1 ). Multivariable logistic regression analysis shows that maternal age and embryo grade are potential factors that influence HCG levels ( Table 2 ).

Characteristics Vitrified-warmed blastocyst transfer ( n = 341) Fresh blastocyst transfer ( n = 789) P -value (95% CI)
Maternal age (years) 34.2 ± 4.3 34.0 ± 3.8 NS (−0.00 to 1.00)
Paternal age (years) 38.5 ± 7.0 37.5 ± 5.6 0.03 (0.00 to 2.00)
Body mass index 24.5 ± 5.3 24.6 ± 5.5 NS
Smoking (%) 25/250 (10) 54/635 (8.5) NS
First IVF cycle, n (%) 4 (1.2) 466 (59.1) <0.001 (−0.54 to −0.46)
Mean total FSH dose (IU) 2542 ± 1592
Mean total LH dose (IU) 725 ± 995
Fertilization rate 0.8 ± 0.14 0.78 ± 0.16 NS (−0.07 to 0.12)
ICSI, n (%) 602 (76.3)
Good-quality embryos (grade 2), n (%) 257 (75.4) 715 (90.6) <0.001 (0.18 to 0.35)
Assisted hatching, n (%) 282 (82.7) 56 (7.1) <0.001 (−0.81 to −0.71)
Total positive HCG results 341 789 NS (−30 to 20)
Mean ± SD (IU/l) 306.4 ± 241.68 298.66 ± 204.08  
Range 6 – 1359 6 – 1237  
Biochemical pregnancies 108 148 NS (−15 to 13)
Mean ± SD 145.81 ± 178.85 150.14 ± 185.31  
Range 6 – 867 6 – 1029  
Ectopic pregnancies 2 3 NS (15 to 120)
Mean ± SD (IU/l) 101.5 ± 48.79 38.67 ± 19.73  
Range 67 – 136 16 – 52  
Clinical pregnancies 231 638 0.01 (7 to 66)
Mean ± SD 383.26 ± 230.31 334.33 ± 192.04  
Range 30 – 1359 6 – 1237  

a Data presented as mean ± SD, unless noted otherwise.

Table 1

Variable Coefficient estimate 95% CI P -value
Fresh a −5.79 −46.40 to −34.80 NS
Maternal age −4.75 −8.27 to −1.22 0.008
Paternal age 0.73 −1.71 to −3.17 NS
Grade 2 b 54.61 19.65 to −89.57 0.002
Assisted hatching 14.06 −26.80 to −54.92 NS

a Vitrified-warmed as reference.

b Grade 3 as reference.

Table 2


The HCG levels from cycles resulting in a clinical intrauterine pregnancy after the transfer of a single vitrified-warmed blastocyst (383 ± 230 IU/l) were higher than those after the transfer of a single fresh blastocyst embryo (334 ± 192 IU/l; P = 0.01). Pregnancies resulting in a miscarriage after the transfer of a single vitrified-warmed blastocyst were also higher than in the fresh transfer group ( Table 3 ).

  Vitrified-warmed blastocyst transfer ( n = 231) Fresh blastocyst transfer ( n = 638) P -value (95% CI)
Ongoing pregnancies Number 38 58 NS (−125,52)
Mean ± SD 366.05 ± 216.45 390.76 ± 220.26
Range 30–1117 23–971
Miscarriage Number 51 99 0.01 (13 to 139)
Mean ± SD 332.33 ± 212.21 242.53 ± 161.88
Range 42–817 12–875
Unknown Outcome Number 52 104 NS (−18 to 117)
Mean ± SD 410.46 ± 220.6 350.87 ± 177.75
Range 113–1178 65–768
Live births Number 90 377 NS (−7 to 86)
Mean ± SD 403.67 ± 248.89 345.2 ± 191.66
Range 37–1359 6–1237

Table 3


Multivariable logistic regression analysis shows that levels of serum HCG are positively correlated with a clinical intrauterine pregnancy for both fresh and vitrified-warmed blastocyst transfers ( P < 0.001) ( Supplementary Table S1 ).

The ROC curve based on HCG levels for pregnancies resulting from fresh or vitrified-warmed SBT is shown in Figure 2 . The AUC for single vitrified-warmed blastocyst transfers was 0.83 (95% CI 0.78 to 0.88), and for fresh SBT was 0.80 (95% CI 0.75 to 0.84). The threshold values predicting a clinical pregnancy for a fresh blastocyst embryo was 111 IU/l with sensitivity of 90% (95% CI 88 to 92%) and a specificity of 60% (95% CI 53 to 69%). The threshold values predicting a clinical pregnancy for a vitrified-warmed blastocyst embryo was 137 IU/L, with a sensitivity of 83% (95% CI 78 to 88%), a specificity of 68% (95% CI 58 to 76%), a positive predictive value of 85% (95% CI 80 to 89%) and a negative predictive value of 75% (95% CI 66 to 83%). The two threshold values corresponding to 90% sensitivity are not significantly different.


Figure 2
Receiver-operating characteristic (ROC) curve for serum HCG values for the prediction of clinical intrauterine pregnancy for vitrified-warmed blastocyst group (blue line: 341 cases with 231 clinical intrauterine pregnancies) and fresh blastocyst group (red line: 789 cases with 638 clinical intrauterine pregnancies). False-positive rate is 1; specificity and true positive rate is the sensitivity. Numbers on the lines are a few key cut-off values of beta-HCG values (mIU/l). Area under the curve (AUC) for HCG values are significant with P < 0.0001 (for testing AUC = 0.5). The difference between AUCs of the two ROC curves was not significantly different.



In the present study, the initial HCG levels after the transfer of single fresh or single vitrified-warmed blastocyst were evaluated. Our study shows that the overall beta-HCG levels are comparable after the transfer of a fresh or vitrified-warmed blastocyst. This suggests that the vitrification process most probably does not affect the ability of the embryos to produce beta-HCG ( McCoy et al., 2009; Singh et al., 2013 ). This is further supported by the reports of equivalent and even superior perinatal outcomes of neonates born after vitrified-warmed embryo transfers ( Li et al., 2014; Ozgur et al., 2015; Roy et al., 2014 ).

A similar study by Reljič et al. (2013) reported higher beta-HCG levels after the transfer of a fresh blastocyst compared with after transfer of a vitrified-warmed blastocyst. Yet, when cycles were stratified according to the pregnancy outcome (biochemical pregnancy, miscarriage, ectopic pregnancy or live birth) no difference in HCG levels was observed between the groups. In addition, their study included transfer of one or more blastocysts hampering interpretation of the results.

Previous studies have shown that initial serum HCG levels are dependent on the day of embryo transfer ( Kathiresan et al., 2011; Kumbak et al., 2006; Oron et al., 2015; Papageorgiou et al., 2001; Zhang et al., 2003 ).

In our study, only single embryo transfers of the blastocyst stage were included, all cryopreserved by vitrification, eliminating potential bias of embryo developmental stage and cryopreservation method. The study groups were similar in demographic and treatment characteristics and, as expected, the fresh group had substantially more first cycles than the vitrified-warmed group. In addition, in the fresh blastocyst transfer group, the rate of good-quality blastocysts was higher and the rate of assisted hatching was lower. Yet, the overall mean HCG serum levels between the two groups were comparable. Multivariate analysis shows that maternal age and embryo grade are potential factors that influence HCG levels but not cryopreservation. Maternal age, embryo quality ( Ahlstrom et al., 2011; Balaban et al., 2000; Hill et al., 2013 ) and HCG values ( Alahakoon et al., 2004; Bjercke et al., 1999; Chen et al., 1997 ) have been shown to be correlated with pregnancy outcome. As HCG might be indicative of embryo quality, it is reasonable to assume that these two parameters may affect the ability of the embryo to produce HCG ( Butler et al., 2013 ).

In August 2010, we implemented a change to our IVF practice to mostly single embryo transfers. Single embryo transfers has allowed us to evaluate the true contribution of HCG during early stages of embryo development. Multiple gestations are associated with higher initial levels of HCG compared with singleton pregnancies. In addition, a 10% rate of vanishing twin phenomenon is associated with multiple embryo transfers, undoubtedly affecting initial HCG levels ( Poikkeus et al., 2007; Urbancsek et al., 2002 ).

The important finding in our study is the significantly higher serum beta-HCG levels in pregnancies resulting in a clinical intrauterine pregnancy after the transfer of a vitrified-warmed blastocyst compared with a fresh blastocyst transfer. Keane et al. (2016) recently reported that initial median beta-HCG levels were higher after single embryo transfers of a vitrified-warmed embryo compared with a fresh embryo resulting in a singleton live birth (844.5 IU/l versus 369 IU/l; P < 0.001). The reason for higher initial levels of beta-HCG may be due to the day the test was drawn (day 19 of the luteal phase), the fact that these were all clinical pregnancies resulting in a live birth and from the repeated injections of recombinant HCG given as luteal support in both study groups. The present study, however, included both the transfer of cleavage and blastocyst embryos, with a significantly higher rate of blastocyst stage embryos in the vitrified-warmed group (79.6% versus 20.4%; P < 0.001), most likely accounting for the bigger difference in beta-HCG values between the two groups.

In contrast, Sites et al. (2015) reported that initial median serum beta-HCG levels were higher for fresh embryo transfers compared with cryopreserved embryo transfer. They concluded that cryopreservation may impair the embryo's ability to produce HCG. Their study, however, included both cleavage and blastocyst transfers as well as different cryopreservation methods (both slow cooling and vitrification). In fact, the method of cryopreservation has been shown to affect serum HCG levels among cleavage stage embryos ( Xue et al., 2014 ). Initial HCG levels were lower after transfer of vitrified-warmed cleavage stage embryos compared with slow cooled thawed cleavage embryos. Their study also included transfer of more than one embryo, limiting interpretation of the results ( Xue et al., 2014 ).

The results of our study are in agreement with previous reports of higher beta-HCG production per implantation after the transfer of a vitrified-warmed double blastocyst compared with a fresh blastocyst ( Li et al., 2014; Ozgur et al., 2015 ). The higher HCG levels per implantation and the higher levels in clinical pregnancies are most probably caused by a difference in embryonic regulation of beta-HCG expression and secretion and a greater endometrial competence in cryopreserved embryo transfer cycles ( Braunstein et al., 1980; Licht et al., 2001, 2007; Perrier d'Hauterive et al., 2007; [4] ). It is possible that it may also be correlated with higher neonatal birth weights after the transfer of vitrified-warmed embryos compared with that of fresh embryos ( Ishihara et al., 2014; Pinborg et al., 2010; Wennerholm et al., 2013 ).

Initial beta-HCG levels are predictive of pregnancy outcome; yet, the prognostic thresholds remain unclear ( Homan et al., 2000; Kathiresan et al., 2011; Lawler et al., 2011; Sugantha et al., 2000 ). On the basis of ROC curves, we set a threshold value for HCG levels with a high specificity and sensitivity for the prediction of a clinical pregnancy. We found that higher levels are needed to predict a clinical pregnancy after the transfer of a vitrified-warmed blastocyst compared with a fresh blastocyst transfer.

Our findings provide a broad generalization for interpreting HCG levels and can assist clinicians in counselling patients, taking into account that higher levels are needed after a vitrified-warmed blastocyst transfer to predict a clinical pregnancy. We recommend that each clinic determine its own threshold as HCG thresholds may vary between different clinics. Models for predicting pregnancy have been carried out separately for single fresh blastocyst transfers ( Ochsenkuhn et al., 2009 ), or single vitrified-warmed blastocyst ( Ueno et al., 2014 ) or for both fresh and vitrified-warmed blastocysts ( Homan et al., 2000 ). Our study covered only single fresh or vitrified-warmed blastocyst transfers in a single centre using the same study population, laboratory culture and vitrification conditions. The limitations of our study include its retrospective design and the lack of randomization. The choice to include only blastocysts was a deliberate attempt to reduce any possible effect of embryo culture duration on HCG levels.

In conclusion, our study suggests that higher initial serum HCG values are needed to predict a clinical pregnancy after the transfer of a single vitrified-warmed blastocyst compared with a fresh blastocyst.

Appendix Supplementary material

The following is the supplementary data to this article: Table S1

Multivariable regression analysis to determine the association between clinical intrauterine pregnancy with HCG value and maternal age, for fresh and vitrified-warmed blastocyst transfers.



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Galia Oron, MD, completed her Obstetrics and Gynaecology residency in 2010 and postgraduate training in reproductive endocrinology and infertility, with excellence, at McGill University, Canada, in 2014. Her special research interests are the effect of growth differentiation factors on maturation of human ovarian follicles and IVF pregnancy outcomes.


Key message


Initial beta-HCG levels are comparable after the transfer of a single fresh or vitrified-warmed blastocyst; therefore, the vitrification process probably does not affect the ability of the embryos to produce HCG. Significantly higher beta-HCG levels, however, are required to predict clinical pregnancy after the transfer of a vitrified-warmed blastocyst.