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Keywords:

  • ART births;
  • blastocyst;
  • cleavage-stage embryo;
  • ICSI;
  • IVF;
  • logistic regression;
  • sex ratio at birth;
  • single embryo transfer

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Please cite this paper as: Dean J, Chapman M, Sullivan E. The effect on human sex ratio at birth by assisted reproductive technology (ART) procedures – an assessment of babies born following single embryo transfers, Australia and New Zealand, 2002–2006. BJOG 2010;117:1628–1634.

Objective  To assess the effect on the human sex ratio at birth by assisted reproductive technology (ART) procedures.

Design  Retrospective population-based study.

Setting  Fertility clinics in Australia and New Zealand.

Population  The study included 13 368 babies by 13 165 women who had a single embryo transfer (SET) between 2002 and 2006.

Methods  Logistic regression was used to model the effect on the sex ratio at birth of ART characteristics [in vitro fertilisation (IVF) or intracytoplasmic sperm insemination (ICSI) SET, cleavage-stage or blastocyst SET, and fresh or thawed SET] and biological characteristics (woman’s and partner’s age and cause of infertility).

Main outcome measures  Proportion of male births.

Results  The crude sex ratio at birth was 51.3%. Individual ART procedures had a significant effect on the sex ratio at birth. More males were born following IVF SET (53.0%) than ICSI SET (50.0%), and following blastocyst SET (54.1%) than cleavage-stage SET (49.9%). For a specific ART regimen, IVF blastocyst SET produced more males (56.1%) and ICSI cleavage-stage SET produced fewer males (48.7%).

Conclusions  The change in the sex ratio at birth of SET babies is associated with the ART regimen. The mechanism of these effects remains unclear. Fertility clinics and patients should be aware of the bias in the sex ratio at birth when using ART procedures.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

The sex ratio at birth, also known as the secondary sex ratio (SSR), can be defined as the proportion of males in all live births. Variation in SSR within most human populations is common,1–3 with the SSR deviating from the equality ratio of males to females (50% males) in the population.4 Assisted reproductive technology (ART) to treat infertility has developed rapidly since the first in vitro fertilisation (IVF) baby was born in 1978. The SSR among babies born after ART treatment varies as in any other population.5–7 However, whether the variation in SSR among ART babies is a result of natural causes (i.e. environmental or biological causes) or of the effect of ART is debatable. Concerns about whether ART might alter SSR have led researchers to study SSR in ART babies.

In 1989, Thatcher et al.8 reported that the SSR of babies born following in vitro fertilisation and embryo transfer (IVF-ET) was significantly higher than normal (64.1% males). In 2000, Ghazzawi et al.9 observed a significantly higher proportion of female births (61.7%) following the transfer of embryos fertilised by intracytoplasmic sperm injection (ICSI). Other researchers10,11 have also observed a trend of a higher SSR in babies born after IVF, and lower SSR after ICSI, although the findings were nonsignificant between the observed SSR and the general population. The reduction in SSR after ICSI has often been attributed to male infertility, because ICSI is predominantly used to treat male infertility.12

Both advances in the development of embryo culture media and evidence of higher pregnancy rates by blastocyst transfer (BT)13–16 have provided fertility clinics with the opportunity to offer BT to patients for treatment. In 1994, Pergament et al.17 observed that the transfer of fast growth embryos resulted in more male births than female births. Other studies that have found skewed SSR in favour of males after BT18–20 have attributed the excess of male births to the selection for transfer of fast growth male embryos. The suggestion is that, because there is sex-related differentiation in embryo development, and male embryos show, on average, more blastomeres at the time of transfer, more male embryos may be selected for transfer. Other researchers,21–25 however, have not found a significant association between higher SSR and BT.

One of the limitations of SSR studies in ART babies is the small sample size, which may explain the lack of association between ART procedures and SSR.26,27 The lack of association could also be related to the use of different analytical techniques.28

Another common limitation is the inclusion of multiple births. In the SSR calculation, monozygotic multiples can add additional weight to a particular sex, which may distort the relationship between specific ART procedures and SSR. For example, in a population study, it is not possible to distinguish a set of same-sexed twins as a result of a single embryo (monozygotic) or two embryos (dizygotic) if two or more embryos are transferred. Consequently, a single embryo resulting in a set of monozygotic twins contributes twice in the calculation of a particular sex towards a particular ART procedure, whereas each embryo resulting in a set of dizygotic twins contributes only once in the calculation.

The aims of this study were to address a number of these limitations using ART population data from Australia and New Zealand, and to investigate whether ART procedures alter the SSR of ART babies.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Data sources

Data for this retrospective population-based study were supplied from the Australia and New Zealand Assisted Reproductive Database (ANZARD), a repository of ART treatment cycles and their outcomes from all fertility clinics in Australia and New Zealand. The study population included 13 368 babies born to 13 165 women, who had SET between 2002 and 2006. The size of this study population is sufficiently large to detect differences in SSR between the groups using different ART procedures. As in most studies investigating human SSR, the deviation from the general population SSR is usually small but significant, by about 1–2%.29 For the detection of a difference of 1.5% (two-sided test) with a statistical significance of 0.05, a power of 90% requires a sample size of about 12 000 (SAS Power and Sample Size 3.1; SAS Institute Inc., Cary, NC, USA).

To eliminate the potential bias on the SSR calculation by monozygosity in relation to the ART procedures, we restricted our study population to babies born following SET. ANZARD does not have information on zygosity. For the purpose of this study, the following assumptions were made: that babies in the sets of same-sexed multiples were monozygotic and that no spontaneous conception occurred. Therefore, we included only one baby in each set of multiple births in the SSR calculation and analysis. In the study population, multiples accounted for about 2.9% of total SET babies. After selecting one baby from each set of multiples, the proportion of babies born as multiples in the study population reduced to 1.4%.

Study variables

The data variables included in the study were the baby’s sex, fertilisation procedure (IVF or ICSI), stage of embryo development at transfer (blastocyst or cleavage-stage embryo), type of embryo (fresh or thawed), woman’s and partner’s age at the time of transfer, and cause of infertility. BT was defined as the transfer of a day-4 or older embryo, as specified in the ANZARD collection. Cleavage-stage embryo transfer was a transfer of a day-2 or day-3 embryo.

Sex selection is banned in Australia. In particular, three states, Victoria, South Australia and Western Australia, have specific legislation on the use of ART which bans sex selection.30–32 Furthermore, the Guidelines of the National Health and Medical Research Council (NHMRC)33 in Australia prohibit sex selection in Australia. Fertility clinics in Australia to be accredited must comply with the NHMRC ART guidelines. The sex of the babies analysed in this study was not subject to the process of sex selection.

Statistical analysis

The proportion of male live births is used in this study as the SSR. Data were stratified by categorical variables. Logistic regression analysis with backward stepwise (likelihood ratio) method was used to model the SSR as the dependent variable and other variables as predictor variables. In the tables, the odds ratios (ORs) and 95% confidence intervals (CIs) were given by stratification or by predictors. P < 0.05 was considered to be significant. SPSS v17 (SPSS Inc., Chicago, IL, USA) was used for analysis.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

The SSR for babies born following SET was 51.3%, which did not differ from the Australian population SSR of 51.5% (P = 0.655).34 The majority of women who gave birth(s) following SET were aged between 30 and 39 years (75.1%). The proportion of babies born following ICSI SET increased from 51.5% in 2002 to 59.2% in 2006. Similarly, the proportion of babies born following single blastocyst transfer (SBT) also increased from 26.3% in 2002 to 34.7% in 2006. Table 1 shows the SSRs, crude ORs and 95% CIs, the distribution of babies and their parental mean ages by categorical variables. Among these SET babies, 3.2% were born following the transfer of either a donor embryo or donor oocyte.

Table 1.   Sex ratio at birth by individual assisted reproductive technology (ART) characteristics of single embryo transfer (SET) babies, Australia and New Zealand, 2002–2006
CharacteristicsNumber and %Mean age (years) at the time of treatment (mean ± SD)SSR (%)*OR (95% CI)**
WomanPartner
  1. ICSI, intracytoplasmic sperm insemination; IVF, in vitro fertilisation.

  2. *Sex ratio at birth or secondary sex ratio (SSR) – proportion of males of all live born babies.

  3. **Logistic regression analysis for predicting the odds ratio (OR) and 95% confidence interval (CI) for a male birth by single factor. 1, reference category.

  4. ***Other includes missing cause of infertility.

SET babies13 36833.2 ± 4.336.3 ± 6.151.3 
2002116533.5 ± 4.436.8 ± 6.251.4 
2003156133.1 ± 4.436.4 ± 6.051.2 
2004244833.0 ± 4.336.2 ± 6.251.9 
2005344633.2 ± 4.136.3 ± 6.150.5 
2006474833.2 ± 4.336.3 ± 6.151.6 
Fertilisation procedure
IVF44.6%33.4 ± 4.235.6 ± 5.453.01
ICSI55.4%33.0 ± 4.336.9 ± 6.650.00.89 (0.83, 0.95)
Stage of embryo development at transfer
Cleavage-stage SET67.1%33.2 ± 4.436.4 ± 6.249.91
Blastocyst SET32.9%33.2 ± 4.136.2 ± 6.054.11.18 (1.10, 1.27)
Type of embryo
Fresh SET69.1%32.8 ± 4.236.0 ± 6.251.91
Thawed SET30.9%34.0 ± 4.437.2 ± 6.349.90.92 (0.86, 0.99)
Cause of infertility
Male only40.6%32.8 ± 4.236.9 ± 6.750.61
Female only16.3%33.3 ± 4.135.2 ± 5.251.21.02 (0.93, 1.13)
Male and female7.1%33.3 ± 4.436.2 ± 5.851.11.02 (0.89, 1.18)
Unexplained29.3%33.6 ± 4.436.2 ± 5.752.01.06 (0.97, 1.15)
Other***6.6%33.6 ± 4.836.4 ± 6.453.01.10 (0.95, 1.27)

Comparing the SSRs between the categorical variables, we observed a significant difference between IVF and ICSI groups (P = 0.001), with less male babies born following ICSI SET and more male babies born following IVF SET. We also observed that SSR was significantly higher in babies born following SBT than in babies born following cleavage-stage SET (P < 0.001).

There was a statistically significant univariate association (P = 0.032) between fresh/thawed embryo transfer and SSR. However, this association was not significant after adjusting for other variables in logistic regression analysis. Logistic regression analysis did not detect any contributions to the change in SSR by the partner’s age and the cause of infertility.

Table 2 presents the results from logistic regression analysis. The ICSI procedure, SBT and woman’s age at the time of transfer were the significant factors affecting the probability of having a male birth. Among these factors, SBT was the most significant contributor to the change in SSR, whereas ICSI was the second most significant factor. Although significant, the effect of the woman’s age was much smaller than that of the SBT and ICSI procedure.

Table 2.   Predictors of secondary sex ratio (SSR) of single embryo transfer (SET) babies, Australia and New Zealand, 2002–2006
PredictorWald χ2P valueOR (95% CI)
  1. Logistic regression model for predicting the odds ratio (OR) and 95% confidence interval (CI) for a male birth after adjusting for other factors. Wald χ2 indicates the statistical significance of each predictor in the model.

Fertilisation procedure – intracytoplasmic sperm insemination13.60<0.0010.88 (0.82, 0.94)
Stage of embryo development at transfer – blastocyst21.07<0.0011.19 (1.11, 1.28)
Woman’s age8.970.0030.99 (0.98, 1.00)

To further assess the effect of the fertilisation procedure and stage of embryo development on SSR, we stratified the data by the ART treatment regimen: transfer of IVF blastocyst (IVF SBT), transfer of IVF cleavage-stage embryo (IVF cleavage-stage SET), transfer of ICSI blastocyst (ICSI SBT) and transfer of ICSI cleavage-stage embryo (ICSI cleavage-stage SET). We then calculated the SSRs for the four groups (Figure 1). The highest SSR (56.1% males) was in the IVF SBT group and the lowest SSR (48.7% males) was in the ICSI cleavage-stage SET group. The SSR in the IVF cleavage-stage SET group was similar to the Australian population SSR of 51.5%. As a result of the opposing effect of the ICSI procedure and SBT, SSR in the ICSI SBT group did not differ significantly from that of the Australian population.

image

Figure 1.  Sex ratio at birth of babies born following single embryo transfer by specific assisted reproductive technology (ART) regimens, Australia and New Zealand, 2002–2006. CI, confidence interval; ICSI, intracytoplasmic sperm insemination; IVF, in vitro fertilisation.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

This first population-based retrospective study confirms that a specific ART regimen can alter the SSR of babies born following SET. It shows that the use of ICSI is associated with a reduction in SSR, whereas BT is associated with an increase in SSR. In particular, our results show that an ART regimen for a combination of ART procedures can alter SSR significantly, with the highest SSR among babies born following IVF SBT and the lowest SSR in babies born following ICSI cleavage-stage SET.

A low SSR in ICSI babies has been observed previously by Ghazzawi et al.9 in 2000, Bonduelle et al.10 in 2002, Scott and Ryan11 in 2006 and Hentemann et al.35 in 2009. The effect of decreased SSR by ICSI was first demonstrated by Luke et al.36 in 2009. They reported that there was a 10% reduction in SSR by ICSI in babies born following BT, compared to IVF. In our study, a similar reduction (12%) in SSR was also observed. Our findings are also consistent with the result by Luke et al.36 that a diagnosis of infertility does not contribute to the change in SSR. Although the underlying mechanism of reduction in SSR by ICSI remains unclear, one hypothesis by Luke et al.36 is that the cause may be potentially iatrogenic, because the underlying ratio of X- and Y-bearing sperm is almost equal (50.3% Y-bearing sperm).37,38

The higher proportion of male births after BT seen in our study is consistent with the literature.35,39,40 There are a number of possible reasons. One is the morphological selection criteria. More male blastocysts may be selected for transfer because male embryos cleave faster than female embryos from day 2 and up to blastocyst stage.17,18,22,41,42 Another reason is that male embryos have a faster preimplantation development rate than female embryos.20 However, two recent studies by Weston et al.24 and Csokmay et al.,25 assessing retrospectively the mean number of cells and embryo grades of male and female babies, found no difference in growth and delivery rates between male and female embryos. The authors concluded that BT and the selection of higher grade embryos for transfer did not contribute to the increase in SSR.

Although some early studies observed a trend of higher male births following BT,21–23,43 this higher proportion of male births was not significant. One explanation for a lack of an association between BT and an increase in male births is an underpowered study design. This was evident in the studies by Weston et al.24 (435 births, 51.3% males) and Csokmay et al.25 (498 embryos and 120 births, 51.7% males). To overcome the problem of an underpowered study using data from a single clinic, meta-analyses using data from several similar studies have been employed to assess the effect of BT on SSR by some researchers. For example, Milki et al.22 in 2003 analysed data from seven published articles comparing SSRs between BT (n = 1391) and cleavage-stage ET (n = 1909). Chang et al.40 in 2009, using data from four single centres, assessed the effect of BT on SSR (n = 2711). Both meta-analytic review studies found that, by combining published data, SSR was significantly higher in babies born following BT than in babies born following cleavage-stage ET. One of the difficulties in drawing conclusions from meta-analytic review studies is the heterogeneity of the pooled data used in the analysis, which may lead to bias in assessing the effect of the intervention or treatment.44,45 The data used in Chang et al.40 were obtained from 1995 to 2005. During this period, the technology of BT has evolved dramatically. In particular, the sequential medium for blastocyst culture, which was developed in the late 1990s, has improved the pregnancy and implantation rates in BT.46–48 The effect of BT on SSR in 1995 may differ from that in 2005. Our population-based retrospective study, however, used individual record data from the same clinics in Australia and New Zealand between 2002 and 2006. With an overall power of >90% (n = 13 165), our study has confirmed that, at a population level, BT affects SSR significantly, with a higher probability of male births.

Another possible explanation for the inability to detect a significant change in SSR following BT is the unknown proportion of ICSI embryos involved in most earlier studies. As our results suggest, ICSI has the effect of reducing SSR, which may cancel out the effect associated with BT of increasing SSR. Over the 5-year period of this study, the proportions of SET babies born following ICSI and BT have increased by 15% and 32%, respectively. Consequently, the overall SSR for individual years during the study period did not change (Table 1) because of the opposing effects of ICSI and BT on SSR. Although ICSI was developed initially to treat male infertility, it is now also used to treat patients with other infertilities. In Australia and New Zealand, ICSI cycles increased from 57.6% in 2002 to 59.5% in 2006,5 whereas, in European countries, 66.5% of all cycles were ICSI in 2006.49 In the USA, the use of ICSI increased significantly from 11.0% in 1995 to 57.5% in 2005.50 Around the world, ICSI cycles increased from 47.6% in 2000 to 56.6% in 2002.51 The higher utilisation of ICSI may potentially reduce the overall SSR in ART babies, unless a parallel increase in the use of BT also occurs.

A possible confounder for the difference in sex-related embryo development is the in vitro culture conditions.52 Ray et al.42 have suggested that male embryos have higher metabolic activity than female embryos and, in turn, show significantly higher pyruvate and glucose uptake and lactate production. With the different embryo culture media used, the growth rate of embryos may be slightly different across fertility clinics. How this difference in growth rate affects the selection and implantation of embryos and, in turn, SSR is not clear. There remains a lack of clinical evidence on the alteration of SSR in relation to the embryo culture media.

The use of ART treatment around the world has increased. In Australia and New Zealand, 56 817 ART treatment cycles were started in 2007, an increase of 12.5% from 2006.53 According to the latest report from the International Committee for Monitoring Assisted Reproductive Technology,51 over 601 243 cycles were performed in 53 countries in 2002. It is estimated that between 219 000 and 246 000 ART babies were born following ART treatment in that year. ART babies accounted for between 1% and 4% of total births in some countries. In Australia, about 3.1% of total births were ART babies.53 In 2007, 1.8% babies were born following ART treatment in the UK,54 and over 1% were born in the USA.55 Evidence has shown that one in six couples may experience difficulties in getting pregnant56 in their reproductive lives, and these couples may require ART treatment at some stage of their reproductive lives. The proportion of ART babies born per population will probably continue to increase because of the unmet need for fertility treatment, the falling fertility rate and the development of low-cost IVF in many countries.57–59 In parallel, there is an increasing trend for high-income countries to adopt SET, with the ICSI procedure and BT.49,51,53,55 To date, there has been minimal impact on the SSR at the population level. However, the greater use of ART treatment and the increased use of the ICSI procedure and BT may have a major public health impact on SSR, dependent on future treatment regimens. At the individual patient level, the results from this study may provide an indication of the likelihood of having a male or female baby through the ART procedure proposed or used by clinicians.

The strength of this study is the large sample size with individual record data provided by the same ART clinics in the same period. We are able to demonstrate that the bias towards male births by BT and the bias towards female births by ICSI were statistically significant. However, by using registration data, such as ANZARD, for a retrospective study, we may have potentially introduced some variability in the selection criteria, culture media, and laboratory and clinical parameters. The importance of these factors in relation to the change in SSR has yet to be demonstrated with large multicentre clinical studies or from other regions or countries. The use of SET babies in this study has clearly identified the effect of ART procedures on SSR, and has set a common ground for comparison in SSR studies from other regions or countries.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

On the basis of this retrospective population study, we conclude that ART procedures have a significant effect on human SSR. Further investigation of both biological and environmental factors may take into account the effect of ART factors on SSR. However, in the short term, fertility clinics and patients should be aware of the potential increase in SSR with the transfer of IVF blastocysts and decrease in SSR with the transfer of ICSI cleavage-stage embryos.

Disclosure of interest

All authors report no potential conflicts of interest. MGC is a fertility specialist. He is a director of IVF Australia Pty Ltd. and Clinical Director at St George Hospital Kogarah, Sydney, Australia.

Contribution to authorship

JHD conceived and designed the study, analysed the data and wrote the manuscript. MGC discussed the core ideas, provided clinical insight into the data and revised the manuscript. EAS discussed the core ideas, provided critical comments and revised the manuscript. All authors approved the final version of the manuscript.

Details of ethics approval

Ethics approval for this study was granted by the Human Research Ethics Committee of the University of New South Wales, Australia, and the Ethics Committee of the Australian Institute of Health and Welfare.

Funding

The data used in this study were supplied by the ANZARD collection, which was funded by the Fertility Society of Australia. JHD is a PhD candidate at the University of New South Wales, supported by a half-scholarship, which is funded by the Special Trust Fund, St George Hospital, NSW, Australia.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

The authors are grateful to the staff at the fertility clinics in Australia and New Zealand for providing the data to ANZARD, and the staff at the Australian Institute of Health and Welfare National Perinatal Statistics Unit for validating and collating the ANZARD data and supplying the ANZARD data to this study.

References

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  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References
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