Sibling Embryo Blastocyst Development As a Prognostic Factor for the Outcome of Day-3 Embryo Transfer

Reproductive BioMedicine Online (2013) 26, 486– 490

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ARTICLE Sibling embryo blastocyst development as a prognostic factor for the outcome of day-3 embryo transfer

Antonio MacKenna a,*, Javier Crosby a, Fernando Zegers-Hochschild a,b a Unit of Reproductive Medicine, Department of Obstetrics and Gynecology, Clinica Las Condes, Chile; b Program of Ethics and Public Policies in Human Reproduction, University Diego Portales, Chile * Corresponding author. E-mail address: [email protected] (A MacKenna).

Dr Antonio Mackenna received his MD and MSC degrees from the Chilean University and the Chilean Catholic University, respectively. He obtained his specialization in obstetrics and gynaecology at the Chilean Catholic Universityand completed a fellowship in infertility and reproductive endocrinology at the department of obstetrics and gynaecology, Shefﬁeld University, UK. He works at the Unit of Reproductive Medicine, Clinica Las Condes, Santiago, Chile, and is a professor at the Chilean University.

Abstract This study assessed the development of sibling embryos to blastocyst as a prognostic factor for patients undergoing embryo transfer at day 3. A retrospective analysis of a clinical and embryology database including 353 patients who underwent 393 cycles of intracytoplasmic sperm injection with day-3 embryo transfer and excess embryos, maintained in culture until day 5, was performed. Cycles were divided into group A and group B (with and without blastocyst formation, respectively). Age and basal FSH were similar in both groups. Statistically signiﬁcant differences in clinical pregnancy rates (55.8% versus 40.6%; P = 0.0031), live birth rates (50.0% versus 37.2%; P = 0.012) and implantation rates (34.2% versus 23.7%; P = 0.0035) were observed in groups A and B, respectively. Odds ratios showed women from group A had 1.85- and 1.68-times the odds of patients from group B of achieving clinical pregnancy and a live birth, respectively. Cumulative live birth rate for group A, after one cycle of vitriﬁed–warmed blastocyst transfer, was 66.4%. The development of sibling embryos to blastocyst is a prognostic factor for the outcome of the cycle in which transfer is performed at day 3 and provides valuable information about the prognosis of subsequent cycles. RBMOnline ª 2013, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: blastocysts, cumulative live birth rate, cumulative pregnancy rate, sibling embryos

Introduction For many years these elements have been considered as prognostic factors for becoming pregnant or having a live Multiple factors inﬂuence the outcome of assisted reproduc- birth in a particular treatment cycle (Minaretzis et al., tion treatment, the most relevant being female age, dura- 1998). However, in the last decade the focus has moved tion of infertility, ovarian reserve and embryo quality towards predicting the probability of singleton or twin preg- (Hathaway et al., 1999; van Loendersloot et al., 2010). nancies when two cleaving embryos could be transferred

1472-6483/$ - see front matter ª 2013, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rbmo.2013.02.003 Blastocyst formation from sibling embryos 487

(Hunault et al., 2007), in order to transfer them or to per- global fertilization media (Life Global) in a 6% CO2 incubator form an elective single-embryo transfer (SET) of one (Forma Scientific, USA). Eighteen hours after sperm injec- selected cleavage-stage embryo and avoid a multiple preg- tion, oocytes were inspected for fertilization and those fer- nancy (De Sutter et al., 2003; Practice Committee of Soci- tilized were transferred to fresh global media (Life Global, ety for Assisted Reproductive and and Practice Committee USA). A total of 5020 zygotes from 393 oocyte retrievals of American Society for Reproductive, 2012). Furthermore, were followed from day 1 to day 3. Embryo morphology when enough good-quality embryos are available at day 3, was assessed at day 3 using criteria reported by Veeck an extended culture until day 5 is advisable, in order to (1999), which considers cell number, uniformity of blasto- improve embryo selection and pregnancy rates (Papaniko- mere size and extent of fragmentation. Each embryo was laou et al., 2005, 2008). But patients without four or five designated based on its cell number and grade of uniformity good-quality embryos at day 3 do not have the minimum and fragmentation, as follows; grade 1, symmetric blasto- requirements for extended embryo culture until day 5 and meres without fragmentation; grade 2a, symmetric blasto- must undergo cleavage-stage embryo transfer (Papaniko- meres and <20% fragmentation; grade 2b, symmetric laou et al., 2005; Dessolle et al., 2010), because of the risk blastomeres and 20–50% fragmentation; grade 3, uneven of cycle cancellation due to a lack of blastocysts. size of blastomeres, without fragmentation; and grade 4, Many embryo selection criteria for cleavage-stage uneven blastomeres with fragmentation. embryos have been developed in order to choose the best A total of 960 embryos were transferred at day 3 with a embryos and reduce the number of them to be transferred mean of 2.44 ± 0.55 embryos per patient. Of these, 748 at day 3 (Geraedts and Gianaroli, 2012), although, embryo (77.9%) were classified as good-quality embryos (grades 1 selection at this stage has some limitations. Indeed, Rijn- and 2a). Embryo transfer was performed with an ders and Jansen (1998) reported that only 51% of embryos ultrasound-guided Frydman ultra-soft catheter (CCD, that were transferred on day 5 had been preselected for France). Fourteen days after oocyte retrieval the patients transfer on day 3. Therefore, it is difficult to predict the were tested for pregnancy (b-human chorionic gonadotrophin) result of assisted reproduction treatments based on the and, if pregnant, an ultrasound was scheduled 1 and 2 weeks quality of embryos transferred at day 3, and other criteria later to confirm a clinical pregnancy, register the number of have to be introduced to establish the prognosis of the gestational sacs and the presence of embryos with heart- treatment cycle. These could be previously known clinical beats. Pregnancies were followed until 24 weeks of amenor- criteria of patients (van Loendersloot et al., 2010) or data rhoea, to register those ending in miscarriages, and then coming from the embryology laboratory in the same cycle until the delivery in all remaining cases. in which the transfer is performed. This information can A total of 4060 excess embryos remained in culture and also be considered to define patients that are eligible for were followed until day 5, 459 of them were classified as elective SET or single-blastocyst transfer in future cycles. good quality or grade 1 and 2a (11.3%). Blastocyst formation The main objective of this study is to assess the develop- was evaluated at day 5, using criteria reported by Veeck and ment of sibling embryos to blastocyst stage as a prognostic Zaninovic (2003). A total of 350 embryos reached this stage factor for patients undergoing embryo transfer at day 3. and were vitrified using the Cryotop Kitazato method, ini- tially described by Kuwayama et al. (2005). For warming, the Cryotop was removed from the liquid nitrogen and Materials and methods instantly placed in thawing solution at 37°C; after 1 min, blastocysts were placed in diluting solution at room temper- This is a retrospective analysis of the embryology database ature for 3 min and, finally, a 5-min wash followed by a from January 2006 to December 2011 at the Unit of Repro- 1-min wash was performed with washing solution at room ductive Medicine of the department of obstetrics and gynae- temperature and processed as described by the manufac- cology at Clinica Las Condes, Chile. All patients signed turer (Kitazato). Viability was assessed and transfer of vitri- institutional review board approved consent forms before fied–warmed blastocysts was performed in cycles with initiating and any patient specific identifiers were removed hormone replacement with oral oestradiol valerate and vag- from database prior to the analysis. Retrospective approval inal progesterone. for this study was granted by the ethical committee of For the purposes of this study, the 393 sample cycles Clinica Las Condes on 30 January 2013. were divided into two groups: those who had at least one In total, 353 patients, who underwent assisted reproduc- blastocyst at day 5 (group A) and those who did not have tion treatment in 393 cycles, had embryos transferred at blastocysts at day 5 (group B). For statistical analysis Stata day 3 and had excess embryos that remained in culture until 12 software (Stata Corporation, College Station, Texas, day 5 were included in this study. The age of women was USA) was used. The independent variable was blastocyst (mean ± SD) 34.5 ± 3.8 years and their basal FSH was and primary outcomes were clinical pregnancy rate (trans- 7.1 ± 2.7 mIU/ml. vaginal ultrasound confirmation of a gestational sac inside Intracytoplasmic sperm injection was performed in all the uterine cavity) and live birth rate. Secondary outcomes cases. The oocytes were stripped of their cumulus cells were implantation rate (number of gestational sacs after incubation in hyaluronidase solution (80 IU/ml; Life observed divided by the number of embryos transferred), Global, USA) for 60 s. Spermatozoa were selected by the multiple pregnancy rate and miscarriage rate (number of swim-up procedure and sperm injection was performed with spontaneous clinical pregnancy losses divided by the num- holding and injection micropipettes (Humagen, USA) under ber of clinical pregnancies). Continuous variables were an Olympus inverted microscope and a Narishige microma- described by mean ± standard deviation (normal distribu- nipulation system. Injected oocytes were incubated in tion) and categorical variables by proportions and rates, 488 A MacKenna et al. and to assess differences between the two groups, Student’s Discussion t-test and chi-squared test were used, respectively. To determine significant association between the presence of Without having the possibility of an extended culture to day blastocysts on day 5 and primary outcomes, odds ratios with 5, for blastocyst transfer, the assessment of day-3 embryo 95% CI were calculated by logistic regression. P-value <0.05 characteristics is a key factor to improve the results of was considered significant for purposes of assessing differ- assisted reproduction treatment. However, as has been ences between both groups. demonstrated, it is not accurate enough to predict the outcome of a treatment cycle (Rijnders and Jansen, 1998). Results Therefore, when excess embryos are available, subsequent blastocyst formation could be a good prediction factor for The analysis of the 393 cycles performed in 353 patients the outcome of the current cycle. Sibling embryos achieving who underwent embryo transfer on day 3 showed 46.6% blastocyst stage were available in 39.2% of the cycles in this (183/393) clinical pregnancy rate per cycle, 26.6% study, which is higher than blastocyst formation rate from (255/960) implantation rate, 36.1% (66/183) multiple preg- excess embryos reported by Dayal et al. (2006), regardless nancy rate and 8.7% (16/183) miscarriage rate. In 154 of the cause of infertility. cycles, at least one remaining embryo reached the blasto- It has to be considered that the first signal that the cyst stage (39.2%). results in group A would be better than in group B was the Group A comprised 149 patients undergoing 154 cycles proportion of good-quality embryos among those that were with subsequent blastocyst formation and group B com- transferred (86.7% versus 72.7%) and within the sibling prised 212 patients undergoing 239 cycles without embryos (35.8% versus 5.4%). Although, this preliminary subsequent blastocyst formation. A comparison of the gen- indicator can be obtained at day 3, a more powerful one eral characteristics of groups A and B is provided in Table 1. is the success of sibling embryos to develop to the blasto- Table 2 shows the outcome of patients in groups A and B. cyst stage at day 5. Indeed, results in this study suggest There were statistically significantly higher clinical preg- development of sibling embryos to blastocyst is a strong nancy rate per cycle (P = 0.0031), live birth rate (P = 0.012) prognostic factor for the results of the cycle in which and implantation rate (P = 0.0035) in group A than in group embryo transfer has to be performed at day 3. Statistically B. Odds ratios (logistic regression) showed women from significant higher pregnancy rates (55.8% versus 40.6%), live group A had 1.85- and 1.68-times the odds as patients from birth rates (50.0% versus 37.2%) and implantation rates group B of achieving a clinical pregnancy and having a live (34.2% versus 23.7%) were observed in cycles in which blas- birth, respectively. On the other hand, no differences were tocyst stage was reached by one or more of excess embryos observed in multiple pregnancy rates and miscarriage rates remaining in culture until day 5 (group A) compared with between both groups. cycles without blastocyst formation (group B). Actually, In group A, 98/106 patients underwent one cycle of the chance of becoming pregnant and having a live birth vitrified–warmed blastocyst transfer within 2 years of the was much higher in group A than in group B (OR 1.85, 95% first treatment cycle with a 92.5% survival of at least one CI 1.22–2.78 versus OR 1.68, 95% CI 1.11–2.54, respec- vitrified blastocyst per patient. The number of blastocysts tively). These findings, also reported by Sjogren et al. transferred was 1.87 ± 0.66, with 25.5% (25/98) clinical (1992) and Fisch et al. (1999) in smaller series, suggest that pregnancy rate per cycle, 15.2% (28/184) implantation rate, sibling embryo blastocyst formation may reflect superior 10.7% (3/28) multiple pregnancy and 17.9% (5/28) miscar- embryo quality and the fact that, at least, one of these riage rates. Cumulative clinical pregnancy rate and live embryos reaches the blastocyst stage after performing the birth rate for group A, after one fresh embryo transfer at transfer at day 3 can predict better implantation and preg- day 3 and one vitrified–warmed blastocyst transfer, were nancy rates in the current cycle compared with cycles with- 74.5% (111/149) and 64.4% (96/149), respectively. out embryos developing to blastocyst. Furthermore, results

Table 1 General characteristics of patients who underwent day-3 embryo transfer with (group A) or without (group B) subsequent blastocyst formation. Group A (n = 149) Group B (n = 212) P-value

Cycles 154 239 – Age (years) 34.3 ± 3.6 34.7 ± 3.9 NS Basal FSH (mIU/ml) 7.0 ± 2.3 7.2 ± 2.9 NS Transferred embryos on day 3 2.33 ± 0.47 2.51 ± 0.58 0.0023 Proportion of good-quality embryos transferred on day 3a 86.7 (312/360) 72.7 (436/600) <0.00001 Sibling embryos 788 3272 – Proportion of good-quality sibling embryosa 35.8 (282/788) 5.4 (177/3272) <0.00001

Values are n, mean ± SD or % (n/total). aProportion of embryos classiﬁed as grade 1 and 2a on day 3. Blastocyst formation from sibling embryos 489

Table 2 Outcomes for patients who underwent day-3 embryo transfer with (group A) or without (group B) subsequent blastocyst formation. Group A (n = 149) Group B (n = 212) P-value Odds ratio (95% CI)

Cycles 154 239 – – Clinical pregnancy rate per cycle 55.8 (86/154) 40.6 (97/239) 0.0031 1.85 (95% CI 1.22–2.78) Live birth rate 50.0 (77/154) 37.2 (89/239) 0.012 1.68 (95% CI 1.11–2.54) Implantation rate 34.2 (123/360) 23.7 (142/600) 0.0035 – Multiple pregnancy rate 38.4 (33/86) 34.0 (33/97) NS – Miscarriage rate 10.5 (9/86) 7.2 (7/97) NS –

Values are n or % (n/total).

obtained in group A gives a 74.5% cumulative pregnancy rate the prognosis of the current cycle but also to prospectively and a 66.4% cumulative live birth rate when one fresh predict the cumulative pregnancy rate when sibling embryos embryo transfer at day 3 and one vitrified–warmed blasto- develop to blastocysts and also to make better decisions for cyst transfer were performed, reinforcing the issue that this subsequent assisted cycles. Indeed, the capability of group of patients has good-quality embryos and very good remaining embryos to develop until day 5 may provide valu- prognosis for assisted reproduction treatment. able information about the potential for a future transfer of There is still controversy about the practice of allowing fresh embryos at day 3. The fact that implantation rate was sibling embryos to develop until day 5 and cryopreserving 34.1% in group A suggests that SET gives acceptable preg- blastocysts versus cryopreserving them at day 3 in order nancy rates almost without multiple pregnancies in patients to transfer frozen–thawed blastocysts, frozen–thawed with the characteristics of this group, achieving success cleavage embryos or blastocysts from frozen–thawed cleav- rates similar to those reported for elective SET (Tiitinen age embryos. Pantos et al. (2001) reported a 20.6% implan- et al., 2003; Thurin et al., 2004; Veleva et al., 2006). Fur- tation rate in the group transferring blastocysts from day-3 thermore, patients having blastocyst development from sib- cryopreserved embryos against only 5.3% in the group trans- ling embryos may be considered as eligible for fresh ferring cryopreserved blastocysts. However, vitrification blastocyst transfer in subsequent cycles, improving live has improved results (Sifer et al., 2012), and in the present birth rates for future treatments (Glujovsky et al., 2012). study, implantation rate transfering vitrified-thawed blasto- This applies to 39.2% of all cycles in which embryo transfer cysts was 3-fold (15.2%). Wang et al. (2010, 2011) suggested at day 3 was considered the best choice for the patients in that transfers of thawed blastocysts and thawed cleavage this study. Moreover, patients in group A could also be embryos had significantly lower odds of live birth than trans- future candidates for single-blastocyst transfer, a practice fer of blastocysts from thawed cleavage embryos; however, that ends in higher pregnancy rates than elective SET (Gard- this retrospective study was also performed with slow freez- ner et al., 2004). Actually, the implementation of methods ing and live birth rate for thawed blastocysts was only for deciding which patients are suitable for elective SET and 16.3%. More recently, Eftekhar et al. (2012), in a prospec- single-blastocyst transfer should be a goal in centres having tive study, reported 30% implantation rate transferring blas- multiple pregnancy rates higher than 30% and this study tocysts coming from cleavage-stage frozen–thawed offers a new alternative for making this decision in women embryos compared with only 17% transferring day-3 fro- undergoing further treatment cycles. zen–thawed embryos. The question is whether sibling embryos should be cultured until day 5 to vitrify them as Acknowledgement blastocysts or vitrified at day 3 for subsequent culture after warming. Further prospective randomized case–control The authors would like to thank Magdalena Castro RN, MSc, studies using vitrification techniques have to be performed for her valuable statistical advice and assistance in perform- to assess this issue. However, it has to be considered that ing the analysis. the practice of allowing sibling embryos to develop until day 5 before vitrifying them reduces the number of useless embryos stored, which is an extra advantage for assisted References reproduction centres and patients undergoing treatment. On the other hand, despite the fact that the lower qual- Dayal, M.B., Kovalevsky, G., Patrizio, P., 2006. Rate of blastocyst ity of available embryos at day 3 in group B was compen- development from excess embryos remaining in culture after sated in patients included in this study by an increase in day 3 embryo transfer. Int. J. Fertil. Womens Med. 51, 136–139. De Sutter, P., Van der Elst, J., Coetsier, T., Dhont, M., 2003. Single the number of embryos transferred, in order to improve embryo transfer and multiple pregnancy rate reduction in the outcome, this practice was not effective enough to IVF/ICSI: a 5-year appraisal. Reprod. Biomed. Online 6, achieve the goal. Eventually, patients from group A had a 464–469. better prognosis than those from group B. Dessolle, L., Freour, T., Barriere, P., Daraı¨, E., Ravel, C., Jean, M., This study highlights the value of blastocyst formation Coutant, C., 2010. A cycle-based model to predict blastocyst from sibling embryos, not only to retrospectively assess transfer cancellation. Hum. Reprod. 25, 598–604. 490 A MacKenna et al.

Eftekhar, M., Aflatoonian, A., Mohammadian, F., Tabibnejad, N., Practice Committee of Society for Assisted Reproductive, T. and M. 2012. Transfer of blastocysts derived from frozen-thawed and Practice Committee of American Society for Reproductive, cleavage stage embryos improved ongoing pregnancy. Arch. 2012. Elective single-embryo transfer. Fertil. Steril. 97, Gynecol. Obstet. 286, 511–516. 835–842. Fisch, J.D., Milki, A.A., Behr, B., 1999. Sibling embryo blastocyst Rijnders, P.M., Jansen, C.A., 1998. The predictive value of day 3 development correlates with the in vitro fertilization day 3 embryo morphology regarding blastocyst formation, pregnancy and embryo transfer pregnancy rate in patients under age 40. Fertil. implantation rate after day 5 transfer following in-vitro fertilization Steril. 71, 750–752. or intracytoplasmic sperm injection. Hum. Reprod. 13, 2869–2873. Gardner, D.K., Surrey, E., Minjarez, D., Leitz, A., Stevens, J., Sifer, C., Sermondade, N., Dupont, C., Poncelet, C., Ce´drin- Schoolcraft, W.B., 2004. Single blastocyst transfer: a prospec- Durnerin, I., Hugues, J.N., Benzacken, B., Levy, R., 2012. tive randomized trial. Fertil. Steril. 81, 551–555. Outcome of embryo vitrification compared to slow freezing Geraedts, J.P., Gianaroli, L., 2012. Embryo selection and IVF. Hum. process at early cleavage stages. Report of the first French birth. Reprod. 27, 2876–2877. Gynecol. Obstet. Fertil. 40, 158–161. Glujovsky, D., Blake, D., Farquhar, C., Bardach, A., 2012. Cleavage Sjogren, A., Sjoblom, P., Hamberger, L., 1992. Culture of human stage versus blastocyst stage embryo transfer in assisted spare preembryos: association between blastocyst formation reproductive technology. Cochrane Database Syst. Rev 11, 7, and pregnancy. J. Assist. Reprod. Genet. 9, 41–44. CD002118. Thurin, A., Hausken, J., Hillensjo, T., Jablonowska, B., Pinborg, A., Hathaway, P., Croucher, C., Lass, A., 1999. Predictive value of the Strandell, A., Bergh, C., 2004. Elective single-embryo transfer results of a first IVF cycle. Hum. Reprod. 14, 2417–2418. versus double-embryo transfer in in vitro fertilization. N. Engl. Hunault, C.C., te Velde, E.R., Welma, S.M., Macklon, N.S., J. Med. 351, 2392–2402. Eijkemans, M.J., Klinkert, E.R., Habbema, J.D., 2007. A case Tiitinen, A., Unkila-Kallio, L., Halttunen, M., Hyden-Granskog, C., study of the applicability of a prediction model for the selection 2003. Impact of elective single embryo transfer on the twin of patients undergoing in vitro fertilization for single embryo pregnancy rate. Hum. Reprod. 18, 1449–1453. transfer in another center. Fertil. Steril. 87, 1314–1321. van Loendersloot, L.L., van Wely, M., Limpens, J., Bossuyt, P.M., Kuwayama, M., Vajta, G., Kato, O., Leibo, S.P., 2005. Highly Repping, S., van der Veen, F., 2010. Predictive factors in in vitro efficient vitrification method for cryopreservation of human fertilization (IVF): a systematic review and meta-analysis. Hum. oocytes. Reprod. Biomed. Online 11, 300–308. Reprod. Update 16, 577–589. Minaretzis, D., Harris, D., Alper, M.M., Mortola, J.F., Berger, M.J., Veeck, L.L., 1999. An atlas of Human Gametes and Conceptuses: an Power, D., 1998. Multivariate analysis of factors predictive of Illustrated Reference for Assisted Reproductive Technology. successful live births in in vitro fertilization (IVF) suggests Parthenon, New York; London. strategies to improve IVF outcome. J. Assist. Reprod. Genet. 15, Veeck, L.L., Zaninovic, N., 2003. An Atlas of Human Blastocysts. 365–371. Parthenon, Boca Raton; London. Pantos, K., Stefanidis, K., Pappas, K., Kokkinopoulos, P., Petrout- Veleva, Z., Vilska, S., Hyden-Granskog, C., Tiitinen, A., Tapanai- sou, K., Kokkali, G., Stavrou, D., Tzigounis, V., 2001. Cryopres- nen, J.S., Martikainen, H., 2006. Elective single embryo transfer ervation of embryos, blastocysts, and pregnancy rates of in women aged 36–39 years. Hum. Reprod. 21, 2098–2102. blastocysts derived from frozen-thawed embryos and Wang, Y.A., Chapman, M., Costello, M., Sullivan, E.A., 2010. Better frozen-thawed blastocysts. J. Assist. Reprod. Genet. 18, perinatal outcomes following transfer of fresh blastocysts and 579–582. blastocysts cultured from thawed cleavage embryos: a popula- Papanikolaou, E.G., D‘Haeseleer, E., Verheyen, G., Van de Velde, tion-based study. Hum. Reprod. 25, 1536–1542. H., Camus, M., Van Steirteghem, A., Devroey, P., Tournaye, H., Wang, Y.A., Costello, M., Chapman, M., Black, D., Sullivan, E.A., 2005. Live birth rate is significantly higher after blastocyst 2011. Transfers of fresh blastocysts and blastocysts cultured transfer than after cleavage-stage embryo transfer when at least from thawed cleavage embryos are associated with fewer four embryos are available on day 3 of embryo culture. A miscarriages. Reprod. Biomed. Online 23, 777–788. randomized prospective study. Hum. Reprod. 20, 3198–3203. Papanikolaou, E.G., Kolibianakis, E.M., Tournaye, H., Venetis, C.A., Declaration: The authors report no financial or commercial Fatemi, H., Tarlatzis, B., Devroey, P., 2008. Live birth rates conflicts of interest. after transfer of equal number of blastocysts or cleavage-stage embryos in IVF. A systematic review and meta-analysis. Hum. Received 2 December 2012; refereed 31 January 2013; accepted 5 Reprod. 23, 91–99. February 2013.