HATCHING OF BLASTOCYSTS IN VITRO ON DAY 6 OF EMBRYO CULTURE IS CORRELATED WITH INCREASED PREGNANCY, IMPLANTATION AND LIVE BIRTH RATES IN HUMAN IVF-ET

L.K. Smith, E.H. Roots and M.J. Odom DorsettThe Centre for Reproductive Medicine, Lubbock, TX

IntroductionRecent advances in ART have made it possible to routinely

culture human embryos to the blastocyst stage. Blastocyst

culture and transfer has been reported to increase pregnancy

and implantation rates and to reduce the risk of higher order

multiple pregnancies in human IVF-ET (1-4). Blastocyst

transfers are performed primarily on Day 5 of embryo

culture, with the most advanced embryos presenting as

expanded blastocysts. Recent studies in our laboratory

suggest that Day 6 blastocyst transfers increase pregnancy,

implantation and live birth rates by increasing the efficacy

of embryo selection. The most advanced embryos present

as hatched zona-free (HZF) blastocysts or hatching

blastocysts on Day 6. We have investigated what effect

the hatching of blastocysts in vitro has on pregnancy,

implantation and live birth rates in IVF-ET.

ObjectiveThe objective of this study was to determine if the hatching

of blastocysts in vitro was correlated with pregnancy,

implantation and live birth rates in IVF-ET.

DesignA three-year retrospective analysis of all IVF-ET cases

with Day 6 blastocyst transfers from January 1, 1999

to December 31, 2001.

Materials/MethodsBlastocyst culture was performed on all IVF-ET cases.

Oocytes were co-incubated with 200,000 motile sperm/mL

in 100 µL drops of S1 or G1.2 media (Vitrolife) under

mineral oil (Squibb). All cultures were performed in a

37ºC incubator with 5% CO2. Denuding and fertilization

checks were performed at 18 hrs. Embryos were washed

and moved to 30-100 µL drops of S1 or G1.2 media under

mineral oil and cultured until Day 3. The embryos were

then washed and transferred to 30-100 µL drops of S2

or G2.2 media under mineral oil and cultured until Day 6.

Blastocyst transfers were performed on Day 6 with an

18 cm Wallace transfer catheter. Statistical analysis

of the data was performed using Chi square.

ResultsBlastocyst Classification Criteria:

Early BlastocystBlastocoele formation has begun.Cavitation is < 100%.

Expanding BlastocystCavitation is 100%.Blastocyst expansionhas begun.

Hatching BlastocystBlastocyst has herniated the zona.

Hatched Zona-Free(HZF) BlastocystBlastocyst has completelyevacuated the zona.

Blastocysts were classified as describe above. Results areexpressed as a percent of the total blastocysts on Day 6.

Table 1: Effect of Blastocyst Hatching In Vitro on Pregnancy, Implantation and Live Birth Rates

Blastocyst Stage: Early Expanding Hatching HZFBlastocyst Blastocyst Blastocyst Blastocyst

-------------------------------------------------------------------------------------------------------# Transfers 41 42 71 33

Positive HCG/Transfer 26.8%* 61.9% 83.1%* 97.0%*

Positive US/Transfer 19.5%* 42.9% 69.0%* 75.8%*

Live Birth/Transfer 19.5%* 40.5% 67.6%* 72.7%*

% Implantation 13.7%* 34.7% 51.0%* 61.8%*

Transfers were classified based on the developmental stage of the best embryoat transfer on Day 6. * Results differ from control (Expanding Blastocyst) atp < 0.01 Chi square.

ConclusionsThe hatching of blastocysts in vitro was correlated with an increase inpregnancy, implantation and live birth rates in IVF-ET cases with Day 6blastocyst transfers. These results suggest that the hatching of blastocystsin vitro may be used as a positive selection criterion for embryo transfers.IVF-ET cases that present 1 HZF or hatching blastocysts on Day 6 haveimplantation rates that make them excellent candidates for single embryotransfers.

References1. Gardner DK, Vella P, Lane M, Wagley L, Schlenker T, Schoolcraft WB. Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers. Fertil Steril 1998;69:84-88.2. Gardner DK, Schoolcraft WB, Wagley L, Schlenker T, Stevens J, Hesla J. A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization. Hum Reprod 1998;13:3434-3440.3. Behr B, Pool TB, Milki AA, Moore D, Gebhardt J, Dasig D. Preliminary clinical experience with human blastocyst development in vitro without co-culture. Hum Reprod 1999;14:454-457.4. Marek D, Langley M, Gardner DK, Confer N, Doody KM, Doody KJ. Introduction of blastocyst culture and transfer for all patients in an in vitro fertilization program. Fertil Steril 1999;72:1035-1040.

Graph 1: Day 6 Blastocyst Distribution

36.4%

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25.3%

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Early Blastocyst Expanding Blastocyst Hatching Blastocyst HZF Blastocyst

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