ASSISTED HATCHING OR BLASTOCYST TRANSFER

Assisted Hatching or Blastocyst Transfer

                                                                                                                          Narendra Malhotra

                                                                                                                          Jaideep Malhotra

                                                                                                                          J. P. Shah

                                                                                                                                                                  MALHOTRA TEST TUBE BABY CENTRE, AGRA

Introduction

      ART has come to stay. Today there are solutions to all factors of infertility but still world over the results are not improved to what expected.

      Infertility was conquered in July 1978 by the birth of Louise Brown by the pioneering efforts of Patrick Steptoe & Robert Edwards. Since 1978 rapid advances have occurred in the field of assisted reproductive technology. However, live birth rates have still not reached to our full expections.

      The major factors of success are attributed to poor embryo quality and endometrium. Even good fertilized embryos show an embryonic block after 4 cell stage to reach morulla & blastocyst. The embryos which do not have an embryonic block will ultimately become good blastocysts and hatch and implant.

      The culturing of embryo’s to blastocyst stage will overcome the problem of choosing which embryo to transfer for the best results. On the other hand older age group women show implantation failure due to thickened zona and here it has been proposed to go for assisted hatching techniques by either mechanical, chemical or laser methods to help in implantation rates.

      Various studies all over the world have shown that blastocyst culturing and transfer will yield much better pregnancy rates because good-quality blastocysts are crucial for the development of human embryonic stem cells.

Why Results Not Yet Good

Major Compromising Factors

      1. Poor embryo viability.

      2. Poor endometrial receptivity.

Not So Major Factors

      1. Lab conditions and equipments.

      2. Media and disposables.

      3. Stimulation and PT. Selection.

      4. Smooth embryo transfer.

Poor Results

·    Low fertilization.

·    Sperms with poor motility.

·    Chromosome anomaly in oocyte and embryo.

·    Hostile stimulated uterus.

·    A synchronous transfer (Day 2).

·    Reduced viability of embryo cultured in vitro.

·    Embryo viability questionable on Day 2.

·    Other factors age, stimulation, smooth transfer, luteal support etc.

Normal Physiology

·    Oocytes are fertilized

·    Embryo undergo cleavage to compacted or cavitated stages (Day 4-5).

·    (In fallopian tube)

·    Enters uterus - further cleavage.

·    Dissolution of zona (Day 5).

·    Hatching blastocyst (Day 6)

·    Implantation (Day 7).

Why Blastocyst Culture ?

·    To improve pregnancy and take home baby rates.

·    Overcome and cell embryonic block.

·    Blastocyst/Uterine synchrony

·    Genetically abnormal embryos will be weeded out.

·    In vitro blastocyst has a very high TCN (Total Cell Number) hence better results.

·    PGB and freezing.

·    Development of human embryonic stem cells though Inner Cell Mass (ICM) by immunosurgery (Future cure of diseases and genetic therapy).

Choosing Viable Embryo

      Selecting embryos with a high pronuclear (2PN) scores (bongso) and polarity (edwards) and transferring such embryos after the embryonic block at the 8 cell to blastocyst stage wil help overcome the problem of choosing the best embryo and most viable one.

Embryo Assessment

      1.   Scoring

      2.   2 PN embryo quality (Pronucelar score)

      3.   Cleavage

      4.   Biochemical/metabolic markers

      5.   Blastocyst scoring

      6.   TCN of blastocysts

Other Uses of Blastocyst

·    Field of ART to improve results.

·    P.G.D.

·    Research

·    Study of early embryogenesis.

·    Study of development of human embryonic stem cells.

·    Treatment of a variety of genetic and incurable diseases.

Assisted Hatching Where and Why ?

·    Women age > 35 yrs.

·    Thick Zona.

·    Before P.G.D.

·    Previously failed implanation.

Assisted Hatching How ?

·         Mechanical    —Zona thinning

                              —Zona Drilling

·         Chemical - Acid Tyrode

·         Laser

Advantages of Blastocyst

·         Viable hardiest embryo for transfer.

·         Weeding genetically defective embryos.

·         Physiological environment.

·         Avoid inheritance of abnormal paternal genome after ICSI.

·         Non invasive assessment of TCN.

·         Zona free blastocyst by enzymes, better anchorage of trophoectoderm.

·         Post freeze PN embryos cultured post thaw.

·         Embryo repair process after ICSI.

·         AID embryo biopsy.

·         AID in assisted hatching.

·         Reduce time of embryo-maternal dialogue.

·         ICM for development of stem cells.

Culture System

·         Conventional culture media.

·         Human tubal fluid.

·         Co-cultures.

·         Sequentional complex supercompley media (IVF-to-S2, G 1.2 and G 2.2)

G 1.2^TM  and G 2.2^TM

      For a fertilised egg to develop into a blastocyst in-vitro is an achievement. But that does not necessarily mean it is viable and will continue to grow. The new sequential media, G 1.2TM and G 2.2TM have been optimised for blastocysts development according to the latest research at the Colorado Center for Reproductive Medicine. Clinical trials have shown up to 50.5% implantation rate (fetal heart) for blastocysts cultured in G-media*. For more information, contact IVF Science.

Success Package

·         PT selection.

·         Proper stimulation (at least 800 cytes).

·         Appropriate blastocyst culture (68-70% Blastulation rates).

·         Transfer 2/3 blastocysts.

·         Atraumatic transfer (no trauma no blood).

            (Ultrasound Guided and Mock Previous Transfer)

Success Package

                                  Producing Quality Oocytes                          Recovering Quality Sperms

 

Quality Lab Setup

State of the ART Equipments, Consumables

Timed Oocyte Retrival

 

Insemination/ICSI

Culture Systems

Blastocyst Culture

Day 1

Pronuclear scoring

Growth Media (2 PN) (G 1.2/Medicult)

(Each embryo seperate)

Second look at non cleaved 25 hrs

Day 2

Cleavage grading (Fragmentation) (Good/Fair/Poor)

                                    G 1.2 Media (48 hrs)                                    Embryo Transfer (4 cell)

Prepare G 2.2 Dishes (or M3 by medicult)

(Scandinavian)

 

Day 3

Assess cleavage

                        Assisted hatching (8 cell)             Transfer to G 2.2            Embryo transfer

Day 4

Assess cleavage culture in G 2.2 (48 hrs)

Blastocyst culture

Day 4

Priority to highest 20 N score

prepare one ET dish, one freeze dish & day 6 growth dish (overnight incubate)

Day 5

Blastocyst assess & scoring

                                 Transfer                                     Freeze                              Slow growing

                                   Good                                        Spare

                             2 Blastocysts                             Blastocysts

                              (Zona intact)                                                                       Fresh G 2.2 media

                          Zona free transfer                                                                            Day 6

                100 µl droplet of 101 U Pronase                                                                 Freeze

                             For 1-11/2 hrs

                             Wash 4 times

                                 Transfer

                        Zona free blastocyst

Pregnancy outcomes (Bongso)

Day 3-5 Transfer

      Clinical preg per ET with USG detectable

      Cardiac activity at 7-8 wks   50-57%

Day 2 Transfer

      37%

      Take Home Baby Rates

      Day 2         15-25%

      Day 3         29-42%

      Day 4         33%

      Day 5         52%

      (Singapore Group Bongso ET AL)

Blastocyst Transfer

      1.   no. of Patients                             39

      2.   no. of Embryos for blastulation     53

      3.   no. enbryos reached blastocyst    21 (39.62%)

      4.   no. of blastocyst transferred         13

      5.   no. of ET done                             11

      6.   no. of clinical pregnancies            3 (27%)

      (Fertility Clinic Mumbai) (1998-99)

MTTBC Agra Results (1998-2000)

      1.    Total cycles                                     318

      2.    Day 2 transfers                                285                                                  (Pregnancy 23%)

      3.    Day 3/4 transfer                               25                                                    (Pregnancy 28%)

      4.    Day 5 transfer                                  8                                                      (Pregnancy 51%)

      5.    No. of Embryos for Blastulation is 8 cases = 31                                                                                              

      6.    Blastulation sem is 10 embryos                                                               

      7.    No. of Blastocyst E.T. (cases 2)      

Bottom line & take home message

·         Do assisted hatching at 8 cell stage

·         Micromanipulator guided blastomere biopsy

·         P.G.D.

·         Culture to blastocyst

·         Transfer only P.G.D.—re & blastocyst

·         Overcomes problem of embryonic block

·         Healthy embryos (Genetically healthy)

·         Very good pregnancy rates (> 50%)

·         Very good take home baby rates (> 40%)

Conclusion

      The techniques of micromanipulator have greatly improved the fertilization rats upto 90% with Blastocyst culture the pregnancy rate in experted to go upto 40-50%. A very ideal situation for best pregnancy rates would be to make holes in zona pellucida at the day 3, 8 cell stage by assisted hatching technique by micromanipulator and doing pre-implantation genetic diagnosis and then subsequently growing the healthy breached embryos to the blastocyst stage and then doing a smooth blastocyst transfer on day 5.

n n