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Year : 2016  |  Volume : 3  |  Issue : 3  |  Page : 124-127

Could zona-free blastocyst transfer be the next step in optimizing in vitro fertilization outcomes? A case report of successful outcome after zona-free fresh embryo transfer with preimplantation genetic screening

Lab.Director, Chief Embrylogist, Akanksha Hospital and Research Institute, Lambvel, Anand, Gujarat, India

Date of Web Publication21-Apr-2017

Correspondence Address:
Harsha K Bhadarka
Akanksha Hospital and Research Institute, Lambvel, Anand, Gujarat
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2348-2907.204670

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The objective of this study was to study successful outcome after zona-free fresh embryo transfer with preimplantation genetic screening (PGS) in assisted reproductive technology. The study design was a case report. The study was conducted at in vitro fertilization (IVF) clinic. Patients with multiple IVF failure and miscarriage were included in the study. Zona-free fresh embryo transfer after PGS was intervened. The main outcome measure was live birth after IVF. In a PGS-indicated patient, zona-free chromosomally normal embryo was transferred and live healthy female baby weighing 2.7 kg was delivered.We report a case of natural complete hatching of an embryo after PGS, which was aided by mechanical breach of zona for trophectoderm biopsy. Carefully transferred zona-free embryo in a surrogate resulting in successful pregnancy and live birth which indicates that a good human blastocyst must have ability to expand in vitro and completely hatch out from zona after transfer for implantation.

Keywords: Laser-assisted hatching, preimplantation genetic screening, trophectoderm, zona pellucida, zona-free blastocyst

How to cite this article:
Bhadarka HK, Patel NH, Patel KB, Jadeja YD. Could zona-free blastocyst transfer be the next step in optimizing in vitro fertilization outcomes? A case report of successful outcome after zona-free fresh embryo transfer with preimplantation genetic screening. IVF Lite 2016;3:124-7

How to cite this URL:
Bhadarka HK, Patel NH, Patel KB, Jadeja YD. Could zona-free blastocyst transfer be the next step in optimizing in vitro fertilization outcomes? A case report of successful outcome after zona-free fresh embryo transfer with preimplantation genetic screening. IVF Lite [serial online] 2016 [cited 2021 Dec 8];3:124-7. Available from: http://www.ivflite.org/text.asp?2016/3/3/124/204670

  Introduction Top

Assisted zona hatching has been used in in vitro fertilization (IVF) programs for several years. Successful hatching of the embryo from the zona pellucida is a prerequisite for implantation in the uterus. Assisted zona hatching came into limelight in IVF cycles to breech the zona pellucida and promote the natural process of hatching.[1],[2] Embryo transfer programs started off with day 2 transfer; success rates were then usually much lower (10%–20%). IVF had a major breakthrough when its successful blastocyst culture dramatically increased the success rates to around 40%.[3] Blastocyst transfer was not only more physiological but also the strongest of embryos which bypass the 4–8 cell block and survived blastocyst and hatching stages were transferred. Although human blastocyst expands readily in vitro, its blastocyst rate very heavily depends on laboratory environment, culture media, and techniques.[4] Majority of blastocyst that have hatching problems are unable to completely hatch out of their zona pellucida and subsequently degenerate by day 6 or even day 7.[1] The role of zona includes initial binding of sperm, induction of acrosome reaction, and prevention of multiple sperm penetration and of blastomere scatter with avoidance of direct contact with toxic by-products.[5] It has been demonstrated that the zona is no longer essential for continuing of normal development in vitro, once compaction has been achieved.[6] It has been theorized that protease-like enzyme is released in vivo either by the blastocyst itself or by the endometrium which aids hatching and subsequent implantation.[1],[7] In view of the above-mentioned theory, assisted hatching was practiced by drilling small holes in the zona pellucide with acid Tyrode mechanically and more recently with laser (laser-assisted hatching [LAH]). In current times with advent of preimplantation genetic screening (PGS), which requires laser drilling, it was thought to improve IVF results more folds, but somehow PGS and LAH have yet not proven drastically to improve IVF outcomes. Here, we report the first case in our knowledge of a successful outcome after transfer of human blastocyst that had normally escaped completely from the zona which was laser hatched for PGS on day 5 and freshly transferred on day 6 after normal PGS report.

  Case Report Top

A couple presented to us with a history of 6 years of secondary infertility, the female was 34 years, a homemaker and her husband was 38-year-old with private business. The couple had no significant medical or surgical history. She had regular menstrual cycles. She was a known case of polycystic ovary syndrome with endometriosis and complete uterine septum on hysterosalpingography; laparoscopy showed moderate degree of endometrium with pelvic adhesion which was removed and uterus had a complete septum which was respected in 2012. Subsequently, she underwent IVF cycles at other clinics, out of which three were fresh and three were frozen. Twice the transfer was done in a surrogate elsewhere. The last cycle of surrogate had a positive result but ended up with missed abortion at 6 weeks. The couple approached our clinic for IVF cycle with surrogacy. On evaluation, blood investigations were normal with anti-mullerian hormone of 7.28 and antral follicle count of 15. Her uterus showed bilateral endometriosis cyst of around 1.5 cm on both sides. Male factor evaluation showed normal semen parameter as per the WHO 2010 criteria. After thorough evaluation and counseling, decision for IVF with own eggs with PGS and a fresh transfer in a surrogate was made. The patient was stimulated after downregulation with oral contraceptive pills by flexible antagonist protocol. The patient was given human chorionic gonadotropin (hCG) trigger and oocyte retrieval was performed after 36 h. Eleven retrieved oocytes were washed and incubated in continuous single culture (CSC) (Irvine Scientific, CA, USA) for 2 h at 37°C in an atmosphere of 5% CO2 in Heracell incubator. Spermatozoa were washed using a flushing medium (MediCult, Denmark) followed by swim-up method. Prewash count was 40 million/ml and 60% motility; postwash count was 8 million/ml and 98% motility. The oocytes were denuded of their surrounding cumulus cells approximately 2 h after retrieval using hyaluronidase 80 IU/ml in HEPES-buffered with gentamicin (Irvine Scientific, CA, USA) for 10–15 s. The oocytes were then transferred to same CSC medium for complete mechanical removal of the cumulus cells with sterile-pulled glass Pasteur pipettes. The oocytes were rinsed and incubated for 1 h in CSC medium under light mineral oil (Irvine Scientific, CA, USA). Out of 11 oocytes retrieved, 10 were mature and one was germinal vesicle. ICSI was done and oocytes were incubated in embryoscope (Unisense FertiliTech, Denmark) containing 6% CO2, 5% O2, and 89% N2 with 37°C. On day 5, 2 PN oocytes were seen. On day 3, at cleavage stage, fresh medium was replaced in dish. Four embryos reached up to blastocyst stage. All four blastocyst embryos were placed in 10 μ L of Quinn's advantage medium with HEPES (Ca/Mg free) (sage, CT, USA) under light mineral oil and subjected to biopsy. A hole was made in the zona pellucida exactly opposite to the inner cell mass of the each blastocyst using the LYKOS, (Hamilton Thorne, Beverly, MA, USA) applying gentle suction with the biopsy pipette (Origio, Denmark). Trophectoderm cells were encouraged to herniate from the zona pellucida; 5–7 trophectoderm cells were dissected from each of the blastocyst. The biopsied cells were placed in DNase-RNase-free PCR tubes containing 2.5 μ L phosphate-buffered saline. All four biopsied embryos were then placed into CSC medium and incubated overnight in HERA cell incubator. PGS was done using next genome sequencing method. On the next day, PGS report showed three embryos to be abnormal and one embryo to be normal [Table 1]. The normal blastocyst had completely hatched out; it was extremely carefully loaded in embryo transfer catheter (Cook Medical, USA) and transferred in a surrogate uterus. Endometrium was prepared on pretreatment with 6 mg estradiol valerate for 10 days and progesterone for 5 days before transfer.
Table 1: Detail characteristics of biopsied embryos

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  Results Top

Fifteen days posttransfer, the β-hCG was 3342.04 IU/ml indicating a positive result which doubled to 6669.77 IU/ml after 48 hrs. First scan at 7 weeks showed active cardiac activity with fetal growth corresponding to gestational sac. 11–13 week scan and double marker were also normal with diabetes mellitus showing low risk. At 22 weeks, her anomaly scan was normal. Her entire antenatal period remained uneventful. At 38 weeks, she delivered a healthy female neonate of 2.7 kg through lower segment caesarean section.

  Discussion Top

In spite of recent advances in IVF procedures and embryology, we have not been able to increase success rate to more than 45%–50%. PGS, ERA, and embryoscope showed a lot of potential initially but yet have not shown robust advantage in randomized controlled trials. Infertility specialist and embryologist worldwide are still exploring ways to increase overall success rates. Assisted hatching by zona drilling is still a major debate with multiple differences of opinions to its advantages in supplementing implantation. The theory of hatching in vivo being absolutely necessary for implantation of embryo might have a way for zona-free embryo to be optimal way of transfer for increasing implantation and off course selection of the most optimal blastocyst, with increasing use of PGS in recent time.[8],[9] The laser breech for the removal of trophectoderm and then waiting for the embryo to hatch out of the zona completely could be a sign of higher embryo quality. In this case, the embryo was completely hatched out till the PGS report was achieved and was transferred zona-free, provided it came normal in the PGS test and a positive outcome was delivered. It has been speculated that the in vitro hardened zona may interfere with implantation after transfer in vivo. There are very few reported cases of viable pregnancy of after transfer zona-free blastocyst transfer in humans.[10] Our hypothesis being that the removal zona clears one more hurdle in implantation and that direct contact between trophectoderm and endometrium could bring better implantation thus increasing success of IVF as in the reported case.

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Conflicts of interest

There are no confl icts of interest.

  References Top

Cohen J, Elsner C, Kort H, Malter H, Massey J, Mayer MP, et al. Impairment of the hatching process following IVF in the human and improvement of implantation by assisting hatching using micromanipulation. Hum Reprod 1990;5:7-13.  Back to cited text no. 1
Cohen J, Alikani M, Trowbridge J, Rosenwaks Z. Implantation enhancement by selective assisted hatching using zona drilling of human embryos with poor prognosis. Hum Reprod 1992;7:685-91.  Back to cited text no. 2
Coskun S, Hollanders J, Al-Hassan S, Al-Sufyan H, Al-Mayman H, Jaroudi K. Day 5 versus day 3 embryo transfer: A controlled randomized trial. Hum Reprod 2000;15:1947-52.  Back to cited text no. 3
Fujiwara M, Takahashi K, Izuno M, Duan YR, Kazono M, Kimura F, et al. Effect of micro-environment maintenance on embryo culture after in-vitro fertilization: Comparison of top-load mini incubator and conventional front-load incubator. J Assist Reprod Genet 2007;24:5-9.  Back to cited text no. 4
Fong CY, Bongso A, Ng SC, Anandakumar C, Trounson A, Ratnam S. Ongoing normal pregnancy after transfer of zona-free blastocysts: Implications for embryo transfer in the human. Hum Reprod 1997;12:557-60.  Back to cited text no. 5
Modlinski JA. The role of the zona pellucida in the development of mouse eggs in vivo . J Embryol Exp Morphol 1970;23:539-47.  Back to cited text no. 6
Fong CY, Bongso A, Sathananthan H, Ho J, Ng SC. Ultrastructural observations of enzymatically treated human blastocysts: Zona-free blastocyst transfer and rescue of blastocysts with hatching difficulties. Hum Reprod 2001;16:540-6.  Back to cited text no. 7
Balaban B, Urman B, Sertac A, Alatas C, Aksoy S, Mercan R. Blastocyst quality affects the success of blastocyst-stage embryo transfer. Fertil Steril 2000;74:282-7.  Back to cited text no. 8
Dokras A, Sargent IL, Barlow DH. Human blastocyst grading: An indicator of developmental potential? Hum Reprod 1993;8:2119-27.   Back to cited text no. 9
Mansour RT, Rhodes CA, Aboulghar MA, Serour GI, Kamal A. Transfer of zona-free embryos improves outcome in poor prognosis patients: A prospective randomized controlled study. Hum Reprod 2000;15:1061-4.  Back to cited text no. 10

  Authors Top

Harsha Bhadarka is an IVF lab. Director of Akanksha Hospital and Research Institute, Anand, Gujarat. She has 19 year of experience in clinical embryology. She is pursuing Ph.D. in embryology. She has applied for patent in male infertility "A method of in-vitro maturation of spermatid". She has served as a secretory in embryology ISAR 2016, Anand, Gujarat.


  [Table 1]


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