Retrospective study showing improved pregnancy rates with laser assisted hatching on Day 5 blastocysts in FET cycles in Indian population

Inclusion and exclusion criteria

The study includes 492 patients ages 21 to 43 who were treated with frozen-thawed ICSI-FET at Arihant Hospital, India, between March 2021 and December 2022. Patients with donor cycles (n = 62), poor endometrium (n = 10), and poor-quality blastocysts (n = 15) were excluded from the study. Finally, the evaluation of 338 patients with self-ICSI-FET cycles, good-quality vitrified blastocysts, and a triple-line endometrium with a thickness of more than 8 mm was done.

Patient identification is done on the basis of the final digits of the registration number, which was assigned to them by the administrator at the reception desk on their initial visit to our centre. The whole selected population of 405 patients was divided into two groups: the LAH group (n = 169) and the non-LAH group (n = 236). These groups were further subdivided into two groups: Group I with patients’ ages <35 years and Group II with patients’ ages >35 years.

Ovarian stimulation protocol

All females were subjected to controlled ovarian stimulation using an antagonist regimen with 225 IU as the average dose of gonadotropins on days 2 or 3 of periods. A fixed antagonist was added on day 6. Follicular development was monitored using vaginal ultrasonography. The gonadotropin dose was increased from 150 to 375 IU and then adjusted based on the response of the ovarian follicular development, which was monitored using vaginal ultrasonography. A trigger, either an recombinant human chorionic gonadotropin (HCG) injection or an GnRH agonist trigger, was given when most of the follicles had reached 18 mm in size. The drugs incorporated were triptorelin 0.1 mg and hCG 7500 IU. Transvaginal egg collection was performed 36 hours after the trigger administration using a 17-gauge ovum pick-up needle.

Embryo development and vitrification

The oocytes collected were then fertilized with her husband's semen sample only through intra-cytoplasmic sperm injection (ICSI) Resulting embryos underwent LAH procedure. Fertilized oocytes on Day 1 were then cultured until Day 5 for blastocyst formation. Regular checks were done to monitor embryonic development on Days 2, 3, and 5, respectively.

Embryos were frozen on Day 5 using Kitazato embryo vitrification media. Only those embryos were frozen that had shown proper blastocyst formation in 120 hours with cohesive trophectoderm and prominent and tightly packed inner cell mass (ICM).

Thawing procedures

Each straw was defrosted separately, and the entire process was carried out at room temperature. The thawing process was carried out using the Kitazato Thaw-Kit. Prior to usage, all solutions are pre-adjusted to room temperature except the thawing solution, which needs to be warmed to 37°C. Embryos were thawed for at least 2 hours before frozen embryo transfer (FET).^[13]

Randomization 169 of patients and LAH

The LAH operation was done just before the embryo transfer, and embryo thawing was done at least 2 hours before the embryo transfer. Only embryos that survived after freezing underwent LAH. Cryo-survival evaluation procedures followed those outlined by Rienzi et al.^[14] Embryos that had been frozen and then thawed were deemed to have survived if the blastocyst had coehesive tropectoderm with compact ICM without any degenerations.^[15]

LAH was carried out using a 1480-nm diode laser in a computer-controlled non-contact mode (IVF Workstation and Zona Laser Treatment System, RI Saturn 5 Instruments) installed in an inverted microscope used in the IVF laboratory.

Each embryo underwent quarter laser-assisted hatching (Q-LAH), as previously mentioned.^[16] Laser drilling was used to thin the zona pellucida, starting at one place and continuing until 25% of the zone was drilled. Embryos chosen for laser therapy were first transfused in a 10-μL drop of pre-equilibrated culture media supplemented with human serum albumin (HSA) in a Falcon culture dish, overlaid with paraffin oil, to finally give the laser shots. Just after the LAH treatment, the embryos were again shifted to an embryo transfer dish.

Frozen embryo transfer

Frozen embryo transfer was performed by hormonal replacement therapy. A baseline scan was done in all cases, and tablet Progynova (Bayer Zydus Pharma) was started on day 2 once the endometrial thickness was favourable (≥8 mm) with triple line echogenicity. The embryo transfer was done gently with a Labotech catheter. Luteal support was started by administering 100 mg of oil-based progesterone or vaginal progesterone (Capsule Susten, Sun Pharma) daily. Chemical pregnancy was defined by the analysis of the β-hCG hormone on the 14th day after embryo transfer, and clinical pregnancy as a distinct intrauterine gestational sac seen on transvaginal ultrasound.

Statistical analysis

The data were logistically regressed using TS-PLUS 2000. We also calculated power and reported a 95% confidence interval (95% CI) for the odds ratios (OR) associated with the model's contributing factors.^[15] A P-values of <0.05 was considered statistically significant.

Zscore

The z score is tabulated on the basis of comparative analysis of positive outcomes that is drawn on the basis of population group divided into LAH and non-LAH (Table 2).

Limitations

The limitation of this study are retrospective study and small sample size of the patient as IVF is new to India normal and are extent unaware about it.

Financial support and sponsorship

Nil.

Retrospective study showing improved pregnancy rates with laser-assisted hatching on day 5 blastocysts in frozen embryo transfer cycles in Indian population

The Vienna Consensus meeting of embryologists in 2017 recommended exercising caution and care when introducing new techniques in the in vitro fertilization (IVF) laboratory. Although the first pregnancy using mechanical force to partially zona dissect mature oocytes was reported in 1988 by Cohen et al. for male-factor couples, the utilization of assisted hatching (AH) in IVF laboratories has not undergone a proper systematic evaluation. Under the “add-ons” traffic light system, implemented by the Human Fertilisation and Embryology Authority (HFEA), AH is listed in the red category, indicating that a regular cycle of proven fertility treatment is effective without any treatment add-ons. Although reproductive scientists have gathered important information on the composition, assembly, and structure of zona pellucida (ZP) and its role in human oogenesis and egg–sperm interaction, it is important to note that information on preimplantation development notoriously lags behind; hence, any potential benefits of manipulation should only be considered in this context.

Over the years, three techniques for AH have been introduced. Mechanical, chemical, or laser manipulations of ZP has been used to facilitate the creation of holes, slots, and thinning of the zona of different sizes. Tadir (1991) and Palanker et al. (1991) first reported laser-mediated zona drilling, a precise microsurgical breach in the ZP. Two modes available: are contact mode, laser-guided through optical fibre employing ultraviolet (UV) or infrared (IR) wavelength. In a randomised prospective study, Tucker and coworkers (1991) reported no significant improvement in clinical pregnancy in frozen/thawed embryos. Although the procedure initially led to encouraging clinical outcomes, even after 35 years after the first mechanically manipulated pregnancy, the use of AH remains controversial due to heterogeneity among published studies and the absence of a meta-analysis of randomized controlled trials (RCTs) favoring AH over no AH.

The study reported deserves attention as this is the first to assess the effect of laser AH on clinical and implantation rates using vitrified/warmed blastocysts in the Indian population. Similar studies have been reported elsewhere with promising outcomes.

In their comparative study, Sharma et al. reported improved clinical pregnancy rates favoring AH in the age group of <34 years compared to the group without AH in the same age group. Although the study is promising, the number of patients included and allocated to each group is significantly low to draw any statistically significant conclusion. The stimulation protocols are consistent with those used in the European clinics; however, data on the number of oocytes collected, fertilized, blastocyst conversion rate, number vitrified, and the number that survived the warming process with their grades pre- and post-vitrification would have made this study even more informative. The methodology of the study lacks clarity on how the consistency of the quarter zona thinning was maintained. Equally, the site of ZP thinning, either near the inner cell mass (ICM) or the opposite side, is a piece of vital information (Matsubayashi et al., 2010). To avoid multiple pregnancies, most clinics in the West now follow elective single-embryo transfer (SET). Therefore, in this study, outcome data on multiple pregnancies and live births rates are equally important. Furthermore, any additional data on the percentage of congenital abnormalities registered at the time of delivery would confirm the safety of AH and encourage the use of AH in IVF cycles, in particular, the vitrified/warming cycles.

The commentary highlights the need for detailed methodology, data collection, presentation, discussion, and conclusion statement. The authors are encouraged to reflect on my comments.

Mehta Jayant, HFEA Person Responsible, Subfertility Laboratory Director and Quality Control Manager, Queen's Hospital, Rom Valley Way, Romford, Essex RM7 0AG, UK.