One center experience with a personalized frozen-thawed embryo transfer in patients with recurrent implantation failure

Study design and population

The present study is a retrospective analysis at the interdisciplinary Fertility Centre of the University Hospital Duesseldorf, Germany (UniKiD). The observation period was between March 2016 and June 2021. The study was approved by the University Ethics committee. The trial registration number is 2019–797.

In total, the endometrial receptivity analysis was performed in 134 patients with implantation failure during the study period. The indication to perform the test was left to the treating physician.

Inclusion criterium for the statistical analysis was RIF. RIF was diagnosed when no clinical pregnancy occurred after at least two embryo transfers with a total of three good quality embryos. The definition of RIF is derived from the center’s own data, which show that for women younger than 40 years of age, there is a cumulative clinical pregnancy rate of 93.9% after the transfer of three ideal embryos. All patients who did not meet the criteria for RIF were excluded from further analysis (n = 4). Further exclusion criteria were: no consecutive personalized embryo transfer (n = 11), double embryo transfer (n = 25), age older than 40 years at the time of oocyte retrieval (n = 20), only one oocyte fertilized (n = 4), recurrent miscarriages with ≥ 3 abortions (n = 1), uterine malformations (uterus bicornis unicollis with non-communicating atretic left uterine horn in the state after metroplasty, according to Strassmann, n = 1), and slow freezing (n = 1). Finally, 67 RIF patients were considered for the analysis (case group). See Fig. 1.

Fig. 1figure 1

Study flow chart of the group of patients who performed an endometrial receptivity analysis. RIF: recurrent implantation failure, ERA®: endometrial receptivity analysis, pET: personalized embryo transfer

For the control group, which consisted of 32 subjects, only patients with RIF who had not undergone endometrial receptivity analysis were selected. Inclusion and exclusion criteria of the control group correspond to those of the case group.

Reproductive outcomes of the case group after personalized embryo transfer were compared to reproductive outcomes in the control group after standardized embryo transfer.

Prior to endometrial receptivity mock cycle, a vaginal ultrasound was performed for all patients. There was no evidence for adhesions, hydrosalpinx, submucous polyps, or fibroids in any patient. Our routine hormonal check-up showed normal values for each parameter examined (thyroid-stimulating hormone, prolactin, follicle stimulating hormone (FSH), estradiol).

Protocol and sampling

The mock cycle for performing the endometrial receptivity analysis was performed following a hormonal replacement protocol: estradiol patches (Estramon®; Hexal AG, Holzkirchen, Germany) were started from the 1st day of the menstrual cycle in an increasing dosage from 100 μg on days 1 to 7, 200 μg on days 8 to 11, and 400 μg from the 12th day. Patches were changed every 48 h.

On cycle days 12 to 14, a vaginal ultrasound examination was performed, combined with estradiol and progesterone serum analysis.

When the endometrial lining was ≥ 6 mm and the progesterone level was < 0.5 ng/ml, progesterone supplementation was started. For the progesterone supplementation, progesterone vaginal gel (Crinone® 8% 90 mg, Merck KGAA, Darmstadt, Germany), 50 mg daily of oral dydrogesterone (Duphaston®, Mylan Germany GmbH, Troisdorf, Germany), 600 mg of vaginal micronized progesterone (utrogest®, Besins Healthcare, Berlin, Germany or Famenita®, Exeltis Germany GmbH, Ismaning, Germany), or subcutaneous progesterone (Prolutex®, Marckyrl Pharma, Papenburg, Germany) were used.

Following our FET cycle standard regimen, the endometrial biopsy was performed approximately 108–120 h after starting progesterone supplementation. Endometrial biopsy was performed with a Pipelle catheter (Gynetics®, Lommel, Belgium) and the sample proceeded as indicated by the manufacturer.

The test result differs in receptive and non-receptive. Non-receptive is divided into early receptive (endometrial receptivity 12 h later), pre-receptive (endometrial receptivity > 12 h delayed), and post-receptive results. If the test result was pre-receptive and the receptivity could not be assumed to be in the following 24 h after the initial biopsy, a second biopsy was recommended by the manufacturer. The same scheme was applied with an endometrial sampling 24 h later to predict a precise displacement and therewith a needed duration time of progesterone exposure to reach the individual endometrial receptivity.

Quality of embryos

The quality of the embryos was assessed by using morphological characteristics. A cleavage stage embryo with good quality includes four or five blastomeres on day 2, and at least seven blastomeres on day 3, with less than 20% fragmentation and no signs of multinucleation [27]. For the classification of blastocysts, we used the alphanumeric system published by Gardner and Schoolcraft [28]. A high-quality blastocyst was defined as a blastocyst, whose grade of expansion is ≥ 3 and inner cell mass and trophectoderm is characterized as grade A and B. PGT-A could not be performed due to the German embryo protection law.

Embryo transfer

Only cycles in which a single embryo transfer was performed were selected for the study. For the analysis, only the subsequent FET cycle after receiving the test result was included in the study. Endometrium preparation in the group of patients with a personalized embryo transfer was performed according to the patients’ mock cycle. Embryo transfer of cleavage stage embryos (day 2 or day 3) and blastocysts was performed after the recommended time of progesterone exposure. Endometrial preparation was performed in the control group congruently to the group with receptivity testing.

Definitions of outcomes

The definitions of the terms used to describe clinical outcomes were based on the International Glossary on Infertility and Fertility Care [29]. Our primary outcome was clinical pregnancy rate (CPR), secondary outcomes were pregnancy rate, live birth rate (LBR), rate of biochemical pregnancy loss, and clinical miscarriage rate. Pregnancy was determined as a positive serum level of beta-human chorionic gonadotropin (hCG) at 9 or 10 days after tranfer of a blastocyst, and 11 or 12 days after transfer of a cleavage stage embryo. Clinical pregnancy was the ultrasonographic visualization of one or more intrauterine gestational sacs. Biochemical pregnancy loss was defined as the short-term detection of a beta-hCG without sonographic evidence of a gestational sac. Clinical miscarriage was specified as the spontaneous loss of a clinical pregnancy. Pregnancy rate, CPR, and LBR were expressed as rate per embryo transfer. Clinical miscarriage rate was expressed as rate per clinical pregnancy. Biochemical pregnancy loss rate was expressed as rate per pregnancy. Age, body mass index (BMI), anti-müllerian hormone (AMH), number of previous failed cycles, and days between last biopsy and embryo transfer were reported as mean ± standard deviation. The proportion of embryo transfer using blastocyst and embryo of good quality, as well the proportion of patients with primary sterility, are reported in percentage. We performed further clinical outcome analyses in which we subdivided the case group by receptive and non-receptive patients.

Statistical analysis

Data were analyzed using IBM SPSS Statistics 27. For categorical variables, Pearson’s Chi-squared test was used, or Fisher’s exact test when one of the expected counts was less than 5. Continuous variables were tested for normal distribution using Shapiro–Wilk-test. In independent samples t-test was used for continuous variables with normal distribution. Mann–Whitney U test was used for continuous variables without a normal distribution. A p-value of < 0.05 was considered significant.

An a priori power analysis was performed. Assuming an increase in CPR from 30% after standardized embryo transfer to 40% after personalized embryo transfer, 356 subjects in both groups must be assessed to provide evidence of significance.

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