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Table of Contents
CASE REPORT
Year : 2019  |  Volume : 2  |  Issue : 1  |  Page : 46-49

Why results of endometrial receptivity assay testing should not be discounted in recurrent implantation failure?


1 Department of Reproductive Medicine, Apollo Cradle, New Delhi, India
2 Department of Reproductive Medicine, Mother and Child Hospital, New Delhi, India

Date of Web Publication25-Jun-2019

Correspondence Address:
Dr. Simrandeep Kaur
Department of Reproductive Medicine, Apollo Cradle, Moti Nagar, New Delhi
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/tofj.tofj_9_19

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  Abstract 


Recurrent implantation failure (RIF) is often encountered in patients undergoing ART leading to severe emotional, financial and physical distress. Thorough investigations should be carried out to ascertain whether there is any underlying cause of the condition. Endometrial receptivity (ER) remains a major stumbling block in IVF success as most available tests for diagnosis lack accuracy and predictive value. Microarray technology allows identification of the transcriptomic signature of the window of implantation, hence the development of an ER assay (ERA) for the diagnosis of an altered ER. ERA evaluates 238 genes expressed during WOI using RNA sequencing. Use of ERA test in patients with RIF has shown that window of implantation (WOI) is displaced in a quarter of these patients and use of a personalized embryo transfer (pET) significantly improves reproductive performance. We report three cases of RIF all of whom had failed IVF cycles despite good quality blastocysts transfer with apparently, no identifiable cause of implantation failure (uterine factores, embryonic, immunological factors ruled out). Two patients though had ERA testing done outside which showed displaced WOI, but however pET was not carried out leading to RIF. Subsequently pET carried out at our centre led to successful implantation and clinical pregnancy in both the patients.

Keywords: Endometrial receptivity array, personalized embryo transfer, recurrent implantation failure


How to cite this article:
Kaur S, Naidu P. Why results of endometrial receptivity assay testing should not be discounted in recurrent implantation failure?. Onco Fertil J 2019;2:46-9

How to cite this URL:
Kaur S, Naidu P. Why results of endometrial receptivity assay testing should not be discounted in recurrent implantation failure?. Onco Fertil J [serial online] 2019 [cited 2019 Sep 22];2:46-9. Available from: http://www.tofjonline.org/text.asp?2019/2/1/46/261258




  Introduction Top


Successful embryo implantation is a process which requires synchronous development and interaction between a hatched blastocyst and a receptive endometrium.[1] Failure to implant may be a consequence of embryonic or uterine factors. Repeated or recurrent implantation failure (RIF) is often encountered in patients undergoing assisted reproductive technology leading to severe emotional, financial, and physical distress. RIF is defined as failure of implantation after two embryo transfer cycles, where the cumulative number of transferred embryos is not <4 for cleavage stage embryos and not <2 for blastocyst, with all embryos being of good quality and of appropriate developmental stage.[1]

Thorough investigations should be carried out to ascertain whether there is any underlying cause of the condition.[2] Various uterine pathology including fibroids, endometrial polyps, congenital anomalies, and intrauterine adhesions should be excluded by ultrasonography and hysteroscopy.[2]

Endometrial receptivity (ER) remains a major stumbling block in in vitro fertilization (IVF) success as most clinically available tests for diagnosis, show a lack of accuracy and predictive value. An important breakthrough in this area has been the commercial availability of the ER assay (ERA) test. Identification of the transcriptomic signature of the window of receptivity by microarray technology has led to the development of ERA for diagnosis of ER.[1],[2],[3] Use of this test in patients with RIF has shown that the window of implantation (WOI) is displaced in a quarter of these patients and use of a personalized embryo transfer (pET) on the day designated by ERA, improves reproductive performance.[4] We report three cases of RIF, all of whom had failed IVF cycles despite good-quality blastocysts transfer with apparently, no identifiable cause of implantation failure (uterine factors, embryonic, and immunological factors ruled out). Two patients had an altered ERA, but their physicians after doing the test chose not to go by the findings, as they felt they were inaccurate. A personalized embryo transfer based on ERA subsequently resulted in successful implantation and clinical pregnancy.


  Case Reports Top


Case report 1

Dr. DK, 31-year-old female, married for 5 years, presented to our unit with unexplained infertility. She had undergone four cycles of unsuccessful intrauterine insemination (IUI) and two IVF failures from a reputed local IVF clinic. IVF was done using an antagonist protocol and a combination of recombinant follicle-stimulating hormone (FSH) and human menopausal gonadotropin (HMG) for controlled ovarian stimulation (COS). Twelve oocytes were recovered, nine fertilized and four Grade A blastocysts were frozen. The patient subsequently underwent two unsuccessful frozen embryo transfers in an HRT cycle (Grade A two blastocysts transfer in each FET).

We reevaluated the patient and found that she had adenomyosis-globulous enlargement of the uterus with anterior uterine wall hypertrophy.[5],[6] Uterine cavity assessed using 2D and 3D real-time sonography was found to be normal. Semen analysis and DNA fragmentation tests were normal. We advised the patient to undergo ERA, since it has been reported that patients with adenomyosis have an altered WOI.[7] Hysteroscopy was planned along with ERA. The patient underwent ERA test in a hormone replacement cycle (HRT cycle).

Methodology for endometrial receptivity assay testing

Estradiol valerate (Progynova, Sun Pharmaceutical Industries Ltd, India) was started in a dose of 2 mg, and this was increased to 8 mg till an appropriate endometrial thickness (≥7 mm) was achieved. Progesterone (P4) administration was started after that. Before starting progesterone, S. P4 level was checked as this needs to be below <0.9 ng/mL. Vaginal progesterone (P) suppository 400 mg (Gestone, Ferring Pharmaceutical, Saint-prex, Switzerland) twice a day was then started for a period of 5 days (P + 5). Endometrial biopsy (EB) was collected from the uterine cavity with the use of Pipelle catheter (Gynetics) on day P + 5 (after 5 days of progesterone administration) in a HRT cycle and sent to Igenomix, Valencia, Spain, for analysis.

Patients result was pre-receptive for P + 5 (120 h), and embryo transfer was advised at P + 6 (24 h after the biopsy was taken, 144 ± 3 h).

For IVF, an antagonist protocol was followed and COS was done using recombinant FSH (Gonal-F 225 IU for 5 days [Recombinant follitropin alfa, Merck Serono, Germany]) followed by HMG 225 IU (Menopur [Ferring pharmaceuticals, Saint-Prex, Switzerland]) for 4 days. Antagonist was started on day 6 (Cetrorelix, Intas Pharmaceuticals, India). Ovulation was triggered using hCG 10,000 IU I/M (Fertigyn, Sun Rise International labs Ltd., Hyderabad, Telangana, India). Three Grade A cleavage stage embryos and four Grade A blastocysts were frozen.

Embryo transfer was planned in an HRT cycle similar to the ERA cycle. Two Grade A blastocysts were transferred 6 days after progesterone administration (pET as mentioned in the report). bHCG done 14 days later was positive and twin sacs were identified a week later. Cardiac activity was seen at 6 weeks gestation in both sacs. The patient is currently 28 weeks pregnant (DADC twins), antenatal course till date has been uneventful.

Case 2

Our second patient Ms. T. W, was a 39-year-old female from Pennsylvania, USA, with primary infertility for 3 years. She had a history of thin endometrium; maximum thickness reported was 6.5 mm. She went through two cycles of IUI and subsequently three cycles of IVF with no success.

The first IVF with elective cryopreservation of all embryos was done using an antagonist protocol. The patient underwent a second cycle of IVF for embryo pooling. Embryos obtained from both IVF cycles were subjected to preimplantation genetic testing for aneuploidies (PGT-A) and 5 euploid blastocysts (Grade A) were cryopreserved. The patient underwent two FET's in an HRT cycle, wherein two Grade A euploid blastocysts per cycle, were transferred unsuccessfully. Endometrial preparation for FET was done with injectable estrogen for 4–5 weeks; a maximum endometrial thickness of 6.8–7 mm was achieved before starting progesterone.

The patient was advised ERA test before the third FET, as no other cause for RIF could be determined. ERA testing was done in an HRT cycle using the same regime as in her FET cycles. The test was reported prereceptive at P + 5, and a repeat biopsy was advised at P + 7 or 168 h after progesterone exposure. Patient' s fertility specialist felt the report was inaccurate and instead of repeating the test as advised, went ahead with the third FET using the standard P + 5 protocol. The last single euploid blastocyst was transferred unsuccessfully.

The patient underwent a third failed IVF cycle with elective cryopreservation and transfer of 2 euploid blastocysts. Sperm DNA fragmentation index was done after this unsuccessful cycle, which was normal.

The patient reported to our hospital for further management. She was reevaluated and advised to repeat ERA at P + 7 as advised in her earlier ERA report. A diagnostic hysteroscopy was planned along with ERA. Repeat ERA at P + 7 suggested that there was a shift in the window of implantation (WOI). A personalized ET was advised at P + 6.5 (155 ± 3 h of progesterone administration). Her AMH was 1.8 ng/ml and AFC was 10 in the right ovary and 8 in the left ovary. COS was started with recombinant FSH 300 IU (Gonal-F Recombinant follitropin alfa, Merck Serono, Germany) and HMG 150 IU (Menopur [Ferring pharmaceuticals, Saint-Prex, Switzerland]). HCG 10,000 IU was used to trigger egg maturation. Six MII oocytes recovered and eventually only one Grade A blastocyst could be frozen. FET was done in HRT cycle and embryo transfer was done according to the ERA report, after 155 h of progesterone administration (at P + 6.5).

Urine pregnancy test and beta-HCG done after 14 days of embryo transfer were positive, single gestational sac with good chorionic reaction was noted 3 weeks after ET. The patient is now currently 8 weeks pregnant and antenatal course till date has been uneventful.

Case 3

Mrs. PJ, a 32-year-old patient with PCOS and male factor infertility reported to our clinic with a history of 4 IUI and 2 FET failures, having undergone IVF with elective freezing of embryos. She had 9-day Grade 3 embryos preserved. Her first FET was done in an HRT cycle and 2-day Grade 3 embryos were transferred, which did not result in a pregnancy. The physician advised ERA, and it was done as per protocol given above. The EB was reported to be prereceptive and the advice was to repeat ERA at P + 6. ERA was not repeated and physician decided to go ahead with FET at P + 7, two blastocysts were transferred unsuccessfully.

She underwent an IVF with elective freezing at our center. She did not agree to have a repeat ERA, so we decided to transfer 2-day Grade 3 embryos in an ovulatory cycle which resulted in a biochemical pregnancy. After this failure, the patient agreed to go through another ERA which showed ER at 134 ± 3 h. Personalized FET done at P + 5.5/134 h resulted in a singleton ongoing pregnancy. The patient is currently 12 weeks pregnant.


  Discussion Top


Human implantation is a complex process requiring a healthy embryo, a receptive endometrium, and a synchronized molecular dialogue between the two.[3],[4] It has been a challenge to diagnose ER and most tests available are subjective and lack accuracy and predictive value. Microarray technology has allowed identification of the transcriptomic signature of the window of implantation, hence the development of an ER assay (ERA) for the diagnosis of an altered ER.[8] ERA evaluates 238 genes expressed during WOI using RNA sequencing.[9] Following analysis, the samples are classified as receptive or nonreceptive (NR). The NR endometrium is further classified as pre- or post-receptive meaning that the endometrium has not reached the receptive phase yet or has already passed it, respectively.[4],[7] ERA has been found to be accurate and consistent, and the results are reproducible up to 3 years after the first ERA test.[10],[11] This test determines the duration of progesterone exposure required by the endometrium to reach a receptive status and thus aids in creating a personalized WOI for frozen embryo transfer (ET), thereby improving reproductive performance in patients with RIF.[4],[7] ERA has a sensitivity and specificity of 0.99758 and 0.8857, respectively.[9]

Ruiz-Alonso et al. 2013, in a prospective multicentric trial, first proposed the clinical application of this test to improve implantation in RIF. She investigated patients with more than three IVF or IVF-OD failures and showed that a changed WOI existed in 25.9% of RIF patients suggested that a “pET” or transferring an embryo based on the woman's personalized WOI[12] improved results. Mahajan et al. showed an endometrial factor in 27.5% of RIF patients in the Indian population, which was significantly greater than the non-RIF group 15% (P = 0.04).[4] After pET, the overall ongoing pregnancy rate was 42.4% and implantation rate was 33%, which was at par with their IVF results over 1 year.[4] In a recent study, a significantly increased incidence of a displaced WOI (nonreceptive ERA) was demonstrated in adenomyosis patients. Patients with adenomyosis had a nonreceptive ERA in 47.2% compared to controls 21.6%, (P < 0.001, CI-8.7%-42.5%) making risk ratio of displaced WOI in adenomyosis versus controls to be 2:1.[7]

Considering the above facts, we advised ERA to our first patient who had adenomyosis[7] and two IVF failures (with transfer of four Grade A blastocysts) and repeat ERA to our two patients despite the ERA report being refuted by their fertility specialists. All patients had displaced WOI and hence pETs led to successful implantation and continuation of pregnancies.

The three cases reported here highlight the value of ERA in patients with RIF and in adenomyosis. It should be our aim to mitigate the physical, emotional, and financial stress, not to mention the embryo wastage associated with repeated implantation failure. It is advisable to use ERA in patients with RIF,[4],[12] IVF failures with the transfer of two good-quality embryos and adenomyosis or when there is limited number of cryopreserved embryos left for transfer. Its use in patients with adenomyosis and endometriosis may prove beneficial, as these conditions are associated with an altered ER.[4],[7]


  Conclusion Top


The ERA should be used in patients of RIF to identify the patients personalized WOI. This will mitigate the associated physical, emotional, and financial stress and embryo wastage. Limitations include cost of the procedure and limited studies available in the literature regarding efficacy of ERA to improve implantation. More studies are required to improve physician's acceptance of the procedure.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Polanski LT, Baumgarten MN, Quenby S, Brosens J, Campbell BK, Raine-Fenning NJ. What exactly do we mean by 'recurrent implantation failure'? A systematic review and opinion. Reprod Biomed Online 2014;28:409-23.  Back to cited text no. 1
    
2.
Coughlan C, Ledger W, Wang Q, Liu F, Demirol A, Gurgan T, et al. Recurrent implantation failure: Definition and management. Reprod Biomed Online 2014;28:14-38.  Back to cited text no. 2
    
3.
Timeva T, Shterev A, Kyurkchiev S. Recurrent implantation failure: The role of the endometrium. J Reprod Infertil 2014;15:173-83.  Back to cited text no. 3
    
4.
Mahajan N. Endometrial receptivity array: Clinical application. J Hum Reprod Sci 2015;8:121-9.  Back to cited text no. 4
[PUBMED]  [Full text]  
5.
Puente JM, Fabris A, Patel J, Patel A, Cerrillo M, Requena A, et al. Adenomyosis in infertile women: Prevalence and the role of 3D ultrasound as a marker of severity of the disease. Reprod Biol Endocrinol 2016;14:60.  Back to cited text no. 5
    
6.
Devlieger R, D'Hooghe T, Timmerman D. Uterine adenomyosis in the infertility clinic. Hum Reprod Update 2003;9:139-47.  Back to cited text no. 6
    
7.
Mahajan N, Kaur S, Alonso MR. Window of implantation is significantly displaced in patients with adenomyosis with previous implantation failure as determined by endometrial receptivity assay. J Hum Reprod Sci 2018;11:353-8.  Back to cited text no. 7
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8.
Talbi S, Hamilton AE, Vo KC, Tulac S, Overgaard MT, Dosiou C, et al. Molecular phenotyping of human endometrium distinguishes menstrual cycle phases and underlying biological processes in normo-ovulatory women. Endocrinology 2006;147:1097-121.  Back to cited text no. 8
    
9.
Díaz-Gimeno P, Horcajadas JA, Martínez-Conejero JA, Esteban FJ, Alamá P, Pellicer A, et al. Agenomic diagnostic tool for human endometrial receptivity based on the transcriptomic signature. Fertil Steril 2011;95:50-60, 60.e1-15.  Back to cited text no. 9
    
10.
Díaz-Gimeno P, Ruiz-Alonso M, Blesa D, Bosch N, Martínez-Conejero JA, Alamá P, et al. The accuracy and reproducibility of the endometrial receptivity array is superior to histology as a diagnostic method for endometrial receptivity. Fertil Steril 2013;99:508-17.  Back to cited text no. 10
    
11.
Garrido-Gómez T, Ruiz-Alonso M, Blesa D, Diaz-Gimeno P, Vilella F, Simón C. Profiling the gene signature of endometrial receptivity: Clinical results. Fertil Steril 2013;99:1078-85.  Back to cited text no. 11
    
12.
Ruiz-Alonso M, Blesa D, Díaz-Gimeno P, Gómez E, Fernández-Sánchez M, Carranza F, et al. The endometrial receptivity array for diagnosis and personalized embryo transfer as a treatment for patients with repeated implantation failure. Fertil Steril 2013;100:818-24.  Back to cited text no. 12
    




 

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