An Randomized Double Blind Controlled Trial of niPGT-A in Women With RPL (niPGTA)

A Randomized Double Blind Controlled Trial of Non-invasive Preimplantation Genetic Testing for Aneuploidy in Women With Recurrent Pregnancy Loss

Objectives: To compare the efficacy in embryo selection based on morphology alone compared to morphology and non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) in women with recurrent pregnancy loss (RPL) undergoing in vitro fertilization (IVF).

Hypothesis to be tested: The embryo selection based on morphology and niPGT-A results in a lower miscarriage rate and a higher live birth rate in IVF as compared with that based on morphology alone.

Design and subjects: Randomized double-blind randomized controlled trial. Women with RPL undergoing IVF will be enrolled.

Interventions: Spent culture medium (SCM) of each blastocyst will be frozen individually. They will be randomly allocated into two groups: (1) the intervention group based on morphology and niPGT-A and (2) the control group based on morphology alone.

In the control group, blastocysts with the best quality morphology will be replaced first. In the intervention group, blastocysts with the best morphology and euploid result of SCM will be replaced first.

Main outcome measures: The primary outcome is the miscarriage rate per the first embryo transfer.

Data analysis: Comparison of quantitative variables will be performed using Student's t, while categorical variables will be compared using a Chi-square analysis. All statistical analyses will be performed with the intention to treat and per protocol, and a p-value <0.05 will be considered statistically significant.

Expected outcome results: The embryo selection based on morphology and niPGT-A results in a lower miscarriage rate and a higher live birth rate in IVF as compared with the control group based on morphology alone.

Study Overview

• Status: Not yet recruiting
• Conditions: Infertility
• Intervention / Treatment: Genetic: Non-invasive Preimplantation genetic testing for aneuploidy status

Detailed Description

Trial Objectives and Purpose The primary objective of this randomized double blind controlled trial is to compare the efficacy in embryo selection based on morphology alone versus morphology and niPGT-A in women with RPL undergoing the first frozen embryo transfer. The secondary objectives are to evaluate the impact of eNK cells on the miscarriage and live birth rates and the prediction of live birth using spheroid/BAP-EB attachment assay.

Main hypotheses to be tested:

1. The embryo selection based on morphology and niPGT-A for aneuploidy results in a higher live birth rate of IVF in women with RPL as compared with the control group based on morphology alone.
2. The embryo selection based on morphology and niPGT-A for aneuploidy results in a lower miscarriage rate following IVF in women with RPL as compared with the control group based on morphology alone.

2. Trial Design This is a randomized double blind controlled trial. Eligible women will be recruited for the study and informed written consent will be obtained after counseling.

Endometrial assessment All women will have an endometrial biopsy using a Pipelle sampler 7 days after luteinizing hormone surge (LH+7) prior to the month of having IVF. Part of the endometrial samples will be fixed in paraformaldehyde for immunohistochemical staining of endometrial uNK cells with CD56 antibody [26,27]. The other part of the endometrial samples will be used for epithelial and stromal cell isolation for spheroid/BAP-EB attachment assay [28], to predict the embryo attachment rate in a laboratory setting. Due to ethical issues and practical difficulty, the use of human embryos for endometrial assessment is not feasible. BAP-EB model [28] will be used as the embryo (blastocyst) surrogate in this study. BAP-EB spheroid model are differentiated from human embryonic stem cells. The BAP-EB after 72h of differentiation (BAP-EB-72h) have molecular signature of Day 7 trophectoderm cells of blastocysts [28]. In this study, BAP-EB will be co-cultured with primary endometrial epithelial cells and the attachment rate will be determined according to our established protocol.

IVF protocol Women will undergo IVF with or without intracytoplasmic sperm injection (ICSI) as indicated. The women will receive ovarian stimulation using the progestin-primed protocol [29] or the antagonist protocol. Ultrasound scanning will be arranged on day 2-3 of menses for the antral follicle counts and to exclude the presence of ovarian cyst. Serum oestradiol and progesterone concentration will be checked and if they are basal, ovarian stimulation with gonadotropin injections (225-300 IU daily depending on the antral follicle count) for 10-12 day is given. Provera 10mg daily will be given from day 2-3 to day of trigger in the progestin-primed protocol to prevent premature LH surge while GnRH antagonist will be started on day 6 of ovarian stimulation to day of trigger. Regular ultrasound monitoring will be performed to monitor the growth of follicles. Adjustment of the gonadotropin dosage is corresponding to the number and size of follicles. When three follicles reach >17 mm in diameter, GnRH agonist (Decapeptyl 0.3mg) or human chorionic gonadotrophin (Ovidrel 0.25mg) will be administered. Oocyte retrieval will be scheduled 34-36 hours after the HCG or agonist trigger under transvaginal ultrasound guidance.

Normal fertilization will be assessed and confirmed by the presence of two pronuclei at 16-18 h after insemination. All cleavage stage embryos will be grown individually to the blastocyst stage, usually day 5 or 6 after oocyte retrieval, in a monophasic medium. On Day 3, the culture medium will be replenished, and culture will be continued at 37oC and 6% CO2 in reduced oxygen tension (5%). No fresh transfer of blastocysts will be performed in the stimulated cycle.

Grading of blastocyst by morphology

Blastocysts are graded according to Gardner's classification (29). The blastocyst grading system assigns 3 separate quality scores to each blastocyst embryo, based on the followings:

1. Blastocyst development stage: expansion and hatching status
2. Inner cell mass score
3. Trophectoderm score

Expansion grade Blastocyst development and stage status

1. Blastocoel cavity less than half the volume of the embryo
2. Blastocoel cavity more than half the volume of the embryo
3. Full blastocyst, cavity completely filling the embryo
4. Expanded blastocyst, cavity larger than the embryo, with thinning of the zona pellucida
5. Hatching out of the zona pellucida
6. Hatched out of the zona pellucida

Inner cell mass grade Inner cell mass quality A Many cells, tightly packed B Several cells, loosely grouped C Very few cells

Trophectoderm grade Trophectoderm quality A Many cells, forming a cohesive layer B Few cells, forming a loose epithelium C Very few large cells

Blastocysts are cryopreserved at developmental stage with expansion score 3 or above. If a blastocyst does not reach expansion stage 3 on day 5, it will be assessed on day 6 whether it is suitable for cryopreservation. Blastocysts with either inner cell mass or trophectoderm scored as B or above are regarded as utilizable blastocysts.

Each blastocyst will be frozen by vitrification individually and its SCM (~8 µl) will be frozen at -800C separately and individually. The embryologist will prepare a sequence of blastocyst transfer based on the best morphology by Gardner's criteria.

Then, on the day of blastocyst freezing, women will then be randomly assigned into two groups in 1:1 ratio using a randomization program by the laboratory staff in the PGT laboratory.

1. the intervention group using morphology and niPGT-A and
2. the control group based on morphology alone.

The women and clinicians will be blinded to the treatment groups they are assigned. Only the laboratory staff in the PGT laboratory will be aware of the group assignment.

niPGT-A of spent culture medium (SCM) In the intervention group, comprehensive chromosome screening using NGS will be performed according to the recommendations of the company in all SCM samples. In the control group, the measurement will be done retrospectively in those SCM samples of blastocysts that are replaced in the first transfer. All SCM samples will be saved for possible future research.

A commercially available NI-PGT kit (PG-Seq Rapid Non-Invasive PGT kit, PerkinElmer) will be used to analyse SCM samples. The protocol has been previously optimized with non-invasive samples from 15 laboratories around the world. The kit follows a single tube workflow, two-steps PCR to whole genome amplify the DNA in SCM and then attaches indexes and sequence-specific adapters to template DNA, resulting in sequencing ready samples.

After purification, concentration of each sample is adjusted into equal molar, pooled (96 samples) and then sequenced on a MiSeq system (Illumina) at 1x75 bp read length. On-board secondary analysis was performed automatically by the MiSeq Reporter (Illumina) followed by PG-Find Software (v 1.0, PerkinElmer). Reads aligning to anomalous, unstructured and highly repetitive sequences are filtered from the analysis. A target bin size of 1,000 kb is used, giving a minimum resolution of 10Mb. All genomic positions refer to the human genome build NCBI 37.

According to the default setting of PG-Find software, classification of aneuploidy is determined by CNV (copy umber variation) value. CNV value >2.7 is considered as gain while CNV value <1.3 is considered as loss. Sample will be concluded as non-euploid when one or more of the chromosomes shows gain/loss.

The niPGT-A report of the SCM sample can be euploid, non-euploid and non-informative. It is used only to prioritize the sequence of embryo transfer. Blastocysts with non-euploid result in the niPGT-A report will not be discarded.

Blinding The embryologist will grade the morphology of blastocysts according to Gardner's criteria stated above and enter the grading of blastocysts into an online database, which will be managed by an IT technician. The laboratory staff in the PGT laboratory will enter the PGT result into a local database when the NIPGT results are available. The IT technician will collect the database from the PGT laboratory and enter it into the online software to compile the sequence of embryo transfer according to a pre-determined algorithm which depends on the day of blastocyst development (day 5 better than day 6), blastocyst morphology and niPGT-A result. The IT technician will issue the sequence of embryo transfer which does not contain information on the grading of the blastocyst and the NIPGT result to the embryologists in the IVF laboratory. Therefore, the subjects recruited, the clinicians and the embryologists will be blinded to the group allocation.

Our preliminary results of niPGT-A Preliminary results of the ongoing NIPGT-A trial involved 1168 SCM. Media cultured in parallel but without contact with embryos were collected as controls (n=238). Amplification was successful in 1158 SCM (99.1%, 1158/1168) and 1141 SCM resulted in conclusive result (97.6%, 1141/1168). All controls showed no amplification.

Frozen embryo transfer (FET) Blastocysts can be replaced in the subsequent natural, letrozole or hormonal replacement cycles, depending on whether the women have regular menstrual cycles or not. Only one blastocyst will be transferred each time. In the control group, blastocysts with the best quality morphology will be replaced first and the sequence of blastocyst transfer is decided prior to randomization. In the intervention group, blastocysts with the best morphology and euploid result will be replaced first as the sequence of blastocyst transfer will be modified after the niPGT-A reports are available.

Pregnancy A urine pregnancy test will be performed 14 days after the transfer. If the pregnancy test is positive, transvaginal ultrasound will be performed two weeks later to locate the pregnancy and confirm foetal viability and the number of fetuses. Subsequent management will be the same as other women with early pregnancy. They will be referred for antenatal care when the ongoing pregnancy is 8-10 weeks.

Follow-up Written consent regarding retrieval of pregnancy and delivery data will be sought from the women at the time of study as in all patients coming for IVF for infertility. The women will be contacted after delivery by phone to retrieve the information of the pregnancy outcomes as a routine after IVF pregnancy. The outcome of the pregnancy (delivery, miscarriage), number of babies born, birth weights and obstetrics complications will be recorded.

Women who fail to get pregnant in the first FET will be unblinded and an euploid embryo will be replaced if available. If no euploid embryo, the women can opt to undergo the second IVF cycle.

• Study Type: Interventional
• Enrollment (Estimated): 152
• Phase: Not Applicable

Contacts and Locations

• Study Contact: Name: Heidi Cheng, MBBS; Phone Number: 852-22553657; Email: chy610a@ha.org.hk

Study Locations

• Hong Kong
  • Hong Kong, Hong Kong
    • The University of Hong Kong
    • Contact: Heidi Cheng, MBBS; Phone Number: 852-22553657

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 18 years to 39 years (Adult)
• Accepts Healthy Volunteers: No

Description

Inclusion Criteria:

1. Women aged less than 40 years at the time of ovarian stimulation and
2. >=two spontaneous miscarriages in the first trimester
3. Unexplained recurrent pregnancy loss after standard investigation
4. At least one blastocyst available on day 5 or 6 after the retrieval.

Exclusion Criteria:

1. Women undergoing PGT for monogenic diseases or structural rearrangement of chromosomes;
2. Use of donor oocytes;
3. Hydrosalpinx shown on pelvic scanning and not surgically treated
4. Uterine anomalies distorting the uterine cavity in three dimensional ultrasound
5. No usable blastocysts on day 5 or 6 after the retrieval

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Screening
• Allocation: Randomized
• Interventional Model: Parallel Assignment
• Masking: Double

Number of Arms

2

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Experimental: Intervention group; the intervention group using morphology and niPGT-A	Genetic: Non-invasive Preimplantation genetic testing for aneuploidy status; In the intervention group, comprehensive chromosome screening using NGS will be performed according to the recommendations of the company in all SCM samples. Sequence of replacement shall be altered by the NiPGT result after morpholgy.
No Intervention: Control group; the control group based on morphology alone.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Miscarriage rate	Miscarriage rate in the first FET and is defined as a clinically recognized pregnancy loss before the 22 weeks of pregnancy and whose denominator is the clinical pregnancy. • Miscarriage rate in the first FET and is defined as a clinically recognized pregnancy loss before the 22 weeks of pregnancy and whose denominator is the clinical pregnancy.	12 weeks

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Live birth	delivery beyond 22 weeks of gestation per the first FET	1 year
positive urine pregnancy test	positive urine pregnancy test	at 2 weeks after embryo tranfer
Clinical pregnancy	presence of intrauterine gestational sac on scanning at gestational week 6	6 weeks
Ongoing pregnancy	presence of a fetal pole with pulsation at 8-10 weeks of gestation	10 weeks
Multiple pregnancy	presence of more than one intrauterine sac at 6 weeks of gestation	more than one intrauterine sac at 6-8 weeks
Ectopic pregnancy	Pregnancy not in the uterus	12 weeks
Number of CD56 cells	no. of CD 56 cells per 10 h.p.f from each biopsy	one month before start of IVF
Spheroid attachment rate	Attachment rate by co-culture assay	one month before start of IVF
Preterm delivery	delivery before 37 weeks of gestation	2 years
Gestational hypertension	development of newly-onset hypertension (blood pressure persistently >=140/90mmHg on two occasions at least 4 hours apart during pregnancy after 20 weeks gestation, labour or the puerperium in a previously normotensive non-proteinuric women	2 year
Pre-eclampsia	gestation hypertension with proteinuria	2 year
Gestational proteinuria	spot urine for initial estimation of total protein excretion of 300mg or more/24 hours	2 year
Gestational diabetes	Using a 75 g 2-hour OGTT, any of the fasting glucose ≥ 5.1mmol/l, 1 hour plasma glucose ≥ 10 mmol/l or 2 hour plasma glucose ≥ 8.5 mmol/l would be diagnostic	2 year
Antepartum haemorrhage	any vaginal bleeding during pregnancy from the 24 weeks to term	2 year
Congenital anomaly	Any congenital anomalies upon ultrasound or delivery	2 years
Perinatal mortality	Stillbirth or death within 1 week of delivery	2 year
Birthweight of newborn	Birthweight of new-born at delivery	2 year
Placental weight	Placental weight at delivery	2 year

Collaborators and Investigators

• Sponsor: Queen Mary Hospital, Hong Kong
• Investigators: Study Director: Ernest HY Ng, The University of Hong Kong

Publications and helpful links

General Publications

• Rubio C, Bellver J, Rodrigo L, Castillon G, Guillen A, Vidal C, Giles J, Ferrando M, Cabanillas S, Remohi J, Pellicer A, Simon C. In vitro fertilization with preimplantation genetic diagnosis for aneuploidies in advanced maternal age: a randomized, controlled study. Fertil Steril. 2017 May;107(5):1122-1129. doi: 10.1016/j.fertnstert.2017.03.011. Epub 2017 Apr 19.
• Lee E, Illingworth P, Wilton L, Chambers GM. The clinical effectiveness of preimplantation genetic diagnosis for aneuploidy in all 24 chromosomes (PGD-A): systematic review. Hum Reprod. 2015 Feb;30(2):473-83. doi: 10.1093/humrep/deu303. Epub 2014 Nov 28.
• Munne S, Kaplan B, Frattarelli JL, Child T, Nakhuda G, Shamma FN, Silverberg K, Kalista T, Handyside AH, Katz-Jaffe M, Wells D, Gordon T, Stock-Myer S, Willman S; STAR Study Group. Preimplantation genetic testing for aneuploidy versus morphology as selection criteria for single frozen-thawed embryo transfer in good-prognosis patients: a multicenter randomized clinical trial. Fertil Steril. 2019 Dec;112(6):1071-1079.e7. doi: 10.1016/j.fertnstert.2019.07.1346. Epub 2019 Sep 21.
• van den Berg MM, van Maarle MC, van Wely M, Goddijn M. Genetics of early miscarriage. Biochim Biophys Acta. 2012 Dec;1822(12):1951-9. doi: 10.1016/j.bbadis.2012.07.001. Epub 2012 Jul 13.
• Spandorfer SD, Davis OK, Barmat LI, Chung PH, Rosenwaks Z. Relationship between maternal age and aneuploidy in in vitro fertilization pregnancy loss. Fertil Steril. 2004 May;81(5):1265-9. doi: 10.1016/j.fertnstert.2003.09.057.
• Assou S, Ait-Ahmed O, El Messaoudi S, Thierry AR, Hamamah S. Non-invasive pre-implantation genetic diagnosis of X-linked disorders. Med Hypotheses. 2014 Oct;83(4):506-8. doi: 10.1016/j.mehy.2014.08.019. Epub 2014 Aug 23.
• Xu J, Fang R, Chen L, Chen D, Xiao JP, Yang W, Wang H, Song X, Ma T, Bo S, Shi C, Ren J, Huang L, Cai LY, Yao B, Xie XS, Lu S. Noninvasive chromosome screening of human embryos by genome sequencing of embryo culture medium for in vitro fertilization. Proc Natl Acad Sci U S A. 2016 Oct 18;113(42):11907-11912. doi: 10.1073/pnas.1613294113. Epub 2016 Sep 29.
• Shamonki MI, Jin H, Haimowitz Z, Liu L. Proof of concept: preimplantation genetic screening without embryo biopsy through analysis of cell-free DNA in spent embryo culture media. Fertil Steril. 2016 Nov;106(6):1312-1318. doi: 10.1016/j.fertnstert.2016.07.1112. Epub 2016 Aug 24.
• Feichtinger M, Vaccari E, Carli L, Wallner E, Madel U, Figl K, Palini S, Feichtinger W. Non-invasive preimplantation genetic screening using array comparative genomic hybridization on spent culture media: a proof-of-concept pilot study. Reprod Biomed Online. 2017 Jun;34(6):583-589. doi: 10.1016/j.rbmo.2017.03.015. Epub 2017 Mar 28.
• Ho JR, Arrach N, Rhodes-Long K, Ahmady A, Ingles S, Chung K, Bendikson KA, Paulson RJ, McGinnis LK. Pushing the limits of detection: investigation of cell-free DNA for aneuploidy screening in embryos. Fertil Steril. 2018 Aug;110(3):467-475.e2. doi: 10.1016/j.fertnstert.2018.03.036. Epub 2018 Jun 28.
• Capalbo A, Romanelli V, Patassini C, Poli M, Girardi L, Giancani A, Stoppa M, Cimadomo D, Ubaldi FM, Rienzi L. Diagnostic efficacy of blastocoel fluid and spent media as sources of DNA for preimplantation genetic testing in standard clinical conditions. Fertil Steril. 2018 Oct;110(5):870-879.e5. doi: 10.1016/j.fertnstert.2018.05.031. Erratum In: Fertil Steril. 2019 Jan;111(1):194.
• Hiby SE, Regan L, Lo W, Farrell L, Carrington M, Moffett A. Association of maternal killer-cell immunoglobulin-like receptors and parental HLA-C genotypes with recurrent miscarriage. Hum Reprod. 2008 Apr;23(4):972-6. doi: 10.1093/humrep/den011. Epub 2008 Feb 8.

Study record dates

Study Major Dates

• Study Start (Estimated): July 1, 2023
• Primary Completion (Estimated): February 27, 2025
• Study Completion (Estimated): February 27, 2026

Study Registration Dates

• First Submitted: January 2, 2023
• First Submitted That Met QC Criteria: January 5, 2023
• First Posted (Actual): January 13, 2023

Study Record Updates

• Last Update Posted (Actual): July 3, 2023
• Last Update Submitted That Met QC Criteria: June 29, 2023
• Last Verified: June 1, 2023

More Information

Terms related to this study

• Additional Relevant MeSH Terms: Urogenital Diseases; Genital Diseases; Infertility
• Other Study ID Numbers: niPGTAinRPL

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No