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Year : 2021  |  Volume : 14  |  Issue : 5  |  Page : 31-47

Indian society for assisted reproduction consensus guidelines on preimplantation genetic testing in in vitro fertilization clinics

Jaideep Malhotra¹, Keshav Malhotra², Gaurav Majumdar³, Ritu Hari⁴, Vijayakumar Chelur⁵, Sayali Kandari⁶, Dayanidhi Sharma⁷, Nishad Chimote⁸, Manjeet S Mehta⁹, Sarabpreet Singh¹⁰, Feseena Sethi¹¹, Vijay S Mangoli¹², Parasuram Gopinath¹³, Krishna Chaitanya¹⁴, Priya Selvaraj^15
1 Managing Director, Rainbow IVF, Agra, Uttar Pradesh, President ISAR (2019), India
² MBBS, MCE, Chief Embryologist & Director-Rainbow IVF, Agra (Uttar Pradesh), India
³ MCE, PHD, Center of IVF and Human Reproduction, Sir Ganga Ram Hospital, New Delhi, India
⁴ Chief Medical Geneticist and PGT Head, Craft Hospital and Research Centre, Kerala, India
⁵ Freelance Consultant, Clinical Embryology, Chief Embryologist (Visiting): NIMS & EVAA Fettility Clinic & Research Center, NIMS University Jaipur, Chief independent auditor & Lab supervisor - (I) Motherhood Women’s Health & Child Care Center Science City Road Ahmedabad (II) Srijan Fertility & Research Center, Kankarbagh, Patna (III) Srishti IVF, Srishti Hospital, Paltan Bazaar Dibhrugarh, India
⁶ Embryology Laboratory Director & Clinical Research Head, Cellsure Biotech Research Centre, Mumbai, India
⁷ Bihar Head and Laboratory Director, Indira IVF group , Bihar State, Patna, Bihar, India
⁸ Scientific Director, Vaunshdhara’s Fertility Centre Pvt Ltd, Nagpur, India
⁹ Reproductive Geneticist, Director - Clinical Genomics & Molecular Diagnostics, Lifenity Wellness International, Mumbai, India
¹⁰ Senior Consultant & Chief Embryologist, Artemis Health Institute, Gurugram, Haryana, India
¹¹ MCE (Leeds University, UK); Senior Clinical Embryologist at ARMC Aegis Hospital, Perinthalmanna ARMC IVF, Calicut, Kerala, India
¹² MSc, PhD., Laboratory Director, Fertility Clinic, Mumbai, India
¹³ MBBS, MS (OBG), Sr Consultant & Scientific Director, CIMAR Fertility Centre, Kochi, India
¹⁴ Scientific Head and Clinical Embryologist, Oasis Fertility, Pune, India
¹⁵ MD DNB MCE MNAMS FICOG, Associate Director, Scientific and Clinical Head, GG Hospital, Fertility Research and Women’s Speciality Center, Chennai, India

Correspondence Address:
Dr. Keshav Malhotra
MBBS, MCE, Chief Embryologist & Director-Rainbow IVF, Agra (Uttar Pradesh)
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/0974-1208.330503

Abstract

Study Question: What are the good practice guidelines for Pre implantation genetic testing applicable in INDIA? What is Already Known: Pre-Implantation Genetic Testing (PGT) is not new in India. It is used to identify euploid embryos for transfer, thus enabling couples to achieve a healthy pregnancy. There has been a lot of controversy surrounding PGT in the international forums; most of these debates have failed to reach a consensus on whether PGT should be offered or its concerns be validated more. Study Design, Size, Duration: This is the report of a 2-day consensus meeting where two moderators were assigned to a group of experts to collate information on Pre implantation genetic testing and embryo biopsy practices in INDIA. This meeting utilised surveys, available scientific evidence and personal laboratory experience into various presentations by experts on pre-decided specific topics. Participants/Materials, Setting, Methods: Expert professionals from ISAR representing clinical, embryological and genetic fields. Main Results and the Role of Chance: The report is divided into various components defining the terminologies, the various requirements, qualifications, recommendations on PGT -A,M,SR, and quality management: the report and recommendations of the expert panel reflect the discussion on each of the topics and try to lay down good practice points for labs to follow. Limitations, Reasons for Caution: The recommendations are solely based on expert opinion. Future availability of data may warrant an update of the same. Wider Implications of the Findings: These guidelines can help labs across the country to standardise their PGT services and improve clinical outcomes. Study Funding/Competing Interest(S): The consensus meeting and writing of the paper was supported by funds from CooperSurgical India.

Introduction

Definition of Terms in Indian scenario

Preimplantation Genetic Testing in India

Infrastructural Requirements for Embryo Biopsy

Figure 1: Overview of in vitro fertilization or preimplantation genetic testing process as per the ESHRE recommendations.[4] ESGRE=European Society of Human Reproduction and Embryology, PGT=Preimplantation genetic testing, PGT-M=Preimplantation genetic testing for monogenic genes, PGT-SR=Preimplantation genetic testing for structural rearrangements, PGT-A=Preimplantation genetic testing for aneuploidy Click here to view

Table 1: Recommendations for a genetic counseling center Click here to view

Table 2: Requirements for registration of a genetic laboratory Click here to view

Table 3: Requirements for registration of a genetic clinic Click here to view

Table 4: Biopsy requirements Click here to view

Qualification and Training

• The clinical embryologist must be knowledgeable in mammalian embryology, reproductive endocrinology, genetics, molecular biology, biochemistry, microbiology, and in vitro culture techniques
• The embryologist must also be familiar with assisted reproductive technology (ART). He/she must be either a medical graduate or have a postgraduate degree or a doctorate in an appropriate area of life sciences. (In the case of a clinic in existence for at least 1 year before the promulgation of these rules, a person with a B.Sc. or B.V. Sc degree but with at least 3 years of first-hand, hands-on experience of the techniques mentioned below and of discharging the responsibilities listed below, would be acceptable for functioning as a clinical embryologist. Such persons would also be eligible to take a test to be designed and conducted by an appropriate designated authority.)
• He/she must be familiar with the following:



• Principles and practice of semen analysis and cryopreservation of semen
• Cytology of mammalian and human oocyte to identify stages of oocyte maturation accurately
• All aspects of embryology including developmental biology
• Cell biological techniques used in cell and tissue culture
• Molecular biology and genetics of human reproduction
• Micromanipulation of sperm and oocytes for carrying out intracytoplasmic sperm injection (ICSI) and single-cell biopsies of embryos for PGD
• Principles and functioning of all the equipment used in the laboratory
• IVF of oocytes after processing the gametes
• Principles and practice of gamete/embryo freezing.



• It is recommended that the PGT laboratory should be directed by a person or persons who have executive accountability and the competence to assume responsibility for the services provided
• A defined training and quality assurance (QA) program needs to be developed by the PGT center to encompass all areas of work, including the use of all equipment, all relevant standard operating procedures (SOPs), data protection, and training in the IVF center, for example.



• Training should be supervised by an appropriate person and should be recorded in the logbook of the individual staff member. All staff should keep a log of their continued professional development to ensure continual recording and updating of their certifications/credentials
• It is acceptable to train staff in both disciplines (fluorescence in situ hybridization [FISH]-based and amplification-based PGD/PGS) if each discipline has a training schedule and assessment program to demonstrate proficiency with all the necessary skills and techniques and ability to work independently in either or both sections.
• Establishing internal continuous training programs and competency assessment of staff is recommended.

Counseling Recommendations for Preimplantation Genetic Testing Monogenic Disorders

• PGD can reduce the risk for conceiving a child with a genetic abnormality carried by one or both parents if that abnormality can be identified. Prenatal diagnostic testing to confirm the results of PGT-M is encouraged because the methods used for PGT-M have technical limitations that include the possibility for a false-negative result.^[6]

• Biopsy practitioner must be proficient in ICSI and micromanipulation procedures such as using laser
• Person who is performing biopsy must be trained and proficient in performing tubing/fixing of biopsied cells depending on type of genetic platform being used
• For training, validation, and QA, the biopsies should be performed on waste embryos under the supervision of an expert and then validated by the genetics laboratory for lack of contamination and DNA quality.

Counseling Recommendations for Preimplantation Genetic Testing for Aneuploidy

Consents and Checklist

• Consent for IVF/ICSI for embryo formation for PGT
• Consent for adjuncts alongside PGT testing to prioritize embryos
• Risk of embryo biopsy
• Information of genetic diagnostic testing and risk of misdiagnosis
• PGT safety results
• Chance of live birth rate
• Risk of untested anomalies occurring in screened embryos (polyploidy, microdeletions, etc.)
• National/international society guideline information
• Legally binding PCPNDT Information on illegal sex determination
• Recommendation for pre-PGT genetic counseling/psychological support
• Alternatives to PGT
• Information on avoiding natural sex
• Consent for research of affected embryos
• Consent for research on stored DNA
• Consent regarding affected embryo fate
• Possibility of no embryo to transfer
• Confidentiality.

• Form A certificate copy for patient's records
• Legally binding PCPNDT Information on illegality of sex determination
• PCPNDT formats Form G (invasive techniques)
• Risk of genetic diagnosis beyond scope and information consent
• Benefits/limitations of PGT, testing method, and used equipment/procedures by genetic laboratory
• Embryo biopsy risks
• IVF center-related risks
• Pregnancy-associated risks
• Follow-up recommendation for PND
• Genetic counseling recommendation
• Consent for additional screening tests (e.g., mitochondrial score)
• Confidentiality.

• Scheduled kit drop-off to ART center checkpoint
• Scheduled pickup check with biopsy practitioner kit of consent/The International Air Transport Association (IATA) regulation documents for sample pickup in dry ice
• Embryo biopsy labeled form from ART center
• Biopsy consents checklist via phone/scan upload check
• Form G scan upload checkpoint
• Form D – maintenance of records checkpoint
• DNA quality check – NanoDrop
• DNA amplification check – whole-genome amplification
• DNA quality reporting/degraded DNA reporting to biopsy practitioner for second biopsy
• Reporting check for delivery
• Genetic counseling requirement checkpoint.

Embryo Biopsy

• The biopsy procedure of preimplantation embryos consists of two main steps: creating an opening in the zona pellucida (ZP) followed by removal or biopsy of embryonic cells such as polar bodies (PBs) or blastomeres or trophectoderm (TE) cells. ZP opening may be performed either mechanically or chemically or using lasers
• Chemical method for creating an opening in the zona using acid Tyrode's solution has been universally abandoned as it is not well tolerated by the oocyte and may interfere with embryonic development thereafter
• Mechanical method uses simple glass tools but involves multiple steps such as dissection, release, and rotation of the oocyte. It requires skill and time
• The use of LASER is the method of choice as it allows maximum precision and safety
• PB biopsy.
• PB biopsy and analysis is an indirect approach for diagnosing the genetic status of the corresponding oocyte. Since both first and second PBs are not involved in fertilization nor embryo development, they make good candidates for performing genetic analysis.

• Removal of the first and second PBs can be done at separate times (day 0 and day 1: sequential approach) or at the same time (day 1: simultaneous approach)
• Sequential biopsy may be preferred if a fragmented PB1 is already present at the time of injection on day 0
• Simultaneous biopsy of the two PBs is “acceptable” for FISH analysis since they can provide distinguishable results
• Sequential biopsy of PBs is “recommended” for PCR analysis to determine recombination events between the first and second PBs.

• The main characteristics of the three biopsy approaches are summarized in [Table 5].

Table 5: The main oocyte and embryo biopsy approaches to conduct preimplantation genetic testing Click here to view

• It is recommended to biopsy 5–10 TE cells for genetic testing (according to the stage of development and number of cells constituting the blastocyst). The impact of removal of more than 10 TE cells on embryo development remains an area of further investigation
• Ca2+/MG2+-free medium should not be used for blastocyst biopsy
• There is a theoretical risk of cross-contamination when the same biopsy pipette is used to biopsy more than one blastocyst. However, it is acceptable to thoroughly rinse the biopsy pipette before each biopsy, thus allowing the same pipette to be used for multiple biopsies, but it should be verified in the laboratory that this suffices to avoid cross-contamination
• It is also recommended that following biopsy, blastocyst is immediately transferred in culture medium or cryopreserved once tubing of the biopsy has been confirmed.

• Results published regarding the survival rate of biopsied blastocysts are conflicting. In a recent study, a lower survival rate was found for cryopreserved biopsied blastocysts compared with intact blastocysts, while vitrification showed a higher survival rate than slow freezing^[7]
• For the time being, it is recommended that each center decides its own policy regarding the cryopreservation of biopsied embryos based on its experience and performance on embryo cryopreservation.

• Rebiopsy incidence is rare
• This practice is “acceptable” in the case of lost or anucleate blastomeres and failed diagnosis.

• Embryo biopsy may be performed at any of the three embryo stages, namely polar body, cleavage-stage, or blastocyst-stage embryo
• It is most beneficial to perform embryo biopsy at the blastocyst stage since embryo viability and implantation potential postbiopsy remains unaffected
• Blastocyst biopsy ensures that only the most competent embryos are selected to undergo genetic screening, which therefore helps in avoiding unnecessary biopsies and the associated costs
• Chromosome mosaicism may be an important limiting factor in the effectiveness of chromosome aneuploidy screening since it may be a source of diagnostic error. Consequently, many groups have been moving away from cleavage-stage biopsy since the incidence of mosaicism is higher at the cleavage stage as compared to the blastocyst stage
• Polar body biopsy may be chosen as an alternative to embryo biopsy only if maternal mutations or aneuploidies are investigated
• Meiotic errors from both parents can be detected, but mitotic errors leading to chromosomal mosaicism cannot be estimated from a single blastomere.

How to Use Euploid Embryos

• Frozen euploid ET is indicated in repeated IVF failure patients, discordant embryos and transport PGT with more than 24-h turnaround time
• Fresh euploid embryo transfer if possible is acceptable in good prognosis patients.

Clinical Consensus on What to do with Aneuploid Embryos

• Aneuploid embryos can be discarded or used for research only after consent from a couple
• No role of repeat biopsy in case of aneuploid embryos
• Aneuploid embryos must not be transferred into the patient
• The clinicians should have a right to deny patient requests in such a case, but this needs to be stated in the consent form beforehand.

What to do with Mosaics?

• If two or more different abnormal cell lines are present in an embryo, they are termed as aneuploid/aneuploid mosaics
• If normal and abnormal cell lines are present in an embryo, they are termed as euploid/aneuploid mosaics.

• Overall occurrence is low
• Mosaicism in products of conception of early miscarriages has been reported to be low^[10]
• Embryos with aneuploid ICM are lost during the first trimester owing to spontaneous miscarriages
• Most successful established pregnancies when mosaic embryo transfer (MET) done/in cases of extensive genetic testing done post-PGS have shown placental mosaicism only.^[11]

• All current methods, single-nucleotide polymorphism (SNP) arrays, comparative genomic hybridization (CGH), next-generation sequencing (NGS), etc., use preimplantation embryos and software which is designed to infer the embryo as euploid/aneuploid. Thus, there are a million points of analysis leading to gray areas for the software, and most likely, we are interpreting the gray area as mosaics
• Thus, for conformity, we might have to run PGS on different platforms to decisively comment on mosaicism prevalent in analyzed cells.

• Mosaic embryos when transferred may not implant
• Miscarry spontaneously if implantation occurs
• Result in intrauterine growth retardation
• Result in birth of child with minor – severe birth defects and/or mental retardation
• Result in birth of a normal healthy child.

Recommendations on the Order of Prioritization of Mosaic Embryos by The Preimplantation Genetic Diagnosis International Society, 2016

1. Embryos showing mosaic euploid/monosomy are preferable to euploid/trisomy, given that monosomic embryos (excepting 45,X) are not viable
2. If a decision is made to transfer mosaic embryos trisomic for a single chromosome, one can prioritize selection based on the level of mosaicism and the specific chromosome involved
3. The preferable transfer category consists of mosaic embryos trisomic for chromosomes 1, 3, 4, 5, 6, 8, 9, 10, 11, 12, 17, 19, 20, 22, X, Y. None of these chromosomes involve the adverse characteristics enumerated below
4. Embryos mosaic for trisomies that are associated with potential for uniparental disomy (14, 15) are of lesser priority
5. Embryos mosaic for trisomies that are associated with intrauterine growth retardation (chromosomes 2, 7, 16) are of lesser priority
6. Embryos mosaic for trisomies capable of live-born viability (chromosomes 13, 18, 21) are of the lowest priority for obvious reasons.

• Incidence is very small
• There is a question mark on clinical relevance of mosaicism
• Technology is at par to detect as low as 20% mosaicism
• PGDIS guidelines to be followed in case transfer of mosaic embryo must be performed.

Consensus on Preimplantation Genetic Testing for Aneuploidy Indications

• As per the recommendations by the experts, the findings from the STAR trial^[15] revealed that there was a statistically significant (P < 0.035) improvement in an ongoing pregnancy rate of 14% for women aged 35–40 years in the PGS arm. These findings are consistent with 2014 SART data and other published studies
• The SART data show that implantation rates hover around 50% for PGT irrespective of maternal age, whereas, without PGT, rates decrease after the age of 35 years
• Similarly, recently Ozgur et al. demonstrated that, in young patients aged <35 years with at least two or more 2BB blastocysts, PGT-A blastocyst selection did not result in an increased live birth rate
• The recommendations of the practice committees of ASRM and SART published in April 2017 and PGT consensus group in March 2018 are as follows
• The use of PGT-A does not improve outcomes in good prognosis patients <37 years of age
• No difference in outcomes for patients with recurrent pregnancy losses when compared to expectant management
• In women aged >37 years, PGT-A resulted in higher live birth rates with single euploid embryo transfer
• An advanced maternal age is a clear indication for PGT-A.

• PGT-A is recommended for



• Advanced maternal age (36–40 years)
• Repeated pregnancy loss – known etiologies.



• PGT-A is not recommended for



• Young, good prognosis patients (<35 years)
• Unexplained RPL
• Low AMH – limited eggs; multiple IVF cycles may be necessary in order to obtain one euploid blastocyst.

• Previous child with a defect
• Couple screened positive for a variant (carriers)
• An alternative to prenatal genetic diagnosis for couples with a significant risk for transmitting the defect
• Avoiding abortions
• Family history of any X-linked, autosomal dominant, and recessive conditions.

• Screening result or mutation report positive is a must before counseling patient for PGT-M
• Number of embryos required for a biopsy depending on the inheritance pattern
• Need for multiple cycles for oocyte/embryo pooling
• PGT-M haplotyping/regular to be decided by the geneticist.

• Can be offered to all patients with single or multiple gene disorders with a positive mutation report
• Cannot be offered for diseases which as multifactorial and nongenetic based diseases
• In case PGT-A is desired, it should be performed on PGT-M screened embryos.

Consensus on Preimplantation Genetic Diagnosis for (Chromosomal) Structural Rearrangement

• One or both parents has a balanced translocation (structural rearrangement in the chromosome)
• Chance of having a pregnancy with an unbalanced structural abnormality (extra or missing genetic material). Typically results in implantation failure/pregnancy loss
• PGT-SR for chromosomal structural rearrangements (previously PGS translocation)
• A genetic test designed to detect inherited rearrangements and increase the chance of a successful pregnancy
• Reduces the risk of having a pregnancy with an unbalanced structural abnormality.

• Chromosomal rearrangements are associated with chromosomal changes in terms of normal size or arrangement
• Many carriers of balanced chromosomal rearrangements are healthy and are unaware of their carrier status until they try to have children
• People with chromosomal rearrangements are at an increased risk of producing embryos with the incorrect amount of genetic material, which typically do not lead to a successful pregnancy.

• Certain rearrangements are seen with greater frequency in the general population
• Hot spots can be found at 11q23, 17q11, and 22q11
• Leading to the recurrent translocations t(11;22) and t(17;22)
• The site on chromosome 22 that is often implicated in translocations is the same locus associated with DiGeorge/velocardiofacial syndrome (22q11 deletion disorder)
• Robertsonian translocations are among the most common balanced structural rearrangements seen in the general population, with a frequency in new-born surveys of about 1 in 1000
• Rob (13q14q) accounts for around 75% of all Robertsonian translocations.

• When one parent has known chromosomal aberration^[17]
• Parental karyotyping to be done to get accurate chromosome breakpoints
• Minimum 850 banding pattern (high-resolution)
• Assay to be carried out on a parental sample to test the probes
• Same probes to be used on biopsy specimens
• Total procedure = 2–10 days
• Same cycle transfers possible in some cases depending on the in-house availability of technology.

• In general, PGT-SR is only recommended if the technique applied can detect all expected unbalanced forms of the chromosomal rearrangement
• When comprehensive testing strategies are applied, it is acceptable to use information on copy number of nonindication chromosomes to refine embryo transfer strategies
• Offered to balanced translocation carrier couple either with repeated implantation failure or repeated miscarriages
• Karyotyping is the gold standard to detect chromosomal abnormalities
• Some couples may be advised to undergo PGT-SR straight away after diagnosis
• FISH-based PGT-SR can be offered for patients where chances of getting blastocysts can be low
• Ample number of embryos required for testing based on female/male translocation carrier
• NGS-based consensus circular sequence only on validated pipeline by the geneticist
• NGS and FISH both are robust and sensitive.

Accreditation and Quality Management

Quality Management Systems

• It is recommended that a quality management system is integrated to the IVF/PGT center. Quality management ensures that an IVF/PGT center and the PGT service it provides, is of consistent quality
• It has four main components: quality planning, QA, quality control, and quality improvement
• Aspects of quality management to be implemented include quality policy, quality manual, document control, compliance to SOPs, risk management, continual improvement, audits, and management review
• Technical requirements include personnel, laboratory conditions and environment, laboratory equipment, all stages of examination procedures, results reporting, and QA.



• Validation of all methods used is recommended
• Written SOPs should be available for all steps of the PGT procedure
• Laboratory staff should have profound knowledge of the SOPs as these are the fundamental backbone of the service
• Deviations from protocols should be recorded.

References

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