Expert Consensus on Preimplantation Genetic Diagnosis/Screening Issued: IVF Embryo Implantation Enters an Era of Standardization and Professionalization

1. Chromosomal abnormalities. Either or both partners carry chromosomal structural abnormalities, including reciprocal translocation, Robertsonian translocation, inversion, complex translocation, pathogenic microdeletion, or microduplication.

2. Monogenic disorders. Couples at high risk of having offspring with autosomal dominant, autosomal recessive, X-linked recessive, X-linked dominant, or Y-linked genetic disorders, where the pathogenic gene mutation or linked genetic markers have been clearly diagnosed within the family.

3. Severe diseases with genetic susceptibility. Either or both partners carry pathogenic mutations in genes associated with genetic susceptibility to severe diseases, such as the BRCA1 and BRCA2 pathogenic mutations linked to hereditary breast cancer.

4. Human leukocyte antigen (HLA) matching. Couples who have previously given birth to a child with a severe hematologic disorder requiring bone marrow transplantation can use preimplantation genetic diagnosis (PGD) to conceive a sibling with an HLA match identical to that of the affected child. Hematopoietic stem cells collected from the newborn’s umbilical cord blood can then be transplanted to treat the affected sibling.

Recent research and development in high-throughput genetic testing technologies (PGS version 2.0) have raised new questions regarding the clinical significance of PGS, including the presence of embryonic chromosomal mosaicism of varying degrees and locations, the precision of clinical detection techniques, criteria for selecting or discarding embryos for transfer, methods for calculating live birth rates with PGS, and its overall application value. These findings suggest that the evidence base for PGS requires further research and validation, and its indications are subject to revision and updating. Currently, PGS can be applied in the following areas:

1. Advanced Maternal Age (AMA): The female partner is aged 38 years or older.

2. Recurrent miscarriage (RM) of unknown etiology: two or more consecutive spontaneous abortions.

3. Unexplained recurrent implantation failure (RIF): Failure after three or more embryo transfer cycles, or after transferring 4–6 high-quality cleavage-stage embryos, or after transferring three or more high-quality blastocysts.

4. Severe teratozoospermia.

PGD technology shall not be performed in any of the following circumstances:

1. Hereditary diseases with currently unknown genetic diagnosis or gene localization.

2. Selection of non-disease traits, such as sex, appearance, height, and skin color.

3. Other conditions unsuitable for PGD implementation.

1. For sex chromosome numerical abnormalities, such as 47,XYY and 47,XXX, the probability of producing offspring with sex chromosome abnormalities is low; therefore, preimplantation genetic diagnosis (PGD) is not recommended. In contrast, for 47,XXY, the risk of chromosomal abnormalities in offspring is increased, and the implementation of PGD may be considered on a case-by-case basis.

2. For common chromosomal polymorphisms, such as 1qh+, 9qh+, inv(9)(p12q13), and Yqh+, PGD is not recommended.

Prior to undergoing PGD/PGS, the couple must receive at least one session of genetic counseling to fully understand their reproductive and genetic risks, be informed of the currently available medical interventions and their respective benefits and risks, voluntarily choose a treatment option, and retain relevant counseling records.

This includes collecting original clinical data and genetic test results from patients and their relevant family members, and constructing pedigree charts; inquiring about the medical history, reproductive history, specialized examination findings, and health assessment results of both partners; for individuals undergoing HLA matching, assessing the child’s current condition and treatment status to determine whether the condition permits a waiting period.

Integrate pedigree analysis and genetic testing results, along with the general patterns of genetic disease onset, to thoroughly assess the couple's risk of recurrence in future pregnancies.

Based on the assessed reproductive risks, inform couples of potential interventions—such as prenatal diagnosis, PGD/PGS, and gamete donation—along with the advantages and disadvantages of different intervention options available at this stage, thereby enabling them to make voluntary choices regarding reproductive interventions.

Before opting for a PGD/PGS cycle, couples must be fully informed of the various risks involved throughout the process. These include risks associated with conventional in vitro fertilization (IVF) procedures; embryo biopsy related to PGD/PGS techniques; damage from cryopreservation and thawing; inconclusive diagnoses for certain embryos; the possibility of having no transferable embryos after testing; uncertainty regarding the developmental potential of mosaic embryos; the inability to routinely identify carriers of chromosomal structural abnormalities; the risk of misdiagnosis due to the inherent biological characteristics of embryos and limitations of current testing technologies; and the necessity of undergoing prenatal diagnostic confirmation if a sustained pregnancy is achieved.

1. For embryos following PGD/PGS testing, single embryo transfer is recommended.

2. When no completely normal transplantable embryos are available in the current cycle, patients may voluntarily choose to transfer aneuploid mosaic abnormal embryos [4] after genetic counseling and informed consent, prioritizing the transfer of mosaic embryos with a lower risk of adverse outcomes in sequential order.

3. When performing PGD for monogenic diseases, embryos carrying pathogenic gene mutations but with a low likelihood of disease onset can be considered as alternative embryos for transfer.

4. Fresh cycle transfer or frozen-thawed cycle transfer can be selected.

Patients who achieve a sustained pregnancy after preimplantation genetic diagnosis (PGD) and embryo transfer must undergo invasive prenatal diagnosis. Non-invasive prenatal screening is not recommended at this stage, and pregnancy outcomes as well as neonatal status should be followed up.

1. Prior to entering a PGD/PGS treatment cycle, couples must undergo at least one session of genetic counseling, and complete records of the consultation, informed consent forms, and medical case files must be retained.

2. Confirm that the clinical indications for couples undergoing PGD/PGS cycles are reasonable and sufficient.

3. For patients who achieved sustained pregnancy after PGD, the concordance rate between prenatal diagnostic results and embryonic testing results was >98%.

4. For the analysis of clinical outcomes of PGD/PGS, it is recommended to use the statistical method of live birth rate per initiated cycle.

1. Intracytoplasmic Sperm Injection (ICSI)

For PGD/PGS cycles, ICSI fertilization is recommended to minimize interference from maternal granulosa cells and paternal sperm on the accuracy of downstream genetic testing, particularly when nucleic acid amplification techniques are intended for such testing.

2. Conventional In Vitro Fertilization (IVF)

When using fluorescence in situ hybridization (FISH) for embryonic genetic testing, blastocysts formed after IVF fertilization can also be employed; however, caution should be exercised to avoid contamination from sperm and other nuclei.

PGD/PGS via polar body biopsy enables the analysis and determination of maternal genetic information.

First polar body biopsy: It can be performed after oocyte retrieval or 0.5–2 hours after ICSI.

Second Polar Body Biopsy: Performed 8–14 hours after ICSI, following the extrusion of the second polar body.

Within 8–14 hours after ICSI fertilization, both the first and second polar bodies can be biopsied simultaneously.

Cleavage-stage biopsy is generally performed 66–70 hours after fertilization, targeting embryos that have developed to the 6–8-cell stage with a fragmentation rate of <30%. Typically, one blastomere is biopsied, with a maximum of two. Following cleavage-stage biopsy, the embryos can continue to develop for 2–3 days to reach the blastocyst stage. If preimplantation genetic diagnosis can be completed within this timeframe, fresh cycle embryo transfer may be performed.

Blastocyst biopsy has a minimal impact on embryonic developmental potential and has become the primary biopsy method for PGD/PGS. Blastocyst-stage biopsy is performed on day 5–6 post-fertilization, after the blastocyst has fully expanded. It is recommended that the biopsied blastocysts have a quality grade of 4BB or higher, with an optimal number of 5–10 cells retrieved. Typically, embryos undergo immediate cryopreservation following blastocyst biopsy; once genetic analysis is completed, euploid embryos are thawed and transferred in a subsequent cycle.

1. Methods for zona pellucida drilling include mechanical drilling, Tyrode's acid drilling, and laser drilling. Currently, the laser method is more commonly used; thermal damage to cells should be minimized during biopsy.

2. Mechanical drilling and laser methods can be used for polar body biopsy prior to fertilization, whereas the Tyrode's acid method may have adverse effects on the spindle.

3. Biopsy at the cleavage stage or blastocyst stage can be performed using mechanical methods, Tyrode's acid method, or laser method.

4. Zona drilling for blastocyst biopsy can be performed on day 3 or day 5 after fertilization, 4 hours prior to biopsy, or at the time of biopsy.

The first or second polar body should be aspirated individually or simultaneously, as required by genetic testing; cleavage-stage biopsy should involve the removal of 1–2 cells. If two cells are to be removed, the embryo undergoing biopsy should contain ≥6 cells; the number of trophectoderm cells collected for blastocyst biopsy should be controlled at 5–10.

Repeated biopsies will affect the developmental potential of embryos. However, when the initial biopsy yields an inconclusive diagnosis, a second biopsy may be considered (including for cleavage-stage embryos and blastocysts); if the embryo has been cryopreserved after biopsy, re-biopsy after thawing may be considered.

During cleavage-stage blastomere biopsy, nucleated and mononuclear cells should be selected for removal; for blastocyst biopsy, the site distant from the inner cell mass should be chosen.

During the waiting period for PGD/PGS genetic testing results, biopsied embryos need to be cryopreserved. Vitrification is recommended; the cryopreservation methods for cleavage-stage or blastocyst-stage embryos after biopsy are the same as those for routine embryo cryopreservation.

For downstream genetic testing using nucleic acid amplification methods, the cells obtained via biopsy should be washed and then placed into a PCR tube containing 2.5 μL of phosphate-buffered saline (or the pre-loaded reagent specified by the genetic testing kit). Simultaneously, transfer a small amount of the wash solution into an empty PCR tube to serve as a blank control.

Satisfactory results can be achieved with various cell fixation methods; appropriate isolation and protective measures for the fixative should be implemented during processing.

Consistent with the standards for ICSI in vitro embryo manipulation laboratories, the procedure is performed in sterile culture droplets covered with sterile mineral oil, within a Class 100 laminar flow environment at a constant temperature of 37°C.

To minimize cross-contamination between embryos, it is essential to ensure that each microdroplet contains only a single embryo, and that both the biopsy needle and the pipette used for transferring biopsied material are free of any residual cellular components.

Embryo biopsy technicians should possess proficient experience in ICSI procedures and strictly adhere to aseptic principles throughout the process to prevent exogenous contamination.

Based on the targets of preimplantation genetic testing, it can be further classified into gene-level testing and chromosome-level testing.

Couples identified as being at high risk for monogenic disorders through standardized clinical counseling; one or both partners carry pathogenic mutations in genes conferring clear genetic susceptibility to severe diseases; HLA matching selection.

To avoid misdiagnosis or inconclusive diagnoses caused by amplification failure, preferential amplification, allele drop-out (ADO), sample contamination, and other factors, it is recommended that preimplantation genetic diagnosis (PGD) for embryos involve both direct analysis of mutation sites and linkage analysis of genetic polymorphic markers.

Genetic polymorphic loci used for linkage analysis can be short tandem repeats (STRs) or single nucleotide polymorphisms (SNPs).

It is recommended to select at least two polymorphic sites capable of providing genetic information within the 1 Mb regions upstream and downstream of the pathogenic mutation site, while avoiding SNP sites with high homology, high GC content in adjacent sequences, or polynucleotide sequences.

For sex-linked genetic disorders, it is recommended to include testing for sex-indicating markers.

For HLA-matched individuals, the genetic polymorphic loci used for linkage analysis need to cover the upstream and downstream regions of HLA-A, HLA-B, HLA-DRA, and HLA-DQB1. At least five polymorphic loci that can provide genetic information should be selected in each region.

When conducting linkage analysis of genetic polymorphic loci, attention should be paid to genomic recombination near the mutation site.

1. Nested PCR Method

In the first round of multiplex PCR, multiple target loci (including mutation sites and linked polymorphic sites) should be amplified.

2. Whole Genome Amplification (WGA)

These methods can be further categorized into PCR-based and non-PCR-based amplification techniques. The products of whole genome amplification (WGA) can be analyzed using various methods to identify mutation sites and genetic linkage loci, including fluorescent PCR, Sanger sequencing, single nucleotide polymorphism microarray chips (SNP array), next-generation sequencing (NGS), and combined detection assays.

1. Prior to performing preimplantation genetic diagnosis (PGD), it is necessary to validate the known pathogenic mutation in family samples.

2. Polymorphic loci upstream and downstream of the mutation site should be selected for linkage analysis in family samples, from which informative loci are chosen to construct haplotype maps.

3. Validate the effectiveness of the PGD testing protocol at the single-cell level.

4. Prior to implementing PGD testing, it is necessary to validate the efficiency of the protocol at the single-cell level, assessing the effectiveness of amplification at the target detection loci as well as the ADO rate.

5. Validation samples may include lymphocytes, granulosa cells, oral mucosal cells, embryonic cells, etc. It should be noted that the cell source may affect amplification efficiency and the allele drop-out (ADO) rate.

6. For cleavage-stage biopsy, each validation sample should contain 1 cell; for blastocyst biopsy, each validation sample should contain 4–10 cells.

7. If the direct PCR amplification method is adopted, it is recommended to conduct pre-validation testing on 50 validation samples (including cells carrying pathogenic mutations and normal cells, if possible).

8. For those using the WGA method for the first-step amplification, it is recommended to conduct pre-validation testing in at least 10 validation samples.

9. The amplification efficiency should be >90%, and the ADO rate should be <10%. If the ADO rate is high, additional linked polymorphic markers upstream and downstream may be included for analysis.

Either or both spouses carry chromosomal abnormalities; sex selection for medical purposes; preimplantation aneuploidy screening.

1. For couples in whom one or both partners carry a chromosomal abnormality, preimplantation genetic diagnosis (PGD) can be performed to detect only the specific chromosomal abnormality, or it can simultaneously analyze other chromosomes for numerical abnormalities.

2. Sex selection can be used for X-linked genetic disorders with unclear genetic diagnoses. However, for families with a clear genetic diagnosis, mutation analysis is recommended, and sex selection alone is not advised.

3. For Y-linked monogenic diseases, sex selection alone may be performed.

4. For chromosomal structural translocations, PGD testing may not identify carriers. Laboratories with the necessary capabilities can offer carrier testing, but the technologies employed must be thoroughly evaluated.

5. For preimplantation genetic testing for aneuploidy, it is recommended to use methods capable of simultaneously analyzing numerical abnormalities across all chromosomes (comprehensive chromosome screening, CCS), while the use of FISH technology is not recommended.

1. Nucleic Acid Amplification

Nested PCR Method: The first round of multiplex PCR should simultaneously amplify specific loci on the target chromosomes, and the second round of quantitative PCR should perform copy number analysis for each chromosomal locus.

WGA Combined with High-Throughput Genetic Testing TechnologiesAfter WGA detection, various high-throughput genetic testing technologies can be employed for chromosome copy number analysis, such as array comparative genomic hybridization (array CGH), single nucleotide polymorphism microarrays (SNP arrays), and next-generation sequencing (NGS).

2. FISH Testing

Different nucleic acid probes should be selected based on the target chromosomes to be detected. When testing embryos from chromosomal translocation carriers, the chosen probe combination must be capable of identifying all possible chromosomally unbalanced embryos associated with the translocation. When the translocated chromosomal segments are small and exceed the effective resolution of high-throughput genetic testing technologies, FISH should be prioritized.

1. Commercially available kits are already available for technologies such as array CGH, SNP arrays, and NGS, featuring standardized operating procedures and quality control parameters. Local laboratories must validate the effectiveness and stability of their testing platforms before initial clinical application. Typically, preclinical pilot testing is not required for each individual case.

2. Chromosome copy number analysis using FISH requires prior verification of the karyotype of metaphase chromosomes from the peripheral blood of the patient and their spouse, as well as assessment of the fluorescence intensity and specificity of the probes on interphase nuclei.

Each clinical PGD/PGS laboratory should independently select its testing technology platform based on its own conditions and characteristics. Standard operating procedures (SOPs) must be established for all testing methods. Different testing technology platforms should set quality control parameters at key experimental stages according to the requirements of specific workflows. This guideline provides the following recommendations only for general quality control measures in PGD laboratories:

1. Laboratories conducting PGD/PGS testing using nucleic acid amplification methods must strictly adhere to the general principles outlined in the "Technical Standards for Clinical Gene Amplification Testing Laboratories."

2. The cells obtained from embryo biopsy and those used throughout the entire testing process must have clear and unique identifiers, with a one-to-one correspondence to the embryos.

3. When using nucleic acid amplification for PGD/PGS testing, the initial amplification step requires setting up a cell wash buffer blank control and an amplification reagent blank control to assess potential contamination and its source.

4. Standard Operating Procedures (SOPs) shall be established, strictly followed, and promptly evaluated and updated for all testing technologies.

5. For detection technologies using commercialized kits, such as array CGH, SNP arrays, and NGS, it is necessary to establish SOPs and quality control methods suitable for the local laboratory.

6. Strict dual-person verification is required throughout all experimental procedures, with real-time documentation and signatures.

7. Test results must be independently analyzed and interpreted by two individuals, with final review and adjudication by a third party. Embryos with discordant interpretations shall be classified as having an indeterminate diagnosis. Embryos with an indeterminate diagnosis in PGD are not recommended for transfer.

8. Internal and external quality control comparisons shall be conducted regularly, with proper records maintained.

The PGD/PGS report must include the names and ages of the patient couple, indications for testing, embryo identification numbers, stage of embryo biopsy, date of biopsy, testing methods and items, test results, names of the tester and reviewer, date of the report, and remarks. Except for medical purposes of sex determination, the report shall not indicate the sex of the embryos.

References:

Expert Consensus on Preimplantation Genetic Diagnosis/Screening Technology (2018 Edition)