The clinical practice of PGT-M for monogenetic disease usually adopted a double-checking strategy, which detect the mutation by Sanger sequencing and meantime construct haplotypes using the DNA sample of the proband so as to avoid the risks of misdiagnosis due to recombination and allele drop out (ADO). When there is no affected parent or offspring to serve as the proband, embryo carriers identified through direct mutation detection can be preferentially taken as probands for subsequent linkage analysis. In cases where none of the embryos are detected as mutant carrier, single sperm or the second polar body (PB2) can be complementally collected in the work-up of haplotype establishment. Our study aims to develop an optimized strategy of haplotype construction using gametes or arrested embryos for PGT-M in pedigrees with single gene diseases and no proband in the setting of difficult cases, which takes into account the expected number of oocytes acquired and the gonadal mosaicism.

Inclusion Criteria:

• The couples intended for PGT-M without proband were recruited from Shanghai JiAi Genetics and IVF Institute, Obstetrics and Gynecology Hospital of Fudan University between January 2023 and December 2024, and were included in the study if they were expected to have difficulties in the identification of an EAP, typically meeting one of the following criteria:

  1. the carrier of the pathogenic variant was without typical clinical symptoms or detected as gonadosomal mosaic;
  2. the asymptomatic parents, who had one or more children affected with the same disorder, did not possess the genomic alterations carried by the children as per Sanger sequencing or targeted deep sequencing;
  3. female partner with diminished ovarian reserve and therefore a low yield of embryos;
  4. the variants are X-linked and the karyotype of the variant carrier is 47, XXX or 47, XXY etc.

Exclusion Criteria:

• (1) Non-PGT-M families; (2) Pedigree of other proband samples can be obtained; (3) Patients seeking for PGT-M who strongly request no haplotype analysis in embryos and only require Sanger testing after being fully informed of the risk.

The de-identified IPD will be published along with the publications of this study