This retrospective study collected data on PGT-M cycles performed between January 2015 and December 2022 at Monash IVF, a large private fertility centre in Australia. Only cycles that resulted in at least one embryo biopsied for PGT for a monogenic indication were included in the analysis. Patients from two Australian states (Victoria and Queensland) were included.

Referral pathway, and clinical and laboratory protocols

The following methodology describes standard clinical procedures associated with PGT-M at the centre over the study period.

Patient population

The centre’s genetic counselling team initially assessed all referrals. All patients received pre-test counselling from their fertility specialist, clinical geneticist, and/or genetic counsellor. Written consent was obtained from all couples before commencing the cycling process. After confirming the genetic status, DNA samples were collected from each couple, as well as from one or more relatives, to facilitate feasibility studies.

Stimulation, retrieval, biopsy, and fertilisation procedure

The ovarian stimulation, oocyte retrieval, and fertilisation procedures followed published protocols [19, 20]. In short, 95% of patients underwent a GnRH antagonist cycle (Orgalutran®; Ganirelix; Merck Sharp & Dohme, Macquarie Park, Australia) with recombinant FSH (Gonal-F®; Merck Serono, Frenchs Forest, Australia; Puregon; Merck Sharp & Dohme, South Granville, Australia). Oocyte retrieval (oocyte pick up (OPU)) was scheduled 35 or 36 h after the human chorionic gonadotrophin (hCG) trigger. All oocytes were fertilised using intracytoplasmic sperm injection (ICSI) 40 h post trigger.

Embryo culture, testing, transfer, and freezing

After ICSI, the clinical embryologists cultured the embryos and assessed fertilisation at 16-18h [20]. Embryos were assessed, developmentally classified and quality graded as previously described [20, 21]. Embryos were considered suitable for biopsy between days 5 and 7 if they contained a clearly defined inner cell mass and a suitable number of healthy trophectoderm cells (≥ 30). Approximately five trophectoderm cells for PGT-M were biopsied using a combination of laser and mechanical biopsy techniques. Whole genome amplification was performed on biopsy samples using the RepliG Single Cell Kit (Qiagen, the Netherlands). Karyomapping protocol was performed at the fertility centre’s genetics laboratory as previously described [22]. The use of karyomapping enables haplotype phasing and simultaneous 24-chromosome screening (known as preimplantation genetic testing for aneuploidy, or PGT-A).

Embryo transfer

On day 5, up to two frozen-thawed embryos were transferred, either in a natural cycle or a hormone replacement cycle [19]. The clinic follows a single embryo transfer policy; however, double embryo transfers were occasionally performed at patient request, in recognition of patient autonomy. Human chorionic gonadotrophin (hCG) testing was undertaken ~ 14 days post embryo transfer to confirm biochemical pregnancy status. A viability ultrasound is performed between 7 and 9 weeks post-embryo transfer to confirm clinical pregnancy status. Following confirmation of the pregnancy, patients are referred by their fertility specialist for obstetric care.

Birth outcomes

Treating obstetricians provided birth outcomes, including date of delivery, gestational age, weight, and any birth complications, or infant health concerns, to the fertility centre in compliance with the licensing requirements of the Reproductive Technology Accreditation Committee (RTAC). The RTAC Code of Practice mandates reporting of all pregnancy outcomes to the Australian and New Zealand Assisted Reproduction Database (ANZARD) for clinical quality monitoring. Postnatal testing is not routinely performed for all monogenic indications. This is particularly true of conditions with adult age of onset. Postnatal testing outcomes were subsequently unavailable, and a misdiagnosis rate was therefore unable to be ascertained.

Definition of data terms

Please see Table 1 for a definition of data terms used within this audit.

Table 1 Definition of data termsData collection

We collected data from the fertility centre’s Regulatory Information Management Software, which stores medical records held as part of clinical care. These records include the results of PGT, which are routinely entered into the software by the laboratory’s genetic scientists. This dataset was searched for all instances of PGT-M/A during the study time frame, and relevant data was extracted. We stored and analysed data using Microsoft Excel and STATA v.18.

We extracted data on PGT-M/A outcomes including the results of monogenic and aneuploidy screening, the number of stimulated cycles resulting in embryo biopsy, the number of embryo transfers, and the number of clinical pregnancies and live births.

Data analysis

We calculated clinical pregnancy and live birth rates using three denominators: total cycles, total transferred embryos, and total embryo transfer cycles. We also stratified data according to the monogenic inheritance pattern, and rates were calculated within these groups.

We also analysed the relationship between binary clinical outcomes (clinical pregnancy and live birth) and fertility covariates (body mass index, maternal age, FSH Dose, and the presence of a subfertility indication) within the PGT-M/A cohort. To account for the potential correlation within patients who have undergone multiple PGT-M cycles, Generalised Estimating Equations (GEE) were used to analyse these relationships. We selected a binomial distribution with a logit link function. We assumed the exchangeable correlation structure to accommodate possible intra-patient correlation. We estimated the GEE model using Stata V.18 [23].

Comparison groups

Two comparison groups were selected to evaluate PGT-M/A outcomes. The first group included PGT-A only tested embryo transfers between 2015 and 2022. The second group consisted of all frozen embryo transfers undertaken at the fertility centre during the same period. This 8-year time frame aligns with the study period of the PGT-M/A data.

The PGT-A only comparison group was selected as all PGT-M tested embryos undergo concurrent aneuploidy screening at our centre, allowing for a comparison that partly controls for aneuploidy. Clinical outcome data for the PGT-A tested embryo transfers, including information on maternal age at the time of treatment, were collected from the fertility centre’s Regulatory Information Management Software. To enable age matching with the PGT-M/A cohort, outcomes were stratified into “under 35” and “35 and over” groups. This stratification was selected as 35 is widely recognised as the age threshold of advanced maternal age. Age stratification was performed to control for the potential confounding effects of maternal age, allowing an assessment of whether other factors may contribute to observed differences between the groups.

The frozen embryo transfer group, which includes both PGT-tested and untested embryos, was selected as the general IVF comparison group. Within clinical practice, general IVF outcome data are often presented to prospective PGT-M/A patients in lieu of PGT-M/A-specific figures. A general IVF comparison group was therefore included to enable the comparison of PGT-M/A outcomes with those typically presented to patients. Clinical outcome data following the transfer of frozen embryos were extracted from annual reports from the Victorian Assisted Reproductive Treatment Authority (VARTA) [24]. This included information on the age distribution of individuals undergoing treatment.

To test for differences between the clinical outcomes of the comparison groups and the PGT-M/A cohort, an N-1 chi-square test was performed using the MedCalc Comparisons of Proportions Calculator.

The group undergoing aneuploidy screening alone during the study period (PGT-A only) had aneuploidy screening using low coverage whole genome sequencing (Illumina Veriseq Solution) as per manufacturer’s instructions. In contrast, the group having aneuploidy screening performed concurrent with PGT-M (in the PGT-M/A group) had aneuploidy screening performed by manual analysis of haplotypes and logR/BAF visualisation using the karyomapping protocol (Illumina) according to an in-house standard operating procedure. Data on the origins of chromosomal aneuploidy were obtained using the karyomapping protocol; however, this was limited to identifying meiotic or high-level mitotic origins, with the distinction between them not reported. As a result, the karyomapping protocol exhibits lower sensitivity for detecting low-level mosaicism. Both methods are capable of detecting segmental aneuploidy to a resolution of 10Mb. While these methods differ in their aneuploidy calling criteria, both are commonly used clinically in the detection of whole chromosome aneuploidy.