A 35-year-old G5P0050 presented with her husband for reproductive endocrinology consultation. Both partners were of Latino heritage and in excellent health. Her blood type/Rh group was A positive; she had no known drug allergies; and the patient took no medication on a regular basis. Between 2001 and 2012, the couple established five unassisted conceptions, all of which were HM and electively terminated in the first trimester. After the initial pregnancy, additional evaluation discovered that the patient had an European Society of Human Reproduction and Embryology Class IIb uterine anomaly/bicornuate uterus although her medical records did not define which uterine compartment contained the pregnancy for all cases. To evacuate all molar tissue, each event was followed by dilation and curettage performed by a gynecologist in an outpatient setting. However for her most recent pregnancy in 2012, serum human chorionic gonadotropin (hCG) levels did not decline normally after surgery and the patient received nine cycles of intravenous. doxorubicin [7] for low risk gestational trophoblastic disease under co-management with an oncology team. She was then placed on oral contraceptive pills, referred to a genetic counselor and advised not to conceive again for 12 months.

The couple expressed interest in IVF and preimplantation genetic screening (PGS) of embryos before transfer. Anonymous donor oocyte IVF was also discussed but this option was declined as first-line treatment for personal reasons. While basic chromosomal data (standard G-band karyotypes) from both partners had been reassuring, further evaluation and counseling was obtained before moving forward with IVF in April 2016. Written informed consent for clinical and research aspects of the proposed treatment were obtained from both partners, and the investigational component was approved by our laboratory's independent institutional review board. The rare nature of recurrent HM [3,8] was discussed and genomic sequencing for the NLRP7 gene was carried out for both individuals. Briefly, genomic DNA was extracted from blood samples collected by peripheral venipuncture and, after evaluating the quantity and quality of the DNA, nucleic acids were disrupted to mean fragment size of 250 bp. Hybrid capture was used to enrich for areas of interest that included the coding and splicing regions of the NLRP7 gene. Pair-ended DNA sequences were obtained by next generation sequencing (NGS) using the NextSeq platform (Illumina, San Diego, CA, USA) at a mean depth of 100X; all regions of interest were characterized by coverage equal to or greater than 20X. Analysis of deletions and duplications was derived from NGS data; potential deletions or duplications were excluded for the index case.

Pre-IVF screening tests were normal for both partners. Ovarian reserve [9] was considered satisfactory by serum anti-Müllerian hormone and antral follicle count estimates (4.63 ng/mL and n=12, respectively). Monophasic oral contraceptive pills were used for suppression in the cycle immediately prior to IVF. No ovarian cysts >15 mm diameter were detected and baseline serum estradiol was <80 pg/mL. The patient received a combined follicle-stimulating hormone +human menopausal gonadotropin regime and gonadotropin-releasing hormone (GnRH)-antagonist commenced on stimulation day 6 [10,11]. Estradiol and follicular monitoring via transvaginal ultrasound continued until at least three follicles had maximum diameter of ≥17 mm. This threshold was achieved after a 12 day follicular recruitment interval, and 2500 IU hCG+80 u GnRH-agonist (leuprolide) was administered subcutaneous. with oocyte retrieval scheduled 36 hours later. Because PGS was planned, fertilization for all oocytes was exclusively via intracytoplasmic sperm injection (ICSI) [12].

Embryos were initially cultured in 10 µL microdrops of G1:3 culture medium (Vitrolife, Goteborg, Sweden), and on d3 post-fertilization placed in 20 µ of G2:3 (Vitrolife) to support blastocyst culture. As all embryos in this study had six or more cells by d3, each was placed in 20 µL of G-MOPS medium (Vitrolife) under mineral oil and subjected to biopsy following zona ablation via noncontact laser (ZILOS-tk; Hamilton Thorne Inc., Beverley, MA, USA). Cells from each embryo were removed using micromanipulation and placed immediately in RNAse–DNAse-free polmerase chain reaction (PCR) tubes containing 10 µL of PCR-grade water. Following addition of 50 µg/mL Proteinase K (Roche Biochemicals, Mannheim, Germany) each sample was placed under oil and transferred to our laboratory. DNA assessment used the Illumina Infinium HumanKaryomap-12 DNA Analysis kit. Beadchips were read by the iScan array scanner. Data were analyzed with BlueFuse Multi software to determine familial haploblocks. The screening protocol involved a first-round multiplex PCR, simultaneously amplifying the coding region of NLRP7 along with two unlinked fluoroscopically labelled (Cy5.5) microsatellite markers, GABRB3, and D13S314. The first round PCR reaction was then subject to (1) analysis of NLRP7 genotypes using real-time PCR protocol and allele-specific hybridization probes using the LightCycler system 1.0 (Roche Diagnostics, Manheim, Germany) and (2) microsatellite sizing on a Visible Genetics OpenGene System automatic DNA sequencer with Gene Objects software (Visible Genetics, High Wycombe, UK) to exclude chance contamination, as described previously [13].

Variants in the NLRP7 gene were annotated following American College of Medical Genetics guidelines for interpretation. A substitution affecting the invariant donor splice site at position c.2810+2T>G of the coding region was identified in the sample obtained from the wife (Figure 1), while no pathogenic or likely pathogenic variants were identified in the husband. This pathogenic variant was shown to alter splicing of intron 9 and result in abnormal retention of the intron, which in turn inserts 54 amino acids in the protein (Figure 2). Of note, this pathogenic variant was previously reported in three patients with recurrent HM, all of Latino-American origin [14]. Our patient was fully informed and counseled about all results when available, and elected to proceed with IVF, PGS, and potential single embryo transfer.

Fifteen oocytes were retrieved and nine progressed to 2 pronuclei (pn) stage following ICSI. An additional oocyte (initially designated as 0 pn) cleaved after 72 hours and joined the others as a provisionally viable zygote. However, arrest of all embryos was noted between 72 and 144 hours in culture (Figure 3), rendering none suitable for transfer. Progression of embryo development and associated morphology is summarized in Table 1. The 10 non-viable embryos were submitted for molecular diagnosis and biparental chromosomal contributions were identified in all embryos. While ploidy error for some embryos was of indeterminate origin, none was of paternal origin while five were of maternal origin (Table 2). A summary of NGS findings and genetic recombination events observed in the two euploid embryos is presented in Figures 4 and 5.