A woman aged 33 years was admitted to our fertility clinic with bilateral tubal obstruction. She was married in 2011 and experienced natural miscarriages between 6 and 8 weeks of pregnancy for 5 times from 2013 to 2019. Since then, she used contraceptive tools for 3 years and had been trying to conceive without contraception for one year. However, she failed to become pregnant. The female patient’s basic endocrine levels are normal (FSH 7.27 mIU/ml, E2 41.71 pg/ml, P 0.55 ng/ml, PRL 12.58 ng/ml, LH 4.7 mIU/ml, T 0.52 ng/ml, AMH 2.6 ng/ml). Both partners had normal karyotypes. Her partner is 41 years old, in healthy conditions and with no family history or significant medical history. Semen analysis revealed normal parameters except mild teratozoospermia (normal sperm morphology rate is 2.5%).
She underwent a long ovarian stimulation protocol with 3.75 mg of GnRH agonist (Leuprorelin Acetate Microspheres for Injection, Shanghai Lirui Pharmaceutical Co., Ltd. China) for down-regulation, followed by initiation with 300 IU recombinant follicle stimulating hormone (rFSH) (Puregen, N.V.Organon, The Netherlands) and one month later an additional 5 IU of growth hormone (GenSci, China) was added daily for 12 days. Recombinant human choriogonadotrophin (hCG) alfa Solution for Injection (Ovidrel, Merck Serono, Italy) was administered when more than three ≥ 18 mm follicles were seen. Oocytes were collected 36 h after hCG administration. On the trigger day, the E2 level was 2858 pg/ml, LH was 0.42 mIU/ml, and P was 1.74 ng/ml.
Sperm was prepared using the swim-up method. Briefly, semen was collected by masturbation. After semen liquefaction at room temperature for 30 min, the sperm sample was checked under the microscope. The sperm volume was 1.5 ml; the sperm density was 10 million/ml and the proportion of forward progression sperm was 30%. 1.5 ml of semen was slowly added onto 2 ml of G-IVF PLUS (Vitrolife, Sweden) medium, and the tube was placed at an obliquity of 45° for sperm swim-up for 45 min. 1 ml of the upper layer liquid was gently aspirated into another 5 ml tube. Total sperm count of forward progression was 1 million. To ensure fertilization, we chose ICSI for fertilization. Five hours after oocyte retrieval, cumulus cells were removed with 80 IU of hyaluronidase and mechanically with a Pasteur pipette with an inner diameter of 150 μm. Oocytes were at metaphase II (MII) with one PB, and subsequently used for ICSI (about 3:00 P.M. D0). After ICSI operation, zygotes were cultured in CSCM (Irvine Scientific, USA) medium individually in a 37 ℃, 5% O2 and 6% CO2 COOK incubator(K-MINC-1000, USA). At 17 h after ICSI operation (about 8:00 A.M. D1), the fertilization status was evaluated and the number of PN and polar bodies were recorded.
The couple was informed of the formation of 3PN zygotes and of the possibility of performing microsurgical enucleation to remove one female PN. The advantages and disadvantages of removing one PN operation were also discussed with the patients. The patients approved the procedure to be carried out and gave their written consent (9:00 A.M. D1). Before proceeding with embryo transfer, this work was overseen and approved by the ethical committee (reference number of the approval: 2024-01).
Microsurgical enucleation (about 10:00 A.M. D1, approximately 19 h after ICSI injection) was conducted with the same equipment used for routine ICSI operations, including a micromanipulator (Narishige, Japan), an inverted microscope (NIKON, Japan), a thermostatic stage (TOKAI, Japan), a clean bench (K-SYSTEMS, Denmark), and a stereo microscope (NIKON, Japan). Zygotes for enucleation were transferred to an ICSI operation dish containing G-MOPS (Vitrolife, Sweden) as the operating solution, and the embryo number was marked on the bottom of the dish. The field of view was centered around the microdrop containing the zygotes, and zygotes were carefully rotated for observing the PB conditions. For ICSI-derived 3PN zygotes, if there are two PBs, microsurgical enucleation is not required (Figure S2); but if there is only one PB, one female PN needs to be removed [8]. The criteria for distinguishing male and female PN are as follows: Firstly, the female PN is closer to the PB [12, 13]. Secondly, the female PN is smaller in size than male PN [14, 15]. Thirdly, the number of nucleolar precursor bodies (NPBs) in the female PN is less than that in the male PN (5.6 ± 1.78 vs. 7.67 ± 1.91, p < 0.001, Figure S1). According to these criteria for distinguishing the male and female PN, the female PN could be precisely selected for removal. The holding needle (Vitrolife, Sweden) was first lowered and the zygote was rotated. After determining the PN to be removed and orient it is towards the 3 o’clock position, a mild negative pressure was applied to fix the zygote from the 9 o’clock position The enucleation needle (I.D. 4.7 μm, K-MPIP-3130,Cook, USA) was adjusted to the same level as the PN to be removed and the equator of the zygote. The enucleation needle then entered the zygotes from the 3 o’clock position, first penetrating the zona pellucida (ZP), then entering the oolemma in vicinity of the PN to be removed. The focus and the position of the enucleation needle were slightly adjusted such that the oolemma and the PN membrane basically overlap. Negative pressure was further slowly applied to aspirate the PN into the enucleation needle without interfering the other two PNs. Once the PN is completely aspirated into the enucleation needle, the enucleation needle was slowly retracted and a mild positive pressure was applied to release the zygote (Movie 1). The aspirated PN, the remaining two PNs and the oolemma were checked again to ensure that the PN was successfully removed. Before proceeding to the next zygote, the enucleation needle was cleaned in a PVP (Irvine Scientific, USA) drop several times. After all extra PNs in the zygotes have been removed, they were transferred to CSCM and washed three times before placing them in a 37.0℃,5%O2, 6% CO2 COOK incubator for further culture. On D3, the embryo development was observed and recorded. On D4, the residue cumulus cells were thoroughly removed with an appropriate stripper (I.D. 140 μm) gently and washed three times before transferring into a new culture dish containing about 20 µl of CSCM medium per drop. These operations were used to effectively remove exogenous cell free DNA (cfDNA) contamination. All embryos were morphologicially evaluated on D5/6 before vitrification. The remaining blastocyst culture media were sent to a gene testing company (Yikang, China) for non-invasive chromosome screening (NICS) of the embryos according to methods published by Huang et al. [16].
Immunofluorescence staining
Oocytes were fixed in 100 mM HEPES (pH 7.0, titrated with KOH), 50 mM EGTA (pH 7.0, titrated with KOH), 10 mM MgSO4, 2% methanol-free formaldehyde, and 0.5% triton X-100 for 15 to 60 min at 37℃. Fixed oocytes were further extracted and blocked in phosphate-buffered saline (PBS) with 0.5% triton X-100 and 5% BSA (PBT-BSA). Oocytes were incubated with rat anti-α-tubulin antibody (MCA78G; Bio-Rad) at 10 µg/mL overnight at 4℃, washed in PBT-BSA five times for 3 min each and incubated with Alexa Fluor 488-conjugated AffiniPure Fab Fragment donkey anti-rat (Jackson ImmunoResearch Europe) at 20 µg/mL and Hoechst 33,342 (Thermo Fisher) for 1 h at room temperature. After five washes in PBT-BSA for 3 min each, oocytes were imaged on LSM980 confocal laser scanning microscope (Zeiss).
Whole exome sequencing and variants analyses
Genomic DNA samples were extracted from peripheral blood using a QIAamp DNA blood mini kit (Qiagen, Germany) following the manufacturer’s instruction. Whole exome sequencing (WES) was performed on an Illumina NovaSeq X Plus platform. Exomes were captured by Agilent SureSelect and sequenced by the strategy of PE150. The human genome (GRCh37/hg19) were used as reference to align reads and ANNOVAR were used to functionally annotated refer to public databases, consisting of population frequencies, HGVS variant descriptions, phenotypes or diseases and variant function predictions. Candidate pathogenic variants were filtered according to the following criteria: (1) coding variants (including nonsynonymous exonic, indel or splice site variants); (2) variants with minor allele frequency < 0.1% in the human population genome datasets; (3) variants predicted to be deleterious by SIFT, PolyPhen2, MutationTaster, etc.; (4) homozygous, compound heterozygous or X-linked hemizygous variants; (5) relevancy for phenotype of patients or not reported [17].
