Preimplantation Genetic Testing for Aneuploidies Improve the Clinical Pregnancy Outcome of Patients With AZFc Microdeletion

xiliang wang China Medical University https://orcid.org/0000-0003-2195-181X Yankun Wang peking medriv academy of genetics and reproduction kaibo hou General Hospital of Northern Theatre command dongmei hao General Hospital of Northern Theatre command jinyan zhang General Hospital of Northern Theatre command chang tang General Hospital of Northern Theatre command wei wei General Hospital of Northern Theatre command jia fei (  feijia@jabrehoo.com ) peking medriv academy of genetics and reproduction yuexin yu General Hospital of Northern Theatre command


Background
Azoospermia factor (AZF) is a region located on the long arm of the Y chromosome (Yq11), including the subgroups AZFa, AZFb and AZFc. Microdeletions of AZF are the most common factor causing male infertility except Klinefelter syndrome, and AZFc microdeletion has the highest frequency, accounting for approximately 80% of all AZF microdeletions [1]. It is generally believed that patients diagnosed with AZFc microdeletion have residual spermatogenesis ability, and AZFc microdeletion patients can become fathers through intracytoplasmic sperm injection (ICSI) [2][3][4].
Generally, the embryos obtained by ICSI were scored based on morphological criteria [5], and the best embryos were selected for transfer [6]. However, it has been reported that ICSI possesses higher aneuploidy rates than conventional IVF procedures [7][8][9]. Moreover, the fertilization rate and embryo quality of AZF microdeletion patients were signi cantly decreased after ICSI [10,11]. Therefore, when AZFc microdeletion patients receive ICSI, we should pay greater attention to the embryos for transfer. Preimplantation genetic testing for aneuploidy (PGT-A) could exclude aneuploid embryos, enable clinicians to select euploid embryos for embryo transfer, and improve the pregnancy rate and live birth rate [12].
The use of PGT-A in AZFc microdeletion patients has not been reported. Here, this study summarized the clinical outcomes of oligospermia patients diagnosed with AZFc microdeletion who received PGT-A before embryo transfer in our centre (Department of Reproductive Medicine, General Hospital of Northern Theater Command) in recent years. We found that the application of PGT-A improved the pregnancy rate and live birth rate compared to conventional ICSI methods.

Results
Among 22 couples, the men were 27-43 years old, and the women were 26-38 years old. Twenty-six ICSI cycles were carried out, of which 19 couples conducted one ICSI cycle, two couples conducted two ICSI cycles, and one couple conducted three ICSI cycles. A total of 248 fertilized oocytes were obtained from the 26 ICSI cycles, and 81 blastocysts from 25 cycles were detected by PGT-A. Among the blastocysts detected, 23 (28.4%) were female euploid blastocysts, 25 (30.9%) were male euploid blastocysts, 30 (37.0%) were aneuploid blastocysts, and the remaining 3 (3.7%) blastocysts failed to pass the quality control after genome ampli cation (Table 1).

Discussion
In this study, PGT-A was performed on ICSI embryos of AZFc microdeletion patients. Finally, the overall pregnancy rate and live birth rate were 100% (14/14) and 86% (12/14), respectively, which were much higher than those in previous reports [13][14][15]. In fact, PGT-A should be carried out for ICSI embryos due to severe male factors to exclude aneuploid embryos [16]. However, PGT-A is commonly used in patients with severe male factors such as DANH1, PAG6 and others [17,18], and PGT-A has not been reported in patients with AZF microdeletions.
Female age is a prominent factor affecting the outcome of ICSI-IVF [19,20]. However, male age had no signi cant effect on the rate of aneuploidy embryos [21]. The female age in this study ranged from 26 to 37, of which 16 were under 35 years old and 6 were over 35 years old. Among the 48 embryos under 35 years old, 28 (58.3%) were aneuploid embryos (16 were female, and 12 were male). Among the 33 embryos over 35 years old, 20 (60.6%) were aneuploid embryos (7 were female, and 13 were male). Overall, there was no signi cant correlation between aneuploidy frequency and female age, which may be because the eldest female age was 37 years old or the sample size was not large enough, and the in uence on embryos was not prominent in this study.
Male offspring of patients with AZF microdeletions using ART inherit AZF microdeletions, which may lead to similar or serious reproductive problems in adulthood [2][3][4]. In this study, after full genetic consultation with patients, for the 14 families who obtained female euploid embryos, most of them (13/14) chose to transfer female embryos, and one family was delayed due to COVID-19; for the 8 families who only obtained male euploid embryos, one family chose to enter the embryo transfer phase, and the other families chose to forgo embryo transfer.
Compared to ICSI using microTESE and ejaculatory sperm, the outcome of later sperm performed better [13,14,22]. In this study, our results show that PGT-A can signi cantly improve clinical outcomes after ICSI with ejaculated sperm in AZFc patients. However, the outcomes of PGT-A in AZFc patients undergoing ICSI with microTESE need further observation.

Conclusions
For patients with AZFc microdeletions, ICSI-IVF with ejaculatory spermatozoa and NGS-based PGT-A can signi cantly improve the chance of obtaining offspring.

Patients And Methods
Patients and AZF microdeletion detection  (Table 1).

Oocyte retrieval and oocyte insemination by ICSI
The patients' wives received 11 days with daily infection of 150 IU recombinant follicle stimulating hormone (Merck Serono). On day 6, 0.25 mg gonadotropinreleasing hormone antagonist (Merck Serono) was given every daily until oocyte maturation by 4 mg of gonadotropin-releasing hormone agonist (triptorelin acetate for injection, Beijing Biote Pharmaceutical) on day 12. Thirty-six hours later, the oocytes were retrieved and cultured in Quinn's Advantage fertilization medium (Origio) at 37°C, 6.0% CO2, 5% O2 and 89% N2. For ICSI, cumulus cells were removed by using hyaluronidase (Origio) four hours after oocyte retrieval, and metaphase II oocytes were injected 5 h after retrieval.
Quality assessment of fertilized embryos and blastocyst biopsy Fertilization status was assessed 18 h after ICSI, and normal fertilization was characterized by two distinct pronuclei and two polar bodies. The embryos developed to the blastocyst stage, and 5-10 trophoblastic cells were collected and transferred into 200-μL sterile PCR tubes for PGT-A; the blastocysts after biopsy were cryopreserved for frozen embryo transfer.
A total of 5 -10 trophoblast cells were used for whole genome ampli cation (WGA) according to the documentation of the SurePlex DNA Ampli cation System (Illumina, America), and WGA products were puri ed and recovered using AMPure XP beads (Beckman Coulter, America). The puri ed WGA products were used for library preparation using the VeriSeq TM DNA Library Prep Kit (Illumina, America) and sequenced on the NextSeq 550 platform. The data were analysed by Peking Medriv Academy of Genetics and Reproduction.
Considering that sons of AZFc microdeletion patients will also inherit the affected Y chromosome [2][3][4], the patients were informed of not only the euploid condition but also the sex of the embryos. After full genetic consultation with the patients, the patients chose the embryo for implantation.