*4.4. Male Factor Infertility*

Although embryonic aneuploidies mainly arise from maternal genome, some aneuploidies may derive from the spermatozoa [21]. Males with abnormal karyotype and Y chromosome deletions tend to produce spermatozoa with an unbalanced chromosome complement. Other several factors, such as varicocele, chemotherapy, age, and lifestyle, may also negatively affect meiotic divisions during spermatogenesis [21]. The rate of abnormal sperm after FISH examination was significantly higher in patients with male infertility (55.8% vs. 15.0%) and teratozoospermia was highly correlated with aneuploidy rate for chromosome 17, as documented by Petousis et al. [111]. Therefore, PGT-A should be considered in ICSI cycles with severe male factor (SMF), including azoospermia (obstructive and non-obstructive), severe oligoastenoteratozoospermia, Klinefelter syndrome (KS), Y-chromosome microdeletion, and even in men whose semen analysis does not fulfill the current World Health Organization (WHO) criteria.

Magli et al. [112] showed that SMF might contribute to a higher rate of aneuploid blastocysts (55% aneuploidy rate with normozoospermia, 62% with oligozoospermia, and 69% with nonobstructive azoospermia (NOA)), as evidenced by the 9-chromosome FISH technique performed on day-3 embryos. In a study by Silber et al. [113], patients with oligozoospermia and azoospermia had an euploidy rate of 41% and 22%, respectively, but the analysis was still conducted with the use of 9-chromosome FISH. Coates et al. [114] performed aCGH on TE biopsies; a significant increase in sexual chromosome abnormalities was observed in oligozoospermic males both with own (6.1% vs. 5.9%) and donor (1.7% vs. 2.0%) eggs.

A cohort study based on 1219 consecutive ICSI cycles performing PGT with trophoectoderm biopsy using rtq-PCR [115] evidenced that the rate of euploid blastocyst per inseminated mature oocyte was only significantly reduced in NOA (11.1%) when compared with normozoospermic patients (16.3%), whereas the euploidy rate per biopsied blastocyst was similar among the two groups. Furthermore, no differences in gestational age and birth weight were reported among the groups. The overall prevalence of congenital malformations (2.1%) was similar to that previously reported after either IVF with and without PGT-A [116,117] or spontaneous conceptions [118,119]. Following all of these considerations, SMF was not considered to be a critical parameter for embryo ploidy status. This could be due to the intrinsic potential of the oocyte to prevent further development of aneuploid embryos before the activation of the embryonic genome [120] or to the fact that sperm-derived aneuploidies may result in an early interruption of embryo development [121].

Mazzilli et al. [115] investigated in their study only 35 cycles in women under 35 years. Kahraman et al. [107] investigated 326 cycles with 741 blastocysts biopsied and analyzed using aCGH and NGS techniques; the couples enrolled were composed of young female (≤35) with SMF partners, ranging from five million/mL to NOA.

The couples undergoing IVF cycles with testicular spermatozoa had higher aneuploidy rate with respect to the patients with SMF and controls. In addition, reduced spermatozoa count (1–5 mil/mL) was associated with aneuploidy rate of 15.6%, suggesting that SMF increases the risk of chromosomal abnormalities, regardless of the female age. The highest mosaicism rate (22.0%) was observed in cycles using testicular spermatozoa and it was significantly higher than in cycles with normal sperm

parameters (9.9%). No significant difference was observed in the clinical outcomes between the control group and the SMF groups after transfer of a single euploid embryo. There was a trend toward a higher miscarriage rate in testicular sperm group.

A retrospective analysis by Tarozzi et al. [122], in which blastocysts from 340 cycles were assessed by aCGH, no differences between male (MF) and non-male factor (NMF) groups were found in terms of euploid blastocysts rate. The MF group showed a significantly higher rate of mosaic embryos (3.6% vs. 0.5%, respectively). A similar pattern of results was observed in all SMF groups considered in their complex, when compared to those without SMF (7.7% vs. 1.8%), suggesting that a compromised semen quality is associated with an increase in the mosaic blastocysts rate. The reason of these differences could be found in the fact that, during spermiogenesis, the transit of the spermatozoa in the epididymal tract favors DNA packaging, through the stabilization of the chromatine structure with protamine dephosphorylation and the formation of intra and intermolecular disulphide bridges between protamines.

The study by Scarselli et al. [123] evaluated whether the duration of ejaculatory abstinence might influence the euploid blastocysts rate. The blastocysts euploidy rate resulted in being 27.5% using spermatozoa from semen sample obtained after 2–5 days of abstinence and of 43.5% using spermatozoa from the second sample obtained 1 h after the first one. The use of potentially higher quality spermatozoa from samples with a shorter abstinence might reduce the aneuploidy rate in blastocysts.

Non-mosaic Klinefelter syndrome is associated with a male genetic disorder that often leads to NOA. The literature reports about 101 children born from non-mosaic KS fathers after successful ICSI, in most cases, healthy and without the use of PGT to select normal embryos [124]. Fetuses or embryos were diagnosed to have 47,XXY karyotype in some cases [125,126]. Due to these contradictory findings, it remains an open question as to whether embryo biopsy should be offered to couples with KS male partner. In the study by Greco et al. [127], a total of 26 ICSI cycles were performed in couples with KS and a total of 11 pregnancies and deliveries were achieved (pregnancy rate 42.3%). All of the fetal karyotypes were normal. PGT-A was not done, and the embryo chromosomal assessment was unknown at the time of transfer. It could be thought that, when the SMF is due to altered karyotype, it does not affect embryo ploidy. However, Staessen et al. [128] published an extremely convincing study on the need for PGT in KS patients. They reported data on PGT-M offered to 20 non-mosaic KS, showing that 54% of the embryos were normal but KS patients had a higher incidence of abnormalities involving chromosomes 18 and 21.

Several studies have also explored the effect of advanced paternal age on sperm aneuploidy rates. Two recent studies [129,130] noted that males above the age of 50 present more sperms with damaged DNA, low blastocyst development rate, higher global aneuploidy rate, and a higher rate of trisomies. Advanced paternal age could lead to several alterations in endocrinal and reproductive phenotypes, leading to an increase in DNA damages over years and a decrease in germ cells capability to repair these damages, inducing the production of aneuploid sperms and, consequently, abnormal embryos [131].

The study by Gat et al. [132] evaluated 177 IVF-ICSI cycles, in which PGT-A was performed on 405 blastocysts and concluded that DNA fragmentation index (DFI) was not associated with impaired embryo quality in both genetic and morphological aspects. Similar DFI-related findings were published by Bronet et al. [133], who reported comparable embryo euploidy rates in different DFI levels.

Overall, approximately 25–55% of males with extreme testicular pathologies, such as hypospermatogenesis, sperm maturation arrest, and Sertoli cell only syndrome (SCOS), and about 5–25% males with severe oligozoospermia or azoospermia present Y chromosome microdeletions. In general, deletions of the entire AZFa region invariably result in SCOS and azoospermia. Patients with deletions of the AZFb region presents maturation arrest, mostly at spermatocyte stage. Men with AZFc deletions have the most variable phenotype, which ranges from complete azoospermia to mild oligozoospermia [134]. Comparisons between the different types of microdeletions and treatments revealed that, except AZFc, cases with AZFa and AZFb microdeletions presented a poor clinical

prognosis. On 125 patients, 25 of them (20.0%) had abnormal karyotypes. Ninety-four cycles with testicular sperm leaded to 19 deliveries. Twenty-nine cycles with ejaculated sperm leaded to eight births (all in couples with AZFc). Patients with such alterations should be informed about the possibility of using preimplantation genetic testing due to the reported heredity of microdeletions and a possible association with men abnormal karyotype [134].
