**7. IMSI: maternal age and pre-implantation genetic screening**

Age-related decline in the quality of the oocytes, which affects the ICSI outcome, is a known phenomenon. Cassuto et al. distinguished between the quality of embryos resulting from oocytes of women younger than 30 years and those from women of 30 years and older, following the injection of spermatozoa selected using IMSI. When class 2 or 3 spermatozoa (moderate and bad morphological score) was injected, a lower rate of best and good embryos developed in the group of the older female patients (>30 years old) in comparison with the rate in the younger group (maternal age ≤ 30). Conversely, when a high-quality spermatozoon (score 4–6) was injected, the age-related quality of the oocyte is negligible; no difference was detected when the ratio of high-quality embryos was compared in young and older women. This is logical because these "top spermatozoa" do not need any repair. According to their findings, these researchers have pointed at the important contribution of a highquality spermatozoon, scored in a high magnification microscope, for the injection of oocytes aspirated from women of 30 years and older [35]. This outcome is not surprising because younger oocytes are capable of "repairing" the DNA of the injected spermatozoon. In ICSI, the direct sperm deposition probably does not have a delay in the cell cycle, as it might happen due to insemination in vitro of the oocytes. The extra time helps to save maternal ribonucleic acid (mRNA), which partially might overcome epigenetic defects [11, 16, 19, 64].

Embryo chromosomal status was examined in couples who underwent their first IVF-PGS cycle for aneuploidy due to advanced maternal age [65]. Couples were randomly addressed to routine ICSI or IMSI (n = 60). All cases of sperm concentration less than 1 × 106 /ml and sperm motility less than 20% were excluded from the study to minimize the influence of male factor infertility.

There was an increased incidence for sex chromosome aneuploidy in ICSI embryos when compared with IMSI (23.5% vs. 15.0%, respectively). IMSI was associated with a lower risk of sex chromosome abnormalities (odds ratio 0.57; confidence interval 0.37–0.90). The incidence of chaotic embryos was also higher with the ICSI procedure in comparison to IMSI (27.5% vs. 18.8%). An unexpected difference in gender incidence rates of euploid embryos was detected. The latest was supported by Setti et al., when a higher incidence of XX embryos derived from IMSI cycles in comparison with ICSI was noticed (66.9% vs. 52.5%, respectively) [42]. It is possible that IMSI-selected "normal" spermatozoa may carry a higher proportion of the X chromosome, which might lead to such findings.

Data also demonstrated a consistent decline in semen quality, as reflected by morphological evaluation by high-power microscope magnification, with increased age, suggesting the use of IMSI as routine in the older group of patients [66, 67].

#### **7.1. IMSI and paternal age**

**6.2. IMSI in cases of severe male factor infertility**

210 Spermatozoa - Facts and Perspectives

Usage of IMSI had a significant contribution to the accumulated knowledge of male infertility. At present, few randomized controlled trials are available assessing the advantages of IMSI over the conventional ICSI procedure. Antinori et al. assessed 446 couples, randomly referred to ICSI or IMSI, with at least 2 previous diagnoses of male factors due to severe oligoasthenoteratozoospermia [53]. Despite their initial poor reproductive prognosis, patients with two or more previous failed attempts benefited the most from IMSI not only in terms of increased pregnancy rate (29.8% vs. 12.9%; *P* = 0.017) but also lower miscarriage rates. Patients diagnosed with poor reproductive prognosis with two or more previous failed attempts ben-

swim-up technique showed a positive influence of IMSI on fertilization, implantation, and pregnancy rates [42, 61]. More reports regarding patients with isolated teratozoospermia or severe oligospermia pointed to the benefits following the selection of injected sperm cell using IMSI. Higher clinical pregnancy and higher implantation rates were observed, in comparison

Age-related decline in the quality of the oocytes, which affects the ICSI outcome, is a known phenomenon. Cassuto et al. distinguished between the quality of embryos resulting from oocytes of women younger than 30 years and those from women of 30 years and older, following the injection of spermatozoa selected using IMSI. When class 2 or 3 spermatozoa (moderate and bad morphological score) was injected, a lower rate of best and good embryos developed in the group of the older female patients (>30 years old) in comparison with the rate in the younger group (maternal age ≤ 30). Conversely, when a high-quality spermatozoon (score 4–6) was injected, the age-related quality of the oocyte is negligible; no difference was detected when the ratio of high-quality embryos was compared in young and older women. This is logical because these "top spermatozoa" do not need any repair. According to their findings, these researchers have pointed at the important contribution of a highquality spermatozoon, scored in a high magnification microscope, for the injection of oocytes aspirated from women of 30 years and older [35]. This outcome is not surprising because younger oocytes are capable of "repairing" the DNA of the injected spermatozoon. In ICSI, the direct sperm deposition probably does not have a delay in the cell cycle, as it might happen due to insemination in vitro of the oocytes. The extra time helps to save maternal ribonucleic acid (mRNA), which partially might overcome epigenetic defects [11, 16, 19, 64]. Embryo chromosomal status was examined in couples who underwent their first IVF-PGS cycle for aneuploidy due to advanced maternal age [65]. Couples were randomly addressed

to routine ICSI or IMSI (n = 60). All cases of sperm concentration less than 1 × 106

factor infertility.

sperm motility less than 20% were excluded from the study to minimize the influence of male

/ml after the

/ml and

efited the most from IMSI. Study of patients with motile sperm less than 0.1 × 106

**7. IMSI: maternal age and pre-implantation genetic screening**

to the conventional aspiration of spermatozoa in ICSI [62, 63].

Regarding the question of sperm quality in correlation to male age, it was described that increased male age is associated with a decrease in semen volume of 3–22%, a decrease in sperm motility of 3–37%, and a decrease in percentage of normal sperm of 4–18%, when comparing 30-year-old men with 50-year-old men, with no consistent effect on sperm concentration. Moreover, with control for a female partner, a relative decrease in pregnancy rates of 23 and 38%, increased risks for subfecundity ranging from 11 to 25%, and relative increase in months to achieve pregnancy up to 20% were found, comparing men <30 years old with men >50 years old, respectively [66]. Recently, IMSI provided remarkable information. Considering assessment of semen samples from 975 men who underwent IMSI, two forms of spermatozoa were considered: normal spermatozoa and spermatozoa with large nuclear vacuoles (LNV) [67]. At least 200 spermatozoa per sample were evaluated and the percentages of normal and LNV spermatozoa were determined. The subjects were divided into three groups according to age: Group I ≤ 35 years old; Group II: 3640 years; and Group III ≥ 41 years. Ratio of normal sperm cells in the older group (Group III) was lower than in the younger groups (I and II; P < 0.05). Percentage of LNV spermatozoa was higher in the older group (III) than in the younger (I and II) groups (*P* < 0.05). Regression analysis demonstrated a decrease in the incidence of normal sperm with increasing age (*P* < 0.05; r = −0.10). There was a positive correlation between the percentage of spermatozoa with LNV and male age (*P* < 0.05, r = 0.10).

These results demonstrated a consistent decline in semen quality, as reflected by morphological evaluation following IMSI with increased age, and support the routine use of IMSI for ICSI as a criterion for semen analysis in older group of patients.
