Study population

The study population consisted of 200 couples, including 1467 ICSI cycles that were referred for assisted reproduction at the Fertility Clinic in the International Islamic Center for Population Studies and Researches, Al-Azhar University, Cairo, Egypt, between September 2019 and October 2021.

The 200 male subjects were divided into four groups according to semen parameters (each group contained 50 cases):

• Group 1: Normozoospermia divided into two subgroups (PVP-ICSI and SpermSlow-ICSI).

• Group 2: Oligozoospermia divided into two subgroups (PVP-ICSI and SpermSlow-ICSI).

• Group 3: Asthenozoospermia divided into two subgroups (PVP-ICSI and SpermSlow-ICSI).

• Group 4: Teratozoospermia divided into two subgroups (PVP-ICSI and SpermSlow-ICSI).

Semen analysis

All male partners were subjected to complete semen analysis. Semen samples were collected by masturbation after a 3–7-day period of sexual abstinence. A physical examination, including volume, colour, odour, and liquefaction, was carried out. A microscope examination was performed to evaluate sperm concentration, motility, morphology, and the presence of other cellular elements using a light microscope (Olympus, C 21, Japan). Sperm were classified into progressive motile, non-progressive and immotile.

Sperm preparation

For the first group that was injected, sperm were prepared using a traditional centrifugation method. Sperm samples for ICSI were processed after microscope examination; 1 ml of sperm gradient medium (PureSperm, Nidacon, Sweden) was added to a fresh sample and centrifuged at 1800 rpm for 10 min (Heraeus 300, Osterode, Germany). The supernatant was removed and 2 ml of sperm washing medium (Sage, Denmark) was then added to sperm in the resulting pellet, followed by centrifugation at 1800 rpm for 10 min to reach and isolate the motile and morphologically normal sperm cells needed for assisted reproduction (Longenecker et al., Reference Longenecker, Cho, Khillan and Kulkarni2021).

For the second group that was injected, sperm were prepared using SpermSlow. On a plastic culture dish (IVF Petri dishes), a 2-ml droplet of a suspension of treated spermatozoa was touched with a pipette tip to a 5-ml droplet of fresh culture medium. At the same time, a 5-ml droplet of SpermSlow was touched with a pipette tip to a 5-ml droplet of fresh culture medium. The spermatozoa on this culture dish were incubated for 5 min at 37^oC under oil (FertiCult Mineral Oil). Spermatozoa bound to HA were slowed at the junction zone of the two droplets (Figure 1). These spermatozoa were selected and collected using an injection pipette (ICSI micropipette) and injected into oocytes.

Figure 1. SpermSlow procedure: preparation

Intracytoplasmic sperm injection (ICSI)

Following semen analysis and sperm preparation as described previously, samples were incubated until the time of injection. Each oocyte was injected with a single gross morphologically normal spermatozoon mechanically immobilized in PVP. Individual sperm for ICSI were examined and evaluated. The injection procedures were carried out in sterilized dishes using a holding pipette and injection needle, and then the best sperm were selected for injection and mechanically immobilized in the PVP droplet. In both procedures (SpermSlow and PVP), after sperm selection sperm, the mature oocyte was put in a 10-µl droplet of global total w/HEPES buffer (LifeGlobal, Europe) at 37ºC in a 90–95% humidity environment, equilibrated, and covered with mineral oil. The oocyte was attached to the holding pipette by slight negative pressure. The injection needle containing the sperm in PVP or SpermSlow was brought into the focal plane and a single sperm was positioned just at the tip of the microinjection needle. The next step was a slow, steady and consistent movement into the cytoplasm of the metaphase 2 (MII) oocytes; the sperm then was released into the cytoplasm with ∼1–3 µl medium. The injected oocyte was then washed and put in global total medium (LifeGlobal, Europe) in a culture dish covered with sterile warm equilibrated global oil (LifeGlobal, Europe) at 37ºC in a 6% CO₂ in a 90–95% and 5% O₂ atmosphere until fertilization (Cohen, Reference Cohen and Gardner2006; Sciorio and Smith, Reference Sciorio and Smith2019).

PVP procedure

A 10% PVP with human serum albumin (HSA) solution (Irvine Scientific, catalogue no. 90123, 5 × 0.5 ml kits) was obtained and intended for use in assisted reproductive procedures that included human gamete and embryo manipulation, and to immobilize sperm for ICSI procedures. PVP was dissolved in mHTF (an isotonic HEPES-buffered medium) to make a 10% (w/v) solution that contained 5 mg/ml HSA.

Other components were as follows. Salts and ions: sodium chloride, potassium chloride, magnesium sulphate, potassium phosphate, calcium chloride. Buffers: HEPES, sodium bicarbonate. Energy substrates: glucose, sodium pyruvate, sodium lactate. Protein source: HSA. Polymer: povidone. Water: water for injection (WFI) quality.

The 10% PVP solution is recommended for normal and high-motility sperm specimens. An aliquot of sperm was placed into the 10% PVP solution. The ICSI pipette containing the PVP solution then captured a single, immobilized sperm according to standard laboratory protocols. The sperm were now ready for the ICSI procedure.

SpermSlow procedure

The intended use of SpermSlow (product no. 1094) is to slow the movement of sperm to allow for the selection of the most mature, viable sperm for ICSI.

Other components were as follows. Hyaluronate, HSA, recombinant human insulin, glucose and derived metabolites, physiological salts, nucleotides, amino acids, vitamins, sodium bicarbonate, gentamicin sulphate 10 μg/ml.

Liquid paraffin was pre-equilibrated according to a standard protocol; the preferred holding medium (e.g. Flushing Medium, QA with HEPES) and an ICSI dish were warmed to 37°C for a minimum of 30 min.

1. 1. An elongated (bridging) drop (5–10 μl) of holding medium was dispensed into the ICSI dish. Depending on the number of oocytes used in the ICSI procedure, a corresponding number of 5–10 μl droplets was pipetted into the holding medium.

2. 2. Pipette 2 × 10 μl of SpermSlow: a primary drop should be close to one end of the elongated bridging droplet. A secondary droplet can be placed where convenient (for use as a handling drop). The pipette tip was used to create a junction between the primary SpermSlow droplet and the bridging droplet.

3. 3. A small amount (1–5 μl) of prepared and washed sperm suspension was introduced close to the opposite end of the bridging drop (for low-quality sperm samples, using a bridging drop can be omitted, and contact can be created directly between the SpermSlow droplet and the sperm suspension droplet). The pipette tip was used to create a junction between the sperm droplet and the bridging droplet.

4. 4. The ICSI dish was covered immediately with pre-equilibrated liquid paraffin and either used immediately or incubated at 37°C for 15 min before use. An oocyte was placed in each droplet of the preferred holding medium.

5. 5. An injection pipette was pre-loaded with SpermSlow from the handling droplet.

6. 6. The primary SpermSlow droplet was located and worked along the edge until the junction zone between the bridging droplet and SpermSlow droplet was reached. This should be evident from the number of sperm trapped in the three-dimensional net of hyaluronate.

7. 7. A mature spermatozoon was carefully selected. The mature spermatozoa expressing HA receptors were bound to the hyaluronate in SpermSlow and were easily identifiable by their beating tails but with limited forward motion. Spermatozoa moving freely in the SpermSlow drop are immature spermatozoa and should not be selected (Figure 2).

8. 8. Sperm MUST also be selected based on their morphology, as for standard ICSI. It may be easier to select sperm and then reassess morphology in the secondary SpermSlow handling drop before injection.

   

   Figure 2. SpermSlow procedure: selection

Assessment of fertilization and embryo quality

Fertilization was assessed 16–18 h after microinjection. The injected oocytes were observed for any sign of damage and for presence of pronuclei. Oocytes were classified as fertilized if two pronuclei (2PN) were present and the second polar body had been extruded. Approximately 72 h after microinjection, adequate numbers of embryos were transferred to recipient subjects. Embryo grading and transfer were performed ∼72 h after injection, and the cell number and morphology of each embryo were scored according to the indicated grading (Hill et al., Reference Hill, Freeman, Bastias, Rogers, Herbert, Osteen and Wentz1989):

• Grade A: equally sized blastomeres without fragmentation.

• Grade B: slightly unequal blastomeres up to 10% cytoplasmic fragments.

• Grade C: unequally sized blastomeres up to 50% fragments and large granules.

• Grade D: unequal blastomeres with significant fragmentation and large black granules.

Day 3 embryos were transferred (471 embryos) to recipient subjects according to the guidelines of the American Society of Reproduction. Excess good-quality embryos were cryopreserved. At 14 days after embryo transfer, serum HCG was determined using a chemical pregnancy test (considered positive if the result was 20 IU/L). In addition, a transvaginal ultrasound scan of the uterus was performed after 6–7 weeks of amenorrhea to determine whether a clinical pregnancy had been established (intrauterine gestational sac visible; Parmegiani et al., Reference Parmegiani, Cognigni and Filicori2014).

Statistical analysis

Data were coded and entered using Statistical Package for the Social Sciences (SPSS) version 26 (IBM Corp., Armonk, NY, USA). Data were summarized using mean, standard deviation, median, minimum and maximum in quantitative data, as well as frequency (count) and relative frequency (percentage) for categorical data. Comparisons between quantitative variables were carried out using the non-parametric Mann–Whitney test (Chan, Reference Chan2003a). To compare categorical data, a chi-squared test was performed. The Exact test was used instead when the expected frequency was less than 5 (Chan, Reference Chan2003b). P-values less than 0.05 were considered statistically significant.