Decreased Sperm Motility Retarded ICSI Fertilization Rate in Severe Oligozoospermia but Good-Quality Embryo Transfer Had Achieved the Prospective Clinical Outcomes

<div><p>Introduction</p><p>Spermatozoa motility is the critical parameter to affect the treatment outcomes during assisted reproductive technologies (ART), but its reproductive capability remains a little informed in condition of severe male factor infertility. This retrospective cohort study aimed to evaluate the effects of reduced sperm motility on the embryological and clinical outcomes in intra-cytoplasmic sperm injection (ICSI) treatment of severe oligozoospermia.</p><p>Patients and Methods</p><p>966 cycles (812 couples) of severe oligozoospermia diagnosed by spermatozoa count ≤ 5 × 10⁶/mL and motile spermatozoa ≤ 2 × 10⁶/mL were divided into four groups in according to the number of motile spermatozoa in one ejaculate on the day of oocyte retrieval (Group B—E). The control (Group A) was 188 cycles of moderate oligozoospermia with spermatozoa count > 5 × 10⁶/mL and motile spermatozoa > 2 × 10⁶/mL. All female partners were younger than 35 years of age. Logistic regression analyzed embryological outcomes (the rates of fertilization, cleavage and good-quality embryo) and clinical outcomes (the rates of pregnancy, implantation, early miscarriage and live birth). Quality of embryo transfer (ET) was divided into three classes as continuous factor to test the effects of embryo quality on clinical outcomes.</p><p>Results</p><p>The reduction in the number of motile sperm in four groups of severe oligozoospermia gave rise to comparable inability of the fertilization (p < 0.001) and a decreased rate of good-quality embryo at Day 3 (p < 0.001) by compared to the control. The cleavage rate of the derived zygotes was similar to the control. ET classes significantly affected the clinical outcomes (p < 0.001). Class I ET gave rise to similar rates of clinical outcomes between five groups, but Class II and Class III ET retarded the rates of pregnancy, implantation and live birth and this particularly occurred in Group C, D and E. The rate of early miscarriage was not comparably different between groups. Overall rates in all groups were 41.26% clinical pregnancy, 25.74% implantation and 36.32% live birth, which gave live birth to 252 girls and 252 boys.</p><p>Conclusions</p><p>The reduction of motile spermatozoa in severe oligozoospermia decreased the rates of fertilization and good-quality embryo. Obtaining and transfer of good-quality embryos was the good prognostic to achieve prospective clinical outcomes regardless of the severity of oligozoospermia.</p></div

Embryological outcomes of ICSI treatment of oligozoospermia.

<p>Embryological outcomes of ICSI treatment of oligozoospermia.</p

RNA expression pattern of the <i>bcl-2</i> family genes in the testis.

<p>The expression of the anti-apoptotic gene <i>bcl-2</i> (<b>A</b>), pro-apoptotic genes <i>bax</i> (<b>B</b>), <i>bid</i> (<b>C</b>), <i>bim</i> (<b>D</b>) and <i>bak</i> (<b>E</b>) in the testis were determined with quantitative real time PCR. Target gene expression levels were normalized with the endogenous control ß-2-microglobulin (ß2M). Data are present as 2^ΔCt, ΔCt = Ct_{target gene}-Ct_ß2M. The Mann-Whitney U test was employed for statistical analysis (* <i>p</i><0.05). Each single symbol (circle and triangle) represents one individual testis sample.</p

Caspase-1, -3, -6 and-8 are not activated in UPEC infected testis.

<p>Total testis protein (20 µg) from four different animals in each group were separated on 15% SDS-PAGE. Immunoblots were probed with anti-caspase-8 (<b>A</b>), anti-caspase-3 <b>(B, upper panel)</b>, anti-caspase-6 <b>(B, lower panel)</b> and anti-caspase-1 (<b>C</b>) antibodies and detected using chemiluminescence. RAW 264.7 cells treated with sodium nitroprusside (SNP) served as a positive control. (<b>D</b>) The intensity of target bands on the films was measured with the ImageJ software (<a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a>). Semi-quantitative results are presented as mean ± SD and Student’s t-test was used for data analysis (Caspase-8, <i>p = </i>0.875; Caspase-3, <i>p = </i>0.686; Caspase-6, <i>p = </i>0.486; Caspase-1, <i>p = </i>0.343).</p

Electron microscopical analysis reveals intact blood-testis barrier (BTB) and blood-epididymis barrier (BEB).

<p>(<b>A</b>) Ultrastructural analysis shows that intercellular tracer penetration does not extend beyond the junctional complex of the BTB (arrow in inset) within the seminiferous epithelium of UPEC infected rats (x3,000 magnification, inset x20,000 magnification). SC = Sertoli cells, orientation of the luminal and basal compartment are highlighted (<b>B</b>) Ultrastructural analysis of a UPEC infected epididymis demonstrates intercellular tracer penetration (x3,000 magnification). Inset is a magnification of the area represented in the black frame showing the tight junctions. (x20,000 magnification).</p

NF-κB pathway is not activated in UPEC infected testis.

<p>(<b>A</b>) Total testis proteins were separated on 10% SDS-PAGE. Immunoblots were labeled with mouse anti-IkBα antibody. ß-actin served as a loading control. (<b>B</b>) The intensity of target bands was measured with the ImageJ software (<a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a>) and results are presented as the relative intensity = intensity of p65/intensity of ß-actin. (<b>C</b>) Testis cryosections were probed with anti-p65 antibody labeled with Cy3-linked secondary antibody (orange) and the nuclei were counterstained with DAPI (blue, images taken with x40 objective). Representative results from at least two independent experiments are shown.</p

The association of embryo transfer quality with clinical outcomes of ICSI treatment of oligozoospermia.

<p>The association of embryo transfer quality with clinical outcomes of ICSI treatment of oligozoospermia.</p

Characteristics of the couples and ICSI cycle number in five groups of oligozoospermia.

<p>Characteristics of the couples and ICSI cycle number in five groups of oligozoospermia.</p

Increase of TUNEL positive cells in UPEC infected testis.

<p>(<b>A</b>) DNA strand breakage in testicular cells from control (upper panel) and UPEC infected (lower panel) rats were analyzed using TUNEL assay. Nuclei were counterstained with DAPI (blue). TUNEL (+) cells (green) with ring-like nuclear stain are indicated with arrows. (<b>B</b>) Numbers of TUNEL (+) cells are presented as mean ± SD/seminiferous tubule. Student’s t-test was used for statistical analysis and the level of significance is indicated as **<i>p</i><0.01. (x20 objective).</p

Morphological changes and histological evaluation of the testis and epididymis.

<p>(<b>A</b>) Testicular weight of control (n = 8) and infected rats (n = 10) are presented as mean ± standard deviation (SD). Student’s t-test was employed for statistical analysis and the level of significance is indicated as **<i>p</i><0.001. (<b>B</b>) Sperm concentration was assessed in seven animals of each group and the results are presented as mean ± SD. Statistical analysis was performed with Student’s t-test and statistical significance is denoted as *<i>p</i><0.05. (<b>C</b>) Tissue sections of paraffin embedded testes were stained with hematoxylin and eosin. Histopathological assessment was performed on control (n = 5) and UPEC infected (n = 9) testes using light microscopy. The images were captured using Axioplan 2 Imaging system at magnification x20 and representative figures are shown. Various forms of impairment of spermatogenesis are visible exemplified by a Sertoli cell only tubule (star) and a hypospermatogenic tubule (triangle). (<b>D</b>) Histopathological images of caput (D top panels) and cauda epididymis (D bottom panels, x20 objective). Representative results from control (n = 5) and infected (n = 9) rats are depicted.</p