Influence of Borderline and Pathological Sperm Morphology on Pregnancy Outcomes following Intrauterine Insemination in Pregnant Women: A Retrospective Study.

Male infertility contributes to approximately half of all cases of couple infertility and is commonly associated with abnormalities in sperm parameters such as motility, morphology, and concentration ( 1 ). The levels of these parameters are essential in guiding the choice of appropriate infertility treatment. Sperm morphology (SM) analysis using the Kruger strict criteria, measures the percentage of morphologically normal sperm in a semen sample. According to the 5th edition of the World Health Organization (WHO) guidelines, the lower reference limit for normal SM is 4%. Samples with less than 4% normal forms are classified as teratozoospermia (TZS) ( 2 ). The impact of SM on intrauterine insemination (IUI) success in male factor infertility has been widely investigated. While some studies reported a significant association of the 4% threshold and clinical pregnancy rates (CPR) ( 3 - 5 ), others have found no such correlation ( 6 - 9 ). Although TZS appears to have limited influence on offspring health following in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) when multiple pregnancies are avoided, its predictive value for pregnancy outcomes remain uncertain ( 10 ). Regarding IUI outcomes, recent meta-analyses have shown no difference in pregnancy success rates based on the presence or severity of TZS ( 11 ). Although some studies downplayed the role of SM ( 12 ), other have reported an association between poor sperm quality including abnormal SM and increased rate of aneuploid embryos, indicating that meiotic events during fertilization may influence chromosome segregation ( 13 , 14 ). SM is increasingly recognized as a key indicator of fertilization potential ( 15 ) and reproductive success ( 16 ). Building on prior research that has focused primarily on the relationship between SM and CPR ( 3 - 9 ), this study aims to determine the influence of SM on important post-conception events, specifically pregnancy complications, abortion (first, Second and third trimester), and live birth rates. Based on the current definition of borderline (4-13% normal forms) and pathological (<4% normal forms) SM ( 17 ), the study compares pregnancy outcomes between borderline and pathological SM following IUI in pregnant women.

Ethical approval for this study was granted by the Medical Ethics Committee of the ACECR in Ahvaz, Iran (IR. AJUMS.REC.1398.779), and informed consent was received from all patients. This retrospective study included couples who underwent IUI between July 2012 and January 2017, in which the female partner had confirmed CP. Based on the 6 th edition of the WHO guidelines for semen analysis, male partners were divided into two groups: those with borderline SM and those with pathological SM. Total motile sperm (TMS), defined as ≥50% progressively motile sperm (consistent with fertile men) ( 17 ), was evaluated in both groups. This study investigated the independent effect of SM on pregnancy complications and outcomes. Participants were excluded if they had undergone testicular sperm aspiration (TESA) or microepididymal sperm aspiration (MESA), or if they presented with any of the following conditions: diabetes and thyroid disease, endometriosis, intrauterine adhesions, polycystic ovary syndrome (PCOS), recurrent abortion, autoimmune diseases, infectious diseases, or malignancies. The following variables were considered as covariates: sex, age, duration of infertility, infertility diagnosis, follicle-stimulating hormone (FSH), and anti-Mullerian hormone (AMH). Subsequently, gestational complications (such as diabetes, blood pressure, fetal growth retardation and multiple pregnancy) and pregnancy outcomes were assessed, including live birth rate, abortion rate, heterotopic pregnancy rate, and the frequency of spontaneous abortion stratified by maternal age. Sperm concentration, TMS —as defined by the methodology outlined in the 5th edition of the WHO laboratory manual for the examination and processing of human semen— and SM were evaluated in each sample. Semen smears were prepared by applying 15 µL of semen to slides. SM was then assessed using the Diff-Quick stain kit (Dianzist Azma, Iran) according to the manufacturer’s protocol. SM was assessed by evaluating 200 sperm per slide according to the Kruger strict criteria. SM was evaluated by examining 200 sperm per slide, using the Kruger strict criteria, and performed by a single trained technician to minimize inter-observer variability. SM was categorized as normal (≥14% normal forms), borderline (4-13% normal forms), or pathological (<4% normal forms) ( 17 ). Two groups (borderline SM and path ological SM) based on normal SM were entered into the present study. All samples were prepared using the direct swim-up method for the IUI procedure ( 2 ). This study included participants undergoing ovulation induction using a sequential Letrozole and human menopausal gonadotropin (HMG), depending on the cause of their infertility. On cycle days 12-14, a mid-cycle transvaginal ultrasound was performed. Follicles with a mean diameter ≥20 mm were considered mature, and Ovitrelle (Merck Serono, Germany) was administered when one to three such follicles were observed. The mean follicle diameter and the number of mature follicles were recorded. IUI was performed 36 hours after the trigger. The luteal phase was supported with daily progesterone vaginal suppository administration for 14 days immediately after the IUI procedure, was done. A urine pregnancy test was conducted 14 days post-IUI. Couples with confirmed CP, defined as the presence of fetal cardiac activity on early ultrasound, were enrolled in the study. All statistical analyses were conducted using SPSS version 23.0 (IBM Corp., Armonk, New York, USA). Continuous variables are presented as the mean ± standard deviation (SD), and categorical variables are presented as percentages. Independent samples t-tests were used to compare continuous variables between the borderline and pathological SM groups, while chi-square tests were used for categorical comparisons. The probability of abortion was modelled using simple logistic regression. Statistical significance was defined as a twosided P<0.05. A total of 111 IUI cycles were included in the study. Mean male and female age in two groups of study were not significant (P=0.619, P=0.092), statistically. Similarly, there were no significant differences in the duration of infertility (P=0.409) or the underlying diagnosis of subfertility (P=0.247) between groups. The baseline characteristics are presented in Table 1. Demographics of the women with clinical pregnancy in the two groups undergoing the IUI cycle Data are presented as mean ± SD or %. IUI; Intrauterine insemination, SM; Sperm morphology, FSH; Follicle-stimulating hormone, AMH; Anti-Mullerian hormone, TMS; Total motile sperm, a ; Student’s t test, and b ; Chi-square test were employed to deter mine the statistical significance of differences in variables between the two groups (P<0.05). The frequency of gestational complications, expressed as % (n/total), in the borderline and pathological SM groups was 8.3 and 17.3, respectively, and the difference was not statistically significant (P=0.255). Evaluation of complication types in this study showed diabetes (4%), fetal growth retardation (2.7%), and multiple pregnancy (8%) in the pathological SM group. These findings are detailed in Table 2. Live birth rate was significantly higher in the borderline SM group (91.7%) compared to the pathological SM group (69.3%, P=0.009). Conversely, the abortion rate was significantly lower in the borderline group (2.8%) than in the pathological group (26.7%, P=0.001). Gestational complication, % (n/total) of women with clinical pregnancy in the two groups undergoing IUI cycle IUI; Intrauterine insemination, SM; Sperm morphology, BP; Blood pressure, and FGR; Fetal growth retardation.Chi-square test was employed to determine the statistical sig nificance of differences in variables between the two groups (P<0.05). The incidence of heterotopic pregnancy was 0% in the borderline SM group and 1.3% in the pathological SM group, and this difference was not statistically significant (P=0.676). While the overall abortion rate differed significantly between the two groups, trimester-specific analysis revealed the following: first-trimester abortions occurred in 5.6% of the borderline SM group and 18.7% of the pathological SM group. Second-trimester abortions were 0% and 8% in the borderline and pathological SM groups, respectively. No third-trimester abortions occurred in either group. These data are summarized in Table 3. Logistic regression analysis indicated that a one-unit increase in the morphological variable was associated with decreased odds of abortion. Full regression results are shown in Table 4. Pregnancy outcomes of women with clinical pregnancy in the two groups undergoing an IUI cycle Data are presents as % or n (%). IUI; Intrauterine insemination, SM; Sperm morphology, a ; Student’s t test, b ; Chi-square test were employed to determine the statistical signifi cance of differences in variables between the two groups (P<0.05), and * ; Statistically significant. Simple logistic regression analyses of sperm morphology parameters predicting IUI abortion rate among women with clinical pregnancy IUI; Intrauterine insemination, OR; Odds ratio, and CI; Confidence interval. P<0.05. This retrospective study investigated the relationship between SM and live birth and abortion rates among couples undergoing IUI with a confirmed clinical pregnancy (CP). Significant differences were found between the borderline SM and pathological SM groups (based on WHO 6th edition guidelines) in terms of abortion rate, frequency of age-related abortions (P=0.011), and live birth rate. First-trimester abortion rates were significantly higher in the pathological SM group compared to the borderline group. These findings align with prior research suggesting a strong association between SM parameter and IUI outcomes ( 16 , 18 ). For instance, a meta-analysis conducted in 2017 investigated the correlation between SM and unexplained recurrent spontaneous abortion in two groups (unexplained recurrent spontaneous abortion and control). This meta-analysis demonstrated that abnormal SM is significantly correlated with the unexplained recurrent spontaneous abortion ( 15 ). This meta-analysis confirms the findings of our study, showing that for each unit increase in the morphological variable, the odds of abortion decrease by a factor of 0.6. This analysis can assist clinicians and embryologists in predicting IUI success based on the SM characteristics. A 2001 meta-analysis assessed SM as a marker of male fertility in IUI, reporting significantly improved pregnancy rates when applying a strict morphology threshold of >4%. A risk difference of -0.07 in CPR was observed between patients above and below this threshold ( 16 ). Infertile couples undergoing their first IUI treatment were the subject of a retrospective study in which semen samples were classified according to standard parameters: normal (sperm concentration ≥ 15 million/mL, TMS ≥ 32%, and normal SM ≥ 4%) and abnormal (below these thresholds). While no significant association was found between these classifications and pregnancy rates, normal sperm concentration was observed to be associated with continued pregnancy among those who conceived ( 19 ), which contrasts with the findings of the present study, which reported an association between continuation of the pregnancy and SM parameter. Furthermore, a 2018 meta-analysis demonstrated that IUI achieves comparable pregnancy success rates using either a 4% or 1% SM threshold. This suggests that isolated abnormal SM should not preclude couples from at tempting IUI ( 11 ). A cross-sectional retrospective review of 1,059 cycles categorized patients into three groups based on sperm morphology: normal (≥4%), mild-to-moderate TZS (3%-2%), and severe TZS (≤1%). No statistically significant difference in CPR or early spontaneous miscarriage rate was observed between the three groups ( 20 ). In contrast, in the present study, there was a significant increase in first-trimester abortion in the pathological compared to the borderline group. Additionally, a prospective cohort study concluded that abnormal SM did not significantly affect CPR after IUI ( 8 ). The discrepancy may be due to differences in study design—our study only included participants with confirmed clinical pregnancy, whereas others evaluated all IUI attempts regardless of outcome. Thus, while SM may not influence CPR ( 8 ), it may be associated with pregnancy continuation beyond the early stages. Butcher et al. ( 21 ) found that normal SM and TMS had the highest outcome in the IUI success rates. A large prospective study of 2,231 participants used three prediction models to examine the relationship between TMS and pregnancy. Two models suggested that confounding factors likely explain this relationship, whereas one model suggested a direct effect of TMS ( 22 ). In the present study, there was no difference in TMS between the two groups (borderline: 51.31, pathological: 57.54). The only difference observed was in SM, which indicated to important role of the SM in continued pregnancy. A notable strength of this study is its focus on live birth and spontaneous abortion, whereas most previous studies concentrated solely on CPR. However, the relatively small sample size is a limitation, likely resulting from our inclusion criteria—only couples with confirmed CP and male partners with borderline or pathological SM. In summary, this study demonstrates a significant as sociation between pathological SM and an increased risk of first-trimester abortion. Specifically, for each unit in crease in the morphological parameter, the odds of abortion decreased by approximately 40% (OR=0.6), indicating a protective effect of higher SM values. These findings highlight SM as a potential predictor of IUI outcomes following clinical pregnancy. Therefore, SM should be considered a critical factor in fertility assessments, and alternative treatment strategies may be more appropriate for couples with pathological SM undergoing infertility treatment.