Sperm Morphology of Post-wash Sample and Its Association with Clinical Pregnancy among the Couples Undergoing Intrauterine Insemination: A Cohort Study.

About 15% of couples experience infertility, and in roughly 50% of these cases, the male factor plays a contributing role. More attention emerged on the significance of sperm morphology in male infertility in the 1980s.[ 1 2 3 ] When a series of studies by Kruger et al . revealed a lower oocyte fertilisation rate when sperm morphology fell below 14%. However, it has been advised to examine sperm morphology since the World Health Organization’s (WHO) first edition was published in 1980.[ 4 5 ] All the guidelines for classifying spermatozoa as abnormal have undergone significant modification in the last four decades. As a result, the most comprehensive emphasis on the methodical evaluation of sperm morphology is needed.[ 6 ] Although morphology has been widely accepted as an essential feature of semen analysis and WHO have undergone multiple revisions, medical professionals have not universally supported this test, partly because of its poor analytical reliability and ambiguous predictive significance. Sperm morphological abnormalities may be associated with functional abnormalities such as altered chromatin condensation, acrosome reaction defects, issues with tail motility or even a rise in apoptosis or necrosis occurrences.[ 7 8 9 10 ] Intrauterine insemination (IUI) is a recognised treatment strategy for mild-to-moderate male factor infertility and unexplained infertility.[ 11 ] It is generally attempted before proceeding to more invasive assisted reproductive techniques such as in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI).[ 5 ] Many past studies investigated the effects of teratozoospermia based on previous semen analysis to classify sperm morphology rather than using semen parameters in the post-wash sample used for insemination. This could be a concern because the semen properties in the samples can vary significantly over any given time period. The impact of sperm morphology on the outcome of IUI in the existing literature may have been confounded because the classification of normozoospermia or teratozoospermia was based on the primary semen analysis rather than the actual sample inseminated.[ 12 ] Therefore, the study aimed to assess the sperm morphology of the post-wash inseminated sample and its association with clinical pregnancy rate (CPR) among couples undergoing IUI.

Semen parameters of the inseminated sample were collected from 127 couples who underwent 246 IUI cycles. The baseline characteristics of the entire cohort are listed in Tables 1 and 2 . The median female age was 30.0 (27.0–33.0) years and male age was 33.0 (30.0–35.2) years. Unexplained infertility and ovulatory dysfunction were seen in 29.3% of couples, and male factor infertility was observed in 22% of couples. Ovulation was achieved with letrozole with gonadotropins (73.6%) or with letrozole alone (12.2%). Among 246 IUI cycles, 47.6% had normal post-wash sperm morphology, and 52.4% had teratozoospermia. There was a significant increase in post-wash sperm morphology compared to pre-wash sperm morphology (11.8% had pre-wash normal sperm morphology and 88.2% had teratozoospermia, listed in Table 3 ). The median sperm morphology (%) of the post-wash inseminated sample was 3.0 interquartile range (IQR) (2.0–5.0), and pre-wash sperm morphology was 2.0 IQR (1.0–3.0). The median post-wash TMSC (10⁶ per mL) was 19.5 IQR (9.0–34.1). The median progressive motility (%) was 92 IQR (85.0–95.0). Clinical pregnancy was noted in 10.6% ( n = 26) of couples, of which 6.1% ( n = 15) had post-wash normal sperm morphology and 4.5% ( n = 11) had post-wash teratozoospermia. When categorised by strict morphology ≤1%, 2%–3%, 4%–5% and ≥5%, the CPR was 0.0%, 12.0%, 12.6% and 12.9%, respectively [ Table 4 ]. Association between post-wash TMSC and progressive motility with clinical pregnancy were listed in Table 5 and 6 . Baseline characteristics of the total cohort *Expressed as median (Q1–Q3), # Expressed as n (%). AMH=Anti-Mullerian hormone, TSH=Thyroid-stimulating hormone, TB=Tuberculosis, HMG=Human menopausal gonadotropin, IQR=Interquartile range Comparison of baseline characteristics *Expressed as median (Q1–Q3) and Mann–Whitney U -test is used, # Expressed as n (%) and Chi-square test is used. AMH=Anti-Mullerian hormone, TSH=Thyroid-stimulating hormone Comparison of pre- and post-wash semen parameters *Expressed as median (Q1–Q3) and Mann–Whitney U -test is used, # Expressed as n (%). TMSC: Total motile sperm count Association between post-wash sperm morphology and clinical pregnancy # Expressed as n (%) and Chi-square test is used Association between post-wash TMSC and clinical pregnancy # Expressed as n (%) and Chi-square test is used. TMSC: Total motile sperm count Association between progressive motility and clinical pregnancy *Expressed as median (Q1–Q3) and Mann–Whitney U -test is used

Current study findings suggest that no significant differences in CPR following IUI in couples with post-wash normal and abnormal sperm morphology with a cut of 4%. Hence, the current study findings suggest that sperm morphology should not be a criterion to exclude couples from undergoing IUI. However, future studies with large sample sizes in different populations are needed with different severity of teratozoospermia. PS: Writing – review and editing, writing – original draft, methodology, investigation, formal analysis, data curation and conceptualisation. KNS: Writing – review and editing, supervision, project administration, methodology, investigation and conceptualisation. SM: Data curation, technical help and manuscript editing. JKS: Data curation, technical help and manuscript editing. SV: Writing – review and editing. AR: Writing – review and editing, methodology, results and statistical analysis. There are no conflicts of interest. The data sets used for this study are available with the corresponding author on request.

Among 246 IUI cycles, 47.6% had post-wash normal sperm morphology and 52.4% had post-wash teratozoospermia. There was a significant increase in post-wash sperm morphology compared to pre-wash sperm morphology. Our study evaluating the morphology of post-wash inseminated samples did not find any significant differences in CPR (6.1%, n = 15 vs. 4.5%, n = 11) following IUI in couples with post-wash normal and abnormal sperm morphology. When categorised by strict morphology ≤1%, 2%–3%, 4%–5% and ≥5%, the CPR was 0.0%, 12.0%, 12.6% and 12.9%, respectively. Even though clinical pregnancy was noted only with >1% sperm morphology, there was no significant association between sperm morphology and CPR. Our results were comparable to a similar study conducted by Stanhiser et al. , to determine the effect of sperm morphology from the actual sample used for IUIs on CPR and found no significant association between sperm morphology and CPR.[ 12 ] Our results support the recent meta-analysis and systematic review of 20 observational studies conducted by Kohn et al . that relied on baseline semen analysis morphology, which concluded that sperm morphology is no longer a reliable indicator of the result of IUI, regardless of the severity of teratospermia.[ 14 ] In a retrospective study conducted by Lockwood et al. , on 408 couples undergoing 856 IUI cycles, no significant difference was noted in CPR between isolated abnormal strict morphology (<5% normal forms), very low strict morphology (0%–1% normal forms) compared to normal morphology group which is comparable to our study.[ 15 ] There are contradictory findings in the literature currently available linking sperm morphology and IUI outcomes. However, a study conducted by Van Waart et al . reported a significant improvement in pregnancy rates after IUI when sperm morphology was more than 4%.[ 16 ] Sun et al .[ 17 ] conducted a retrospective study that suggested that for patients with normal sperm concentration and motility, IUI is recommended for first-line treatment when the woman is younger than 35 years or morphologically normal sperm is more than 5%. IVF/ICSI should be performed when the normal forms are 35 years. The median post-wash TMSC in our study was 19.5 million/mL. There is a significant association between post-wash TMSC and clinical pregnancy with increased clinical pregnancy noted in couples with post-wash TMSC ≥10 million/mL. In the present study, unexplained infertility and ovulatory dysfunction contribute to majority of cases of infertile couples (58.6%) undergone IUI followed by male factor infertility (22.0%). There is no significant association between female age and clinical pregnancy following IUI in our study, which is comparable to a similar study conducted by Marzieh Mehrafza et al. ,[ 18 ] in which the mean age of females was 29.3 (range: 20–42) years and the mean age of males was 33.3 (range: 24–54) years. Stanhiser et al. conducted a similar study in which unexplained infertility and male factors contributed to most infertile couples undergoing IUI, followed by ovulatory dysfunction, probably due to racial and geographical differences, varying socioeconomic status and lifestyle habits.[ 12 ] There were four live births and three abortions noted during the study period, but pregnancy outcomes could not be studied for the study population. Our study has several important strengths. To the best of our knowledge, there are few studies evaluating the impact of sperm morphology of the post-wash inseminated sample on the CPR following IUI. All semen analyses included in this study were performed in the same laboratory with established and proven quality control measures. Our study has several limitations. Different ovulation induction and semen preparation methods, long-term outcomes such as miscarriages, congenital anomalies, pregnancy outcomes and live birth rate could not be studied for all the study population.

This prospective cohort study was conducted in the Obstetrics and Gynaecology Department, Women and Children’s Hospital, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), from March 2022 to January 2024. The Departmental Postgraduate Research Monitoring Committee (No. JIP/OBG/PGRMC/JULY 2021) and the Institute Ethics Committee (JIP/IEC/2021/358) approved the study. The study was registered with the Clinical Trials Registry – India (CTRI/2022/09/045920). Couples undergoing IUI with one or both tubes patent (hysterosalpingography/hystero-contrast sonosalpin gography/hysterolaparoscopy with chromopertubation), ovulatory causes, uterine causes of infertility, unexplained infertility, mild-to-moderate male factor infertility were included in the study. Females aged >38 years, donor insemination and Grade 3 and 4 endometriosis were excluded from the study. Our study included a total of 127 couples who underwent 246 IUI cycles. The sample size was estimated to be an expected proportion of subjects with abnormal morphology in our infertility clinic. As 20% with 5% absolute precision and 5% significance level. After written informed consent, all study participants were subjected to detailed history taking by pre-structured data collection pro forma followed by detailed systemic and genital examination along with routine blood investigations, Pap smear, routine health screening as required, complete blood count, thyroid-stimulating hormone, glucose tolerance test, serology (HIV, hepatitis B surface antigen, hepatitis C virus and venereal disease research laboratory), tests for uterotubal evaluation, blood grouping Rh typing and other necessary tests if clinically warranted. Adherence to the Helsinki Declaration (ethical principles for medical research) was ensured throughout the study process. Semen analysis was performed before each IUI. Before the analysis, semen samples collected from the patients were allowed to liquefy for 20–30 min at 37°C. Using a graduated pipette, the ejaculate’s volume and viscosity were measured. Ten microlitres of pre-washed semen samples were put into Makler chamber covered with a cover slip and analysed as per the WHO Sixth Edition Laboratory Manual.[ 13 ] Generally, for sperm washing, the density gradient centrifugation method was preferred for sperm counts 15 million and less with other abnormalities with motility and morphology. For sperm count, more than 15 million with normal motility and morphological parameters, swim up technique was used. Sperm concentration, total motility, progressive motility, morphology (Kruger’s strict morphology criteria) and total motile sperm count (TMSC) were assessed in the sample. Using a phase-contrast microscope set to ×200, the average number of sperm in two sections of the counting grid of a Makler chamber was used to calculate the sperm concentration. By counting ≥200 sperms at more than five locations within the Makler chamber and dividing them into three categories – rapidly progressive, motile non-progressive and non-motile – the fraction of progressively motile sperms was ascertained. About 10 μL post-wash semen sample was smeared on pre-stained morphology slides (Kwik morph) and covered with a cover slip. According to Kruger’s stringent standards, 200 sperms were assessed per slide at ×1000 using an oil immersion objective. The evaluation of 200 sperms was used to compute the per cent of normal forms. Teratozoospermia is defined as less than 4% of normal forms, mild–moderate teratozoospermia (1%–3% normal forms) and severe teratozoospermia less than 1% of normal forms. The same criteria were used for the wash sample. Abnormalities of the head, midpiece and tail were assessed and recorded. Once the sample is processed, 0.4–0.6 mL is loaded in IUI catheter and administered to the patient. Depending on the cause of their infertility, female partners used either their natural cycles or ovulation induction with letrozole, clomiphene citrate, gonadotropins (human menopausal gonadotropin, with or without letrozole/clomiphene citrate) taken daily on cycle day 2–6. On cycle days 12–14, a midcycle transvaginal ultrasound was carried out. IUI was performed when the leading follicle was 18–24 mm the day and approximately 36 h following 5000 IU human choriogonadotropin for ovulation trigger. The lead follicle was 18–24 mm in mean diameter as measured by ultrasound. Cycle cancellation was considered if 3 or more dominant follicles of more than 14 mm to avoid multiple pregnancies and ovarian hyperstimulation syndrome, if the partner was unable to collect the semen sample despite using the vibrator and if the total motile sperm count was less than one million after the semen processing. After IUI, luteal phase support was given to most of the patients with tablet Dydrogesterone 10 mg/Micronised progesterone 200 mg twice daily for 2 weeks; luteal support practice varies among different consultants in natural or ovulation induction cycles. Patients were instructed to check home pregnancy tests in case of missed periods. After positive pregnancy test, a transvaginal ultrasound was performed between 6 and 7 weeks from the last menstrual period to document foetal cardiac activity (clinical pregnancy). The distribution of categorical variables such as parity, comorbidities, indication for IUI, clinical pregnancy, sperm morphology and sperm motility were expressed as frequency and percentages. The quantitative variables, such as age, duration of infertility and sperm parameters, were expressed as mean with standard deviation or median with range. The association of sperm morphology with clinical pregnancy outcome was done using the Chi-square test or Fisher’s exact test. The comparison of quantitative variables, such as age and sperm parameters, concerning the clinical pregnancy outcome was made using the independent Student’s t -test or Mann–Whitney U test. All statistical analyses were carried out at a 5% significance level, and the P value was calculated.