Optimizing post‐wash TPMSC for intrauterine insemination: A key strategy to improve pregnancy outcomes

Abstract

Objective

To investigate the impact of both pre‐ and post‐ovulation post‐wash total progressive motile sperm count (TPMSC) on pregnancy outcomes in intrauterine insemination (IUI) using husband's sperm (AIH).

Methods

This retrospective cohort study analyzed data from infertile couples who underwent IUI treatment at the Reproductive Medicine Center of Xinxiang Central Hospital from January 2020 to June 2024. A total of 462 IUI cycles were included, all of which had consistent pre‐ and post‐ovulation post‐wash TPMSC values. Cycles were divided into four groups based on TPMSC: Group A (<10 × 10⁶), Group B (10–20 × 10⁶), Group C (20–30 × 10⁶), and Group D (≥ 30 × 10⁶). Comparisons were made regarding baseline data and pregnancy outcomes among the groups. Multivariate logistic regression was employed to assess the impact of both pre‐ and post‐ovulation post‐wash TPMSC on clinical pregnancy rates.

Results

The clinical pregnancy rates differed significantly among TPMSC groups (P = 0.024), with the highest rate in the 10–20 × 10⁶ group (26.13%). Multivariate logistic regression identified this range as an independent predictor of clinical pregnancy (odds ratio [OR] = 1.922, 95% confidence interval [CI]: 1.026–3.600, P = 0.041).

Conclusion

Post‐wash TPMSC influences clinical pregnancy rates in IUI. When both pre‐ and post‐ovulation post‐wash TPMSC are within the range of 10–20 × 10⁶, this may optimize clinical pregnancy outcomes.

1. INTRODUCTION

Intrauterine insemination (IUI) is a first‐line treatment for infertility, commonly applied in cases of mild male factor infertility, anovulation, endometriosis, and unexplained infertility. It is widely utilized in clinical practice due to its simplicity and non‐invasiveness. ¹

Several factors influence IUI success rates, with sperm quality being paramount. It is generally accepted that achieving a satisfactory clinical pregnancy rate requires the post‐wash total progressive motile sperm count (TPMSC) to meet a certain quantitative standard. Nevertheless, the precise impact of post‐wash TPMSC on IUI outcomes remain controversial, and consensus has yet to be reached. Recent studies reflect this ongoing debate; for instance, one retrospective analysis indicates post‐wash TPMSC as the most significant male‐factor predictor of pregnancy rates in IUI cycles. ²

Since IUI only assists sperm in bypassing the cervical barrier, their subsequent migration to the fallopian tubes remains entirely dependent on sperm motility. As a direct measure of forward motility capacity, post‐wash TPMSC serves as a key prognostic factor. However, reported thresholds for post‐wash TPMSC associated with IUI success vary considerably across studies and reproductive centers. Most scholars suggest that a post‐wash TPMSC greater than 3 × 10⁶ is necessary to achieve pregnancy, ³ , ⁴ while some literature indicates that optimal pregnancy rates are achieved only when it exceeds 5 × 10⁶. ⁵ Others argue that a threshold of 10 × 10⁶ is required for more favorable outcomes. ⁶ This threshold is supported by the “Guidelines for Human Assisted Reproductive Technology”, which also recommends an ideal post‐wash TPMSC of 10 × 10⁶ for insemination. Wainer et al. ⁷ demonstrated a significant decrease in pregnancy rates when post‐wash TPMSC fell below 1 × 10⁶ compared to counts above 2 × 10⁶. Using multiple regression analysis, Miller et al. ⁶ found that post‐wash TPMSC was independently associated with pregnancy, with significantly higher counts observed in the pregnant group versus the non‐pregnant group. Because post‐wash TPMSC reflects not only sperm concentration and vitality but also the capacity to reach and fertilize the oocyte, it has been utilized in other studies as a clinically valuable predictive tool. ⁸ , ⁹

This retrospective study analyzed 462 IUI cycles from couples undergoing infertility treatment at our reproductive medicine center. All included cycles had pre‐ and post‐ovulation post‐wash TPMSC values within the same predefined range. The objective was to assess whether TPMSC levels influence IUI pregnancy outcomes and provide evidence‐based guidance for clinical practice.

Analysis of IUI cycle data from our center identified both pre‐ and post‐ovulation post‐wash TPMSC as significant factors affecting pregnancy outcomes. This finding was confirmed by multivariate logistic regression, which established both measures as independent predictors of clinical pregnancy. Consequently, this study provides a detailed investigation of the relationship between pre‐ and post‐ovulation TPMSC and pregnancy success.

2. MATERIALS AND METHODS

2.1. Study design

The present study was a retrospective cohort study conducted at the Center for Reproductive Medicine of Xinxiang Central Hospital. The data collection period was from January 2020 to June 2024, and the study subjects were infertile couples treated with IUI in the center. A retrospective analysis was conducted on 462 IUI cycles in which the patients' pre‐ and post‐ovulation post‐wash TPMSC fell within the same predefined range. Inclusion criteria were: Couples who met the standard indications for IUI and completed all required pre‐treatment examinations. Exclusion criteria: (1) Treatment cycles that were canceled and (2) cycles with incomplete data that precluded statistical analysis.

2.2. Ethical review and informed consent

This study was conducted in accordance with the principles of the Declaration of Helsinki. All participating couples met the indications for artificial insemination with no contraindications, as stipulated in the “Regulations on Human Assisted Reproductive Technology” issued by the Ministry of Health. The study protocol was approved by the hospital's Ethics Committee (approval no.: XYLL‐2021176). As this was a retrospective analysis based on routine clinical data, informed consent was obtained from all couples for the use of their anonymized diagnostic and treatment data in scientific research prior to data collection and analysis. All procedures complied with relevant guidelines and regulations.

2.3. Grouping criteria

Given the retrospective observational nature of this study, no randomization or blinding was implemented, and grouping was based solely on objective TPMSC measurements. Reported thresholds for TPMSC associated with successful pregnancy in IUI vary widely, ranging from 0.15 × 10⁶–60 × 10⁶. ¹⁰ , ¹¹ , ¹² In clinical practice, TPMSC <10 × 10⁶ often prompts recommendation for in vitro fertilization (IVF), while intracytoplasmic sperm injection (ICSI) is indicated for even lower values. ¹³ Based on these controversial thresholds and guideline recommendations, cycles were divided into four groups according to both pre‐ and post‐ovulation post‐wash TPMSC values. Only cycles in which both measurements fell within the same group were included. Detailed grouping is presented in Table 1.

TABLE 1.

Grouping information.

Group	TPMSC count (×106)
A	<10
B	10–20
C	20–30
D	≥30

Abbreviation: TPMSC, total progressive motile sperm count.

2.4. Preparation for IUI treatment

Prior to treatment, all patients underwent comprehensive health screenings to rule out contraindications to pregnancy. Participating couples were required to provide valid identification and marriage certificates, as well as informed consent for both the IUI procedure itself and the potential need for multifetal pregnancy reduction.

2.5. Natural cycle and ovarian stimulation protocol

Based on the underlying causes of infertility, the clinical team determined whether to use a natural or stimulated cycle for IUI. Natural cycles were prioritized for patients with confirmed spontaneous ovulation. In cases where natural cycles were unsuccessful or when patients presented with issues such as abnormal follicular development, ovulatory disorders, or irregular menstruation, a stimulated cycle protocol was employed. In such cycles, the number of dominant follicles was controlled at three or fewer. Follicular growth was monitored by vaginal ultrasound. When the leading follicle reached a mean diameter of ≥18 mm, in conjunction with a positive serum or urine luteinizing hormone (LH) test, 5000–10 000 U of human chorionic gonadotropin (hCG) was administered. The IUI procedure was performed 24–36 h later.

2.6. Semen preparation

On the day of insemination, semen samples were collected via masturbation by the male partner after 2–7 days of abstinence, following WHO‐standards. If necessary, repeated washing at room temperature was performed to facilitate liquefaction. Liquefied samples were processed using density‐gradient centrifugation. Briefly, SpermGrad gradient solution (Vitrolife Sweden AB) was pre‐equilibrated in an incubator. A 5‐mL test tube was prepared by layering 0.6 mL of 45% gradient solution over an equal volume of 90% solution, taking care to maintain a distinct interface. Liquefied semen (3 mL) was gently layered on top and centrifuged at 300 × g for 15 min. The supernatant was discarded, and the sperm pellet was collected, resuspended in wash medium, and centrifuged twice at 200 × g for 5 min. The final pellet was resuspended in culture medium for insemination. Sperm concentration and motility were assessed using a MAKLER counting chamber before and after processing, with at least 200 sperm counted per sample to determine TPMSC and recovery rate.

2.7. Insemination

The patient was placed in the lithotomy position. The perineum and vagina were irrigated with preheated normal saline, and cervical mucus was gently removed. Approximately 0.5 mL of the prepared motile sperm suspension was slowly injected into the uterine cavity using a disposable insemination catheter. The catheter was held in place for 3–5 s before it was carefully withdrawn. Following the procedure, the patient was advised to elevate her hips and remain supine for 20 min.

2.8. Reproductive outcomes

Following the final IUI procedure, patients received a regimen of oral dydrogesterone (Duphaston, Abbott Biologicals B.V., Netherlands, 10 mg/tablet) at a dosage of one tablet three times daily. hCG levels were measured 14 days post‐IUI; a value >5 mIU/mL indicated biochemical pregnancy. Transvaginal ultrasound was performed 28 days after insemination, and the presence of one or more gestational sacs, or other definitive clinical signs, confirmed clinical pregnancy. Miscarriage occurring within the first 12 weeks of gestation was recorded as early pregnancy loss.

2.9. Statistical analysis

Data normality was assessed using the Kolmogorov–Smirnov test. Categorical variables were compared using the Chi‐square test (χ ²‐test) or Fisher exact test, as appropriate. Continuous variables with normal distribution were analyzed using one‐way analysis of variance (ANOVA), and post hoc multiple comparisons were performed using the Tukey HSD correction. Non‐normally distributed data were analyzed using the Kruskal‐Wallis test. Binary logistic regression was performed to identify factors associated with clinical pregnancy. A P value of less than 0.05 was considered statistically significant. IBM SPSS Statistics version 27 was used to analyze data.

3. RESULTS

3.1. Statistical analysis of data for different post‐wash TPMSC groups in IUI cycles

This study included a total of 462 IUI cycles that met the inclusion criteria, all of which had pre‐ and post‐ovulation post‐wash TPMSC values within the same predefined range. Among these, biochemical pregnancy was achieved in 100 cycles, and clinical pregnancy was confirmed in 93 cycles, resulting in rates of 21.65% (100/462) and 20.13% (93/462), respectively. As shown in Table 2, no significant differences were observed in female age, body mass index (BMI, calculated as weight in kilograms divided by the square of height in meters), and infertility duration among the four TPMSC groups (all P > 0.05). However, other baseline characteristics and outcomes varied significantly. Specifically, the biochemical pregnancy rate showed significant overall differences among groups (F = 3.411, P = 0.017), but post hoc Tukey HSD tests revealed no significant pairwise comparisons (all P > 0.05). The clinical pregnancy rate also differed significantly overall (F = 3.188, P = 0.024), with a near‐significant difference between Group B and Group C (P = 0.051); no other pairwise differences were significant. Group B exhibited the highest clinical pregnancy rate (26.13%), while Group C had the lowest (11.59%). For detailed data, please refer to Table 2.

TABLE 2.

Comparison of baseline data and pregnancy outcomes in IUI cycles according to post‐wash TPMSC.

Item	Group A	Group B	Group C	Group D	F/χ 2 value	P value
No. of cases	68	222	69	103
Female age (year)	31.56 ± 4.21	31.21 ± 4.87	32.44 ± 4.86	30.75 ± 4.83	1.846	0.138
Female BMI	23.80 ± 4.11	24.60 ± 4.65	23.83 ± 4.29	23.85 ± 3.81	1.204	0.308
Infertility duration (year)	3.33 ± 2.09	2.92 ± 2.28	2.67 ± 2.18	2.90 ± 2.51	0.992	0.396
Infertility type (%)					0.393	0.758
Primary infertility	60.29 (41/68)	53.60 (119/222)	53.62 (37/69)	52.43 (54/103)
Secondary infertility	39.71 (27/68)	46.40 (103/222)	46.38 (32/69)	47.57 (49/103)
Type of cycles (%)					3.164	0.024
Natural ovulation	51.47 (35/68)	32.43 (72/222)	30.44 (21/69)	33.98 (35/103)
Induced ovulation	48.53 (33/68)b	67.57 (150/222)a	69.56 (48/69)a	66.02 (68/103)ab
Biochemical pregnancy rate (%)	16.18 (11/68)	27.48 (61/222)	14.49 (10/69)	17.48 (18/103)	3.411	0.017
Clinical pregnancy rate (%)	16.18 (11/68)	26.13 (58/222)	11.59 (8/69)	15.53 (16/103)	3.188	0.024
Early spontaneous abortion rate (%)	27.27 (3/11)	27.59 (16/58)	37.50 (3/8)	25.00 (4/16)	0.675	0.567
Live birth rate (%)	11.76 (8/68)	17.12 (38/222)	7.25 (5/69)	10.68 (11/103)	1.907	0.128

Note: BMI, calculated as weight in kilograms divided by the square of height in meters. IUI represents positive number/total number in the brackets; Group A represents TPMSC <10 × 10⁶; Group B represents TPMSC 10–20 × 10⁶; Group C represents TPMSC 20–30 × 10⁶; Group D represents TPMSC ≥30 × 10⁶. Means within the same row followed by the same lowercase letter are not significantly different (P > 0.05), while different lowercase letters indicate significant differences (P < 0.05) based on Tukey's honestly significant difference (HSD) test.

Abbreviations: BMI, body mass index; IUI, intrauterine insemination; TPMSC, total progressive motile sperm count.

3.2. Multivariate logistic regression analysis of pregnancy outcomes with different post‐wash TPMSC in IUI cycles

Multivariate logistic regression analysis, after adjusting for female age, BMI, infertility type, and cycle type, revealed that infertility duration was an independent negative predictor of clinical pregnancy (OR = 0.842, 95% CI: 0.730–0.971, P = 0.018), indicating that a longer duration of infertility was associated with a lower probability of pregnancy. Regarding TPMSC, using ≥30 × 10⁶ as the reference, only patients with TPMSC in the range of 10–20 × 10⁶ showed a significantly increased odds of clinical pregnancy (OR = 1.922, 95% CI: 1.026–3.600, P = 0.041). In addition, female age, BMI, infertility type, and cycle type did not reach statistical significance (all P > 0.05). Detailed results are presented in Table 3.

TABLE 3.

Binary logistic regression of characteristics associated with clinical pregnancy.

Item	B	SE	Wald	df	P value	OR	95% CI
Female age (year)	−0.051	0.029	3.039	1	0.081	0.950	0.898–1.006
Female BMI	0.053	0.027	3.690	1	0.055	1.054	0.999–1.112
Infertility duration (year)	−0.172	0.073	5.576	1	0.018	0.842	0.730–0.971
Infertility type (%)
Primary infertility						1.00	1.00
Secondary infertility	0.213	0.253	0.711	1	0.399	1.238	0.754–2.032
Type of cycles (%)
Natural ovulation						1.00	1.00
Induced ovulation	0.321	0.281	1.309	1	0.253	1.379	0.795–2.391
Post‐wash TPMSC of twice‐before and after ovulation
<10 × 106	0.247	0.438	0.318	1	0.573	1.281	0.542–3.024
10–20 × 106	0.653	0.320	4.166	1	0.041	1.922	1.026–3.600
20–30 × 106	−0.302	0.475	0.404	1	0.525	0.740	0.292–1.875
≥30 × 106						1.00	1.00

Note: BMI, calculated as weight in kilograms divided by the square of height in meters.

Abbreviations: BMI, body mass index; CI, confidence interval; df, degrees of freedom; OR, odds ratio; SE, standard error; TPMSC, total progressive motile sperm count.

4. DISCUSSION

Intrauterine insemination (IUI) is a commonly used assisted reproductive technology that involves the transcervical insertion of optimally processed, high‐motility sperm into the uterine cavity during the female partner's ovulation period. Although IUI has been widely applied in infertility treatment, its success rate is influenced by multiple factors, resulting in significant variability in pregnancy outcomes. Previous studies have indicated that many elements can affect the success of IUI. ¹⁴ While the “Technical Specifications for Human Assisted Reproduction” recommends post‐wash total progressive motile sperm count (TPMSC) >10.0 × 10⁶ for insemination, evidence regarding optimal thresholds remains inconsistent. Some studies support that a TPMSC >10 × 10⁶ yields ideal clinical outcomes, ¹⁵ whereas others reported a TPMSC >2 × 10⁶ may be sufficient for achieving favorable pregnancy rates. ¹⁶

This study investigated the impact of both pre‐ and post‐ovulation post‐wash TPMSC on pregnancy outcomes in IUI cycles. After adjusting for confounders such as female age, BMI, and duration of infertility, multivariate logistic regression analysis identified pre‐ and post‐ovulation TPMSC as a primary independent factor influencing pregnancy outcomes. Our data demonstrate that a post‐wash TPMSC in the range of 10–20 × 10⁶ was associated with more favorable clinical pregnancy and live birth rates, whereas values either below 10 × 10⁶ or above 20 × 10⁶ were linked to lower clinical pregnancy rates. Notably, when post‐wash TPMSC exceeded 20 × 10⁶, clinical pregnancy rates did not further increase with rising sperm numbers, suggesting a non‐linear relationship between sperm count and pregnancy outcome. This finding implies the existence of an optimal sperm concentration range, beyond which merely increasing sperm number may not further improve outcomes—a trend supported by previous literature. ¹¹

The underlying reasons for this phenomenon may be multifactorial. Studies have shown that in addition to total sperm count, other semen quality parameters—such as pre‐wash sperm concentration, post‐wash progressive motile sperm concentration, sperm recovery rate, normal morphology rate, and sperm DNA fragmentation index‐also significantly influence IUI pregnancy success. ¹⁷ , ¹⁸ These factors may interact when sperm concentration is excessively high, collectively affecting the final fertilization outcome. Therefore, when evaluating the likelihood of IUI success, multiple semen quality indicators should be considered comprehensively rather than focusing solely on total sperm count.

Currently, the most commonly used sperm processing methods are density gradient centrifugation and the swim‐up technique. In this study, all IUI cycles employed density gradient centrifugation to isolate motile sperm. While this method has been shown to yield significantly higher sperm recovery rates compared to the swim‐up technique, it has also been associated with a slightly increased risk of early miscarriage. It is hypothesized that the centrifugal force may induce mechanical and physical damage to sperm, compromising their morphology and DNA integrity. Previous studies have reported that the sperm DNA fragmentation index (DFI) is significantly negatively correlated with fertilization, embryo quality, and implantation rates, ¹⁹ , ²⁰ , ²¹ and is also linked to poor preimplantation embryo development, increased early miscarriage, and even long‐term fetal growth concerns. ²² , ²³ , ²⁴

Based on our finding that the 10–20 × 10⁶ range represents an optimal sperm concentration for successful fertilization, we propose a practical clinical strategy: for patients with a post‐wash TPMSC ≥20 × 10⁶, outcomes may be improved by diluting the sample to this optimal range. Additionally, for samples with high sperm concentration, the swim‐up technique could be adopted in future cycles, aiming to achieve a post‐wash TPMSC within the optimal 10–20 × 10⁶. This strategy is anticipated to improve clinical pregnancy rates while reducing the risk of early miscarriage. Our center will prospectively document these clinical data to facilitate further research.

4.1. Limitations

This study has several limitations. Its retrospective design introduces inherent biases and potential residual confounding, despite adjustment for known factors. The small sample size in the subgroups with TPMSC <10 × 10⁶ (n = 68) and ≥ 30 × 10⁶ (n = 69) limits generalizability, and the single‐center setting among couples with unexplained infertility restricts applicability to broader populations. Furthermore, the number of IUI cycles per patient was not considered, which may influence pregnancy outcomes. Additionally, unmeasured factors such as hormonal profiles, lifestyle, and genetic variables may have influenced the results. Therefore, future prospective, multicenter studies with larger sample sizes, appropriate adjustment for repeated cycles, and inclusion of direct biomarkers are warranted to validate these findings and elucidate the underlying biological mechanisms.

5. CONCLUSION

This study concluded that consistent pre‐ and post‐ovulation post‐wash TPMSC values are a primary factor influencing pregnancy outcomes in IUI cycles. The clinical pregnancy rate is closely related to the TPMSC introduced into the uterine cavity, highlighting its significant value in IUI treatment.

Furthermore, patients with both pre‐ and post‐ovulation post‐wash TPMSC values within the range of 10–20 × 10⁶ achieved relatively higher clinical pregnancy and live birth rates. These findings indicate that pre‐ and post‐ovulation post‐wash TPMSC can independently predict the clinical pregnancy outcomes of IUI cycles.

AUTHOR CONTRIBUTIONS

Z. Li: Project development and data collection, S. Ma and Y. Hai: Data analysis and manuscript writing. R. Liang, Y. Fu, N. Feng and J. Hai: Data management and manuscript editing. All authors contributed to the study conception and design. The first draft of the manuscript was written by Z. Li and S. Ma. Material preparation, data collection and analysis were performed by Y. Hai, R. Liang, Y. Fu, N. Feng and J. Hai. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

CONFLICT OF INTEREST STATEMENT

No conflict of interest has been declared by the authors.

DATA AVAILABILITY STATEMENT

The datasets generated and/or analyzed during the current study are not publicly available due to patient privacy concerns but are available from the corresponding author on reasonable request.