The Interval Between Insemination and Ovulation Predicts Outcome After Intrauterine Insemination With Donor Sperm (IUI-D)

Objective To identify whether the time interval from insemination to ovulation (I-O interval) affects outcome after intrauterine insemination with donor sperm (IUI-D). Multiple predictors were identied for LBR. The I-O interval was the predictor for LBR. An I-O interval ≥ 19 hours signicantly decreased CPR (odds ratio [OR], 95% condence interval [CI] =0.285, 0.171-0.475) and LBR (OR, 95%CI =0.322, 0.189-0.549). The presence of at least two follicles ≥ 18mm on ovulation day signicantly increased the LBR (OR, 95%CI =1.274, 1.012-1.602). Women aged 35 years and older had a signicant decreased LBR (OR, 95% CI =0.607, 0.377-0.976). The I-O interval, a new prognostic factor, combination with the women’s age and number of mature follicle, can predict the outcome after IUI-D. IUI-D is best performed within 19 hours of I-O interval for a higher probability of clinical pregnancy and live birth.


Introduction
Infertility is becoming a serious health problem in developed countries. It is estimated that about 8%-32% of all married couples of reproductive age suffer from infertility and sterility worldwide [1,2]. With respect to males, approximately 5%-10% of infertility is due to azoospermia [3]. Intrauterine insemination with donor sperm (IUI-D), is mainly used for couples who have severe semen deficiencies or azoospermia, lesbian couples and single women [4]. For these women, IUI-D could be a better choice because of its simplicity, low cost and fewer complications compared to other assistant reproductive technologies (ARTs) and has been widely accepted as a rst-line treatment for achieving pregnancy [5] .
The timing of insemination is a key factor affecting the outcome after IUI-D [18]. A proper interval from insemination to ovulation (I-O interval) may increase the chance of pregnancy [19]. Several studies have investigated the effects of different insemination time on the pregnancy rate (PR) but have reported inconsistent conclusions [20][21][22]. According to the newest global recommendation, a single IUI for an ovarian stimulation cycle can be performed any time between 24-40h after HCG injection and the IUI for a natural cycle should be performed 1day after the increase in LH [23]. However, these studies did not include an important covariate, the ovulation time, which is key to determining IUI timing and can potentially affect the IUI outcome. The error in the predicted ovulation time could be a confounding factor affecting the clinical outcome after IUI-D. Moreover, in the IUI-D cycle, the processed spermatozoa are injected directly into uterus cavity, which means that the time for successful pregnancy in the IUI-D cycle is shorter than that for natural conception [24] . It is plausible that the insemination time should be scheduled as close as possible to ovulation to obtain a higher PR. In the present study, we retrospectively analyzed 2,091IUI-D cycles from 1,165 couples and used the relative exact ovulation time to calculate the I-O interval to determine whether short I-O interval would contribute to better IUI-D outcomes.

Patient and study design
We retrospectively analyzed the IUI-D cycle performed in the reproductive medicine center of Northwest Women's And Children's Hospital, China, from January 2014 to December 2016. The data were collected from the medical records of couples. The study protocol was approved by the Ethics Committee for the Clinical Application of Human Assisted Reproductive Technology of Northwest Women's and Children's Hospital.
In our center, IUI-D is performed for male factor infertility (azoospermia or severe oligospermia). Before treatment, all women underwent a detailed history and physical examination. All women had a salpingography to con rm at least single tubal patency. Both the natural cycle and stimulated cycle were included. The recorded parameters were mainly related to the woman, including age, duration of infertility, pregnancy history, number of attempts, tubal patency, Cos protocol, endometrium thickness and types, number and diameter of mature follicles on the day of insemination, I-O interval and donor semen quality.

IUI-D Procedure and I-O Interval Evaluation
Transvaginal ultrasound and serum LH and E 2 tests were performed to monitor ovulation. For natural cycles, the ultrasound check started on the eighth day of the cycle. For ovulation stimulation cycles, the test started on the fth day of the cycle. The COS cycle was stimulated by clomiphene citrate, gonadotropins or clomiphene citrate plus gonadotropins. The initial dose was 50 mg/day for clomiphene citrate (CC) (days 5-9) or 75 IU/day for gonadotropins and was modi ed according to the ovarian response. The ovulation trigger was given with an injection of 10,000 IU HCG when the follicle was ≥18 mm but the serum LH was <35 IU/L. An ultrasound check was carried out between 8:00 a.m. and 9:00 a.m. every one to ve days depending on the growth speed of the follicle. When the leading follicle was larger than 14 mm, the patients started the test for urinary LH; if the test was positive or the leading follicle was larger than 18 mm, serum estrogen (E 2 ) and LH were quanti ed (except for those who refused blood tests). If the serum LH was ≥35 IU/L (de ned as a spontaneous LH rise), E 2 and LH were retested three hours later. If the serum LH was <35 IU/L, we continued to test serum LH and E2 the next day for natural cycles, but for stimulating cycles, we administered an HCG injection when the follicle was ≥18 mm. Meanwhile, we increased the frequency of the ultrasound test ( Figure 1).
According to the change in E 2 and LH, the HCG injection time and the ultrasound results, the insemination was arranged at 4:00 p.m. or 9:00 a.m. Thus, the I-O interval could be identi ed according to multiple ultrasound checks and the time of insemination (Table 1). In total, there were three types of I-O intervals: ≥ +19 h, -1 h ~ +19 h, and -19 h ~ -1 h (a minus value means that sperm was delivered after ovulation; Table 1).

Processing of Donor Sperm
The donor sperm samples were supplied by Shaanxi Province Human Sperm Bank and guaranteed by the National Health and Family Planning Commission (NHFPC) of the People's Republic of China. Sperm donors were screened strictly in accordance with NHFPC standards. Generally, eligible sperm had a minimum concentration of 60 × 10 6 per ml, progressive motility of 60%, and normal morphology of 4%. A proper match between patients and donors in racial and ethnic features, as well as blood type, were guaranteed. Before IUI, the frozen sperm sample was thawed fully and then centrifuged at 300g for 20 minutes, using a two-step discontinuous density gradient in a 45% and 90% Pure Sperm-100 platform.
The semen samples were examined after thawing as well as after optimization according to the WHO standard [25]. The volume of washed sperm sample used for insemination was 0.5 mL.

Intrauterine Insemination and Luteal Phase Support
Insemination was performed by one of our center's gynecologists. The prepared sperm was gently inserted within 1 cm of the fundal extend of the uterine cavity using a soft catheter. The patient then rested for 10-15 minutes in a supine position. Daily treatment with 200 mg micronized progesterone or 20 mg dydrogesterone was used for 15 days after IUI-D. Some patients with a history of recurrent spontaneous abortion received an injection of 2000 IU human chorionic gonadotropin three times (every three days).

Diagnosis of pregnancy
The serum β-hCG concentration was quanti ed approximately 16 days after insemination. For women who had positive serum β-hCG, ultrasound con rmation of pregnancy was carried out two weeks later. A clinical pregnancy was de ned after sonographic evidence of the gestational sac was observed. Live birth was de ned as a live-born delivery at least 28 weeks after IUI-D.

Statistical Analysis
The observations of any variables were not completely independent of each other when all IUI-D cycles were included. Consequently, classical statistical analyses with the assumption that samples are independent could not be used for the entire data from all cycles. In this case, generalized estimating equations (GEEs) that allow analysis of correlated observations [26] were used to evaluate the effects of these variables on IUI outcome, as in previous studies [8,14]. The outcome measure response variable was whether pregnancy existed per cycle. The outcome measure used as a response variable was whether pregnancy existed per cycle. For independent samples, two-group comparisons were performed by two-tailed Student's t-test or Mann-Whitney U tests for continuous variables (expressed as the means ± standard deviations (SDs)) or by the χ 2 test for categorical variables (expressed as frequencies and percentages). Moreover, stepwise multivariate logistic analysis was used to construct a predictive model for the pregnancy rate in independent samples. The initial analysis included the variables shown in Tables 2-5. Variables were removed stepwise when the Wald test P-value for a given variable was over 0.05. Only statistically significant variables were included in the final model. All analyses were performed using the software Statistical Package Social Science (SPSS) 22.0. For all statistical tests, P < 0.05 de ned statistical significance.

Results
A total of 2,091 IUI-D cycles from 1,165 patients were included in our analysis, comprising 909 natural, 860 gonadotropin-induced, and 322 clomiphene citrate plus gonadotropin-induced cycles. Patients underwent an average of 3.50 years of infertility (SD: 2.7) and 1.8 (SD: 0.9) treatment cycles. The mean age of the women at treatment was 27.8 years (SD: 3.8). A total of 687 pregnancies occurred among 2,091 treatment cycles, with 592 live-birth deliveries, which represents a 32.9% pregnancy rate and a 28.3% live birth rate per cycle. In addition, 648 patients were pregnant, and 578 patients had live births after IUI-D among 1,165 patients; of the patients who became pregnant, 610 had become pregnant once, 37 became pregnant twice and 1 became pregnant three times during our research period. The characteristics of the included patients are summarized in Table 2.

Univariate Analysis
Continuous variable comparison between the cycles yielding positive and negative outcomes for pregnancy The continuous variables were compared between cycles yielding positive and negative outcomes for pregnancy ( Table 2). The mean age of the women was signi cantly lower in cycles resulting in live birth (P = 0.01). None of the other selected factors (Table 2) signi cantly varied between the cycles with or without clinical pregnancy (P > 0.05).
Univariate analysis of categorical factors related to patient demographic and clinical features We further strati ed the patients by demographic and clinical features and then compared the clinical pregnancy rate (CPR) and live birth rate (LBR) among the different patient strati cations ( Table 3 to Table  5). The results indicated that an interval of ≥ +19 h was associated with a signi cantly decreased CPR and LBR relative to -19 h ~ -1 h (13.0%, 11.6% vs. 34.7%, 29.4%; P <0.01 and <0.01) and -1 h ~ +19 h intervals (13.0%, 11.6% vs. 34.1%, 29.7%; P <0.01 and <0.01). Moreover, we found that the cycles carried out in patients ≥ 35 years old yielded a lower CPR and LBR relative to those in patients who were younger (23.9%, 19.7% vs. 33.4%, 28.8%; P = 0.04 and 0.03). Patients who had only one mature follicle had a lower CPR and LBR than those who had two or more mature follicles (31.7%, 27.2% vs. 37.3%, 32.8%). There were no signi cant associations of CPR and LBR with the other categorical variables included in Table 3 -Table 5.
Multiple GEEs analysis and model building A predictive model for the clinical pregnancy rate and live birth rate was created by GEE analysis based on all 2,091 IUI-D cycles with predictor variables with P < 0.05, as shown in Tables 3-5. The CPR was only associated with the I-O time interval. The LBR was associated with not only the I-O interval but also the patient's age and the number of mature follicles. Compared with the -19 h ~ -1 h interval, the ≥ +19 h I-O interval resulted in a signi cantly decreased CPR (OR 0.29; 95% CI 0.17-0.48; P< 0.01) and LBR (OR 0.32; 95% CI 0.19-0.55; P< 0.01). No signi cant difference was observed between the -19 h ~ -1 h interval and the -1 h ~ +19 h interval. We found a signi cant decrease in the LBR as age increased beyond 35 years (OR 0.61; 95% CI 0.38-0.98; P=0.04), as well as a signi cant increase in women with two or more mature follicles (OR 1.27; 95% CI 1.01-1.60; P=0.04). No signi cant difference was observed in the CPR between women who were younger or older than 35 years or for women with different numbers of mature follicles.

Discussion
In the present study, we correlated the live birth rate with a series of demographic and cycle-speci c factors in 2,091 IUI-D cycles from 1,165 infertile couples. To the best of our knowledge, this was the rst study to investigate the effect of I-O interval on the PR of IUI-D based on the ovation prediction by ultrasound combined with the trend of LH and E 2 regulation or HCG injection time. The overall live birth rate of 28.3% per cycle and the clinical pregnancy rate of 32.9% per cycle are comparable to previous data reported in China [27]. These results enable us to highlight some female prognostic factors for LBR.
Our data showed that the I-O interval, combined with the woman's age and number of mature follicles, can predict the LBR per cycle. The odds of having a live birth signi cantly decreased when the I-O interval was ≥19 h. Detecting spontaneous LH rise is another widely accepted way to schedule IUI-D insemination. However, this method still has some limitations. The urinary LH kit used by most studies has been reported to have a relatively high false-negative rate, which could cause high cancellations and inappropriate insemination time [19]. Only one prospective study [24] used blood samples to detect spontaneous LH rise in the IUI cycle, but the limitation of this study is its neglect of serum E 2 levels. Moreover, too much blood collection for serum LH measurement also decreases its clinical application.
The classic theory of ovulation indicates that the rise in estrogen levels during the late proliferative phase triggers the pre-ovulatory LH surge, which in turn is followed by ovulation [33]. In natural cycle IVF treatment, when the LH surge reaches a peak or a descending slope with a decreasing E 2 , oocyte retrieval is scheduled the next morning or on the same day to achieve the highest oocyte retrieval rate [34].
According to this theory, we modi ed our IUI procedure. Urinary LH was tested for follicles ≥ 14 mm until it was positive or for follicles ≥ 18 mm, and then serum LH and E 2 quanti cation was performed. When LH was ≥ 35 IU/L, we retested LH and E 2 . Based on the regulation trend of LH and E 2 or HCG injection time, insemination was scheduled. This method could minimize the frequency of blood sampling and detect premature spontaneous LH rise as early as possible. Sequential transvaginal ultrasound checks were carried out to monitor ovulation for up to three days, which allowed us to obtain a relatively more exact I-O interval. Using this protocol, we can obtain a better outcome, indicating that this method could be used in scheduling exact and better insemination times in the IUI-D cycle.
As spermatozoa have a longer survival period in the uterus than the ovulated oocyte [35], it is reasonable that the probability of conception increases when spermatozoa are available in the reproductive tract before ovulation occurs; in other words, spermatozoa wait for oocyte [36]. Interestingly, our data indicate that there is an equal probability of conception when the interval length between insemination and ovulation is ≤19 h, regardless of which one occurred rst, suggesting that such an interval is long enough for both spermatozoa and oocyte to maintain their activity and complete fertilization. After 19 hours, the activity of the spermatozoa or the oocyte may sharply decrease, and the LBR would drop signi cantly.
Patient age was another predictor for the CPR and LBR in our study. The CPR and LBR were signi cantly decreased for women ≥35 years old, similar to the results of previous studies [37,38]. This could be attributed to decreasing ovarian reserves with increasing age [39]. Furthermore, women presenting with two more mature follicles on the ovulation day were more likely to achieve pregnancy than women with only one mature follicle.
The main limitations of our study include our relatively small sample size and possible sample selection bias from the retrospective analysis. Studies based on a larger sample size and prospective design should be carried out in the future to con rm these results. In conclusion, our results have shown for the rst time that the time interval between insemination and ovulation highly correlates with the live birth rate in IUI-D cycles. We also identi ed the I-O interval as a new prognostic factor for the outcome of IUI-D and highlighted that within 19 hours of ovulation is the proper time window for fertilization in the IUI-D procedure.

Declarations Ethics approval and consent to participate
This study was performed in accordance with guidelines outlined in the Declaration of Helsinki. All methods and information collection protocols were approved by the Ethics Committee for the Clinical Application of Human Assisted Reproductive Technology of Northwest Women's and Children's Hospital.

Consent for publication
Not applicable.

Competing interests
The authors declare that they have no competing interests.

Availability of data and materials
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.  Therefore, the interval from insemination to ovulation can be classi ed into three types: ≥+19h (ovulation is ≥ 19h after insemination), -1~+19 h (ovulation is <19h after insemination), -19~ -1 h (ovulation is <19h before insemination) ( Table 1).     Figure 1 Algorithm for IUI timing combining dominant follicle diameter and luteinizing and estrogen hormone testing