A paradigm of higher success rate of intracytoplasmic sperm injection - preovulatory human chorionic gonadotropin-day serum oestradiol.

Intracytoplasmic sperm injection (ICSI), as a substitute to in-vitro fertilization (IVF), is now widely used to overcome male and female infertility problems [ 1 , 2 ]. Accurate timing of human chorionic gonadotropin (hCG) administration requires careful cycle monitoring because the success of the cycle depends on it [ 3 ]. Human chorionic gonadotropin administration is on time when the dominant follicle has reached suitable maturity, which is immediately before the spontaneous luteinizing hormone (LH) peak [ 4 ]. The most significant criterion for evaluating follicular maturity is the serum oestradiol (E 2 ) level, so the relationship between the E 2 level on the day of hCG injection and the cycle outcome is not surprising. Many techniques – namely controlled ovarian hyperstimulation (COH) – were used to attain multiple oocytes; nevertheless, this circumstance results in supraphysiologic E 2 . Serum E 2 concentrations during COH are increased 10-fold compared with those of natural cycles [ 5 ]. On the day of hCG administration, high E 2 levels might cause variable ICSI outcomes, caused by unsettled endometrial receptivity [ 6 ]. Serum E 2 plays a basic role in the reproductive system, being synthesized by dominant follicles, and plasma E 2 levels are used as an index to evaluate follicle maturity [ 7 ]. Oestradiol plays a fundamental role in cervical mucus regulation, endometrial propagation for embryo implantation, and initiation of mid-cycle LH surge. During COH, E 2 levels are directly proportional to follicle size, with 200 pg/ml equivalent to a mature ovarian follicle. Nevertheless, high E 2 levels may be incriminated in unwanted adverse outcomes of IVF/ICSI cycles and decreased endometrial receptivity [ 8 ]. Several studies have suggested that high serum E 2 concentration on hCG administration day has a positive effect on pregnancy, however, others have reported contrary to this [ 9 ]. During COH, supraphysiological E 2 levels are assumed to be interrelated with ICSI outcome, and consequently it was meticulously investigated for many years. Nevertheless, such a relationship could neither be established nor repudiated. Endometrial receptivity, embryo viability, and adhesion capacity are the possible mechanisms through which supraphysiological E 2 levels may affect pregnancy rates in ICSI cycles [ 10 ]. The aim of the current work was to assess the relationship between the serum E 2 level on the day of hCG administration and the ICSI outcome.

A total of 150 infertile women were included in the current study; their mean age was 29.4 ±3.8 years and their mean body mass index (BMI) was 26.5 ±4.42 kg/m 2 . Most of the infertile women (58%) had primary infertility, while the rest of them (42%) had secondary infertility. The most common cause of infertility was unexplained (42.7%), while the least common cause was male factor (8.7%) ( Table 1 ). The highest number of retrieved oocytes was 11.4 ±4.6 in group D, while the lowest number was seen in group A (7.2 ±3.2). The highest fertilization rate was in group E (58.1%) and the lowest was 37% in group A. The highest number of transferred embryos was in group E (2.9 ±0.5) while the lowest was 1.6 ±0.71 in group A. The highest pregnancy rate was (21.5%) in group E ( Table 2 ). The median serum E 2 level on the day of hCG administration was highly significant in women who became pregnant when compared to women who did not ( Table 3 ). The best cut-off value of serum E 2 on day of hCG administration was ≥ 3682.3 pg/ml. Serum E 2 at hCG administration ≥ 3682.3 pg/ml was associated with a successful clinical pregnancy outcome, with a sensitivity of 81.3%, specificity of 97.1%, PPV of 83.1%, NPV of 89.5%, and an overall accuracy of 83.2% ( Table 4 ). Demographic data of infertile women Comparison of intracytoplasmic sperm injection outcomes according to serum oestradiol levels on day of human chorionic gonadotropin administration Data presented as mean ± standard deviation, number (percentage). Analysis using one-way ANOVA test and LSD post-hoc test for numeric variables. Analysis using χ 2 test for categorical variables. Significant ( p < 0.05) vs. group A Significant ( p < 0.05) vs. group B Significant ( p < 0.05) vs. group C Significant ( p < 0.05) vs. group D Comparison between women who became pregnant and those who did not, regarding serum oestradiol level on day of human chorionic gonadotropin administration E 2 – oestradiol, hCG – human chorionic gonadotropin, NS – non-significant Analysis using Mann-Whitney U test Validity of the association between serum oestradiol levels on day of human chorionic gonadotropin administration and clinical pregnancy outcome E 2 – oestradiol, NPV – negative predictive value, PPV – positive predictive value

The current prospective cohort study was conducted during the period from January 2019 to September 2021 at Zigzag Obstetrics and Gynecology Department, Al-Azhar Obstetrics and Gynecology Department and private ART centers after approval of research ethics committee. One hundred and fifty women attended the infertility clinic were recruited. Before participation, written informed consent was obtained from all women. Women with submucous fibroid > 5 cm, ovarian cyst > 2 cm, hydrosalpinx, endometrial polyp, and follicular stimulating hormone (FSH) > 15 IU/l were excluded. All women were subjected to detailed history, physical examination, transvaginal ultrasound (TVUS) examination, and laboratory investigations. Transvaginal ultrasound was done on the 3 rd day of the natural cycle for exclusion criteria and for assessment of antral follicular count (AFC). Laboratory investigations included basal serum FSH, basal serum LH, basal serum E 2 , serum prolactin, and serum thyroid stimulating hormone (TSH) on the 3 rd day of the cycle. Controlled ovarian hyperstimulation (COH) was done by the standardized long protocol. The select of it was based on the women’s age, body mass index (BMI), basal FSH, and AFC. 500 µg folic acid was received daily by all women before induction. Combined oral contraceptive pills were received on day 2 of the previous cycle. Then standard mid-luteal protocol started on day 21 of the previous cycle by GnRH agonist triptorelin acetate (Decapeptyl ® , Ferring, Germany) administered daily at a dose of 0.1 mg subcutaneous injection until ovarian suppression was detected by serum E 2 (≤ 50 pg/ml) on the 2 nd day of the menstrual cycle. Then the GnRH agonist was continued daily at a dose of 0.05 mg, and IM daily injections of HP-urinary HMG (Merional ® 75 IU LH and 75 IU FSH, IBSA) were given. Follicular development was started in the 6 th day of ovarian stimulation, and the dose was adjusted according to ovarian response, which was evaluated by TVUS (Mindray ® , 50/60 Hz, Hamburg, Germany). When more than 2 leading follicles reached ≥ 18 mm in diameter, ovulation trigger was done using intramuscular injection of hCG (Choriomon® 10,000 IU, IBSA). Venous samples were obtained from women by venipuncture and processed one hour after withdrawal for E 2 assessment just prior to hCG injection, and serum was stored at 2–8°C for less than 8 hours and assayed for E 2 . The ADVIA Centaur Estradiol-6 assay (a competitive immunoassay using direct chemiluminescent technology). ADVIA measured oestradiol concentrations up to 1000 pg/ml (3670 pmol/l) with minimum detectable concentration (sensitivity) of l0 pg/ml (36.7 pmol/l). The conversion formula was 1 pg/ml = 3.67 pmol/l. The included women in our study ( n = 150) were divided into 5 groups according to the serum E 2 level on day of hCG administration: Group A – serum E 2 < 1000 pg/ml; Group B – serum E 2 1000 to < 2000 pg/ml; Group C – serum E 2 2000 to < 3000 pg/ml; Group D – serum E 2 3000 to < 4000 pg/ml; and Group E – serum E 2 ≥ 4000 pg/ml. Transvaginal ultrasound-guided oocyte retrieval was done 36 hours after hCG injection under general anaesthesia. Oocyte maturation was evaluated by an embryologist. Oocytes were cultured in modified culture media (global total). Luteal phase support by micronized progesterone was given starting the day after hCG until the occurrence of biochemical pregnancy, which, confirmed by serum B-hCG concentration, was > 50 IU/l on the 14 th day after embryo transfer, and clinical pregnancy was defined as the presence of a gestational sac with foetal heart beat on ultrasound performed 7 weeks after embryo transfer. If the patient became pregnant, she continued with progesterone supplementation until 12 weeks of pregnancy. Data were analysed using SPSS ® for Windows ® , version 15.0 (SPSS, Inc, USA). Description of quantitative (numerical) variables was performed in the form of mean, standard deviation, and range. Description of qualitative (categorical) data was performed in the form of number of cases and percentage. Analysis of numerical variables was performed by using the independent Student’s t -test. Analysis of categorical data was performed by using the χ 2 test. The ANOVA test was used to compare multiple variables. The Mann-Whitney U test was used to compare quantitative variables. Diagnostic accuracy was assessed using the following terms: sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and overall accuracy. Receiver operating characteristic curve analysis was constructed for estimation of the association between serum E 2 levels on the day of hCG administration and the clinical pregnancy outcome. A two-sided p -value < 0.05 was considered statistically significant. The significance level was set at 0.05.

It has been described that 17 b E 2 prompts cytoplasmic maturation of germinal vesicle oocytes through increase in intra-cytoplasmic calcium level; this has been linked to better fertilization [ 11 ]. In the present study the causes of infertility of our patients were male factor (8.7%), anovulation and polycystic ovary syndrome (PCOS) (23.3%), tubal factor (10%), and endometriosis (15.3%). This result disagreed with that of others, who found that the causes of infertility were tubal factor (33.2%), male factor (23%), PCOS (18.4%), and endometriosis (11%) [ 11 ]. In the current study the highest number of retrieved oocytes women with E 2 3000 to < 4000 pg/ml (11.4 ±4.6) was statistically significant versus women with E 2 < 1000 pg/ml and women with E 2 2000 to 4000 pg/ml versus women with E 2 4000 pg/ml, while Mittal et al . reported that the mean number of retrieved oocytes increased as the serum E 2 value increased from women with E 2 1700 pg/ml to women with E 2 5600 pg/ml [ 3 , 11 ]. In our study the highest number of transferred embryos was in group E; this result disagreed with Kara et al ., 2012, who concluded that the mean number of transferred embryos (2.5 ±0.6) was statistically significant in women with E 2 > 4000 pg/ml [ 6 ]. In this study the women with E 2 ≥ 4000 pg/ml had a higher fertilization rate (58.1%) compared with other women with different E 2 levels, but there was no significant difference among these women; this result was analogous with Kong et al ., who reported a fertilization rate of 60%, which is not significantly different than that seen in women with dissimilar E 2 levels [ 5 ]. In the present study the highest pregnancy rate (21.5%) was in women with E 2 > 4000 pg/ml; this result is consistent with Kara et al ., who showed that the pregnancy rate was high in women with E 2 > 4000 pg/ml, but the difference among women with different E 2 levels was not statistically significant [ 6 ]. Joo et al . found that the pregnancy rate rises with increasing levels of serum E 2 from women with E 2 4000 pg/ml. This may be justified by the fact that oestrogen increases endometrial proliferation and uterine perfusion, increased possibility of pregnancy. In the current study the best cut-off value of serum E 2 at hCG administration was ≥ 3899 pg/ml. A serum E 2 at hCG administration ≥ 3899 pg/ml was associated with a successful clinical pregnancy outcome, with a sensitivity of 81.3%, specificity of 97.1%, PPV of 83.1%, NPV of 89.5%, and an overall accuracy of 83.2%. However, Wu et al . stated that the serum E 2 cut-off value is 19,124 pmol/ml; this figure has low sensitivity (58.1%) and low specificity (49.2%), which is not implemented for attaining a successful pregnancy [ 10 ].

The serum E 2 level on hCG day may influence a successful ICSI outcome, and this study proposes that the optimal range of E 2 level for attaining a successful pregnancy is > 4000 pg/ml.