Study design

This single-center retrospective cohort study was performed in the Momò Fertilife Private Center for Reproductive Medicine Bisceglie, Italy, in the period between April 2020 and July 2023, in which there were performed 1217 oocyte retrievals. Couples with the following clinical characteristics were excluded from the study:

Karyotype abnormalities.

Male partner with severe oligoasthenospermia.

Female partner over 40 years old.

Female partner with BMI ≥ 30.

Female partner with reduced ovarian reserve.

Female partner with endocrinopathies.

Once ICSI cycles were performed, patients showed serum P levels measured on the trigger day greater than or equal to 1.5 ng/ml were included in the study group. MedITEX – IVF software (CRITEX GmbH; Regensburg; Germany) was then used to identify a matching control group of patients with similar characteristics based on their epidemiologic characteristics and data resulting from anamnesis (age, body mass index (BMI), antral follicle count (AFC), Anti-Müllerian hormone (AMH) and Follicle-Stimulating Hormone (FSH) blood levels, the type of infertility and smoking/non-smoking). To obtain the evaluation of the sample to be analyzed, we applied the formula of the sample size calculator with G*Power software (Heinrich Heine Universitat, Dusserdolf, Germany). We set a dichotomous or binomial endpoint (only two possible results). The parameters used to calculate the sample size were: an expected incidence of 4.5% (this incidence was related to the possibility of having a premature P peak in stimulated cycles) and an expected study group of around 30% (with the prediction that a morphokinetic development anomaly could occur in at least 30% of cases), with a Type I/II error rate of alpha 0.05 and beta 0.05. The power of the study was set at 95%. All these parameters determined the need for a sample of at least 47 patients in the study group. We selected 58 patients to further increase the power of the study. The study design is summarized in Fig. 1.

Fig. 1

Flowchart of study participation

Patients’ selection

Patients were selected based on their serum P levels measured on the trigger day after underwent ICSI cycle. In detail, the study group consisted of 58 women with P levels greater than or equal to 1.5 ng/ml, while the matching control group comprised 58 women with P levels below 1.5 ng/ml selected using MediTEX software as described above. Epidemiological characteristics of the patients, including age, BMI, AFC, AMH and FSH blood levels, the type of infertility and smoking/non-smoking patients were recorded. Additionally, data on the outcomes of the assisted reproductive cycle, such as the number of retrieved eggs, mature (MII) eggs, fertilized eggs, and blastocysts formed were collected.

Progesterone (P) measurement

Serum P levels were assessed through analysis of a blood sample taken from patients on trigger day, which was immediately delivered to the laboratory to complete the measurement by MINDRAY PROG (CLIA) CL-1200i (Mindray Medical International Limited, Shenzhen, China). It is a chemiluminescent immunoassay based on the principle of competitive binding for the quantitative determination of P in human serum. Each determination of P was performed in triplicate order to ensure the best accuracy and the mean was considered the final value. The assessment device kit showed a sensitivity ≤ 0.1 ng/ml. Precision: Intra-assay CV: 5.15%, Inter-assay CV: 7.4%.

Controlled ovarian stimulation protocol and oocyte collection

Patients of both groups were down-regulated with a conventional GnRH antagonist (Cetrotide, Merck Serono, Germany) and stimulated with a recombinant FSH preparation (GONAL-f., Merck Serono, Darmstadt, Germany). Once at least 3 follicles with diameters ≥ 18 mm were reached, a dose of 10.000 IU of hCG was administered to induce ovulation (GONASI, IBSA, Lugano, Switzerland). On the same day, serum P concentration was also determined through a blood sample. The cumulus-oocyte complexes (COCs) were retrieved via transvaginal ovarian pick-up (OPU) under ultrasound guidance (VOLUSON S8, GE Healthcare; Chicago, IL, USA) [25]. This procedure was performed approximately 35 to 36 h after induced ovulation. Three hours after the retrieval of cumulus-oocyte complexes, they were denuded from the corona radiata by repeated pipetting in a solution containing 25 IU/ml hyaluronidase (LifeGlobal Group, Guildford, CT, USA) [19, 25,26,27]. The ICSI procedure was performed on a heated stage at 37 °C under an inverted microscope (Nikon Eclipse TE 200) at 400X magnification. ICSI was performed by an oil-hydraulic microinjection system (Nikon Eclipse TE 200) [19, 25,26,27].

Embryo culture

Injected oocytes were transferred to an EmbryoSlide culture dish (Geri, Genea Biomedx, Australia) prefilled with 80 µl of commercial IVF culture single-step medium (Gtl, Vitrolife, Sweden). The culture dish was then covered with 4 mL of paraffin oil. Embryos were cultured in a time-lapse incubator (Geri, Genea Biomedx, Australia) at 37 °C under a controlled atmosphere of 6% CO2 and 5% O2. The fertilization rate was assessed approximately 16 to 20 h after ICSI and was defined as the presence of two pronuclei and two polar bodies within the matured MII oocytes that were inseminated. Time-lapse images of each embryo were retrospectively analyzed using external image analysis software (Geri Connect & Geri Assess Software, Genea Biomedx, Australia). Embryonic events were annotated with corresponding timing in hours after ICSI. These annotations included key events after insemination such as the extrusion of the second polar body (t2PB), appearance of the two pronuclei in the cytoplasm (t2PN), cleavage to the two- and four-cell stage (t2, t4 respectively), the developmental timelines of the morula (tm - when the blastomeres exhibited compaction forming a continuous outer covering composed of the plasma membranes of the outermost cells), and the time of early blastocyst formation (tb - when the blastocyst began to exhibit the blastocoelic cavity) [28, 29]. Blastocyst morphology was evaluated using conventional scoring criteria on day 5 blastocyst embryos according to Gardner’s classification [30].

Statistical analysis

First, the two selected groups were statistically evaluated to demonstrate that the two samples were homogeneous. For this reason, epidemiological data were studied as mean ± standard deviation for continuous parametric variables or as a percentage for categorical variables. IVF outcomes, embryonic morphokinetic parameters, and blastocyst quality were assessed. Student’s t-test was used to compare age, BMI, AFC, AMH, FSH, type of infertility, smoking and non-smoking patients, number of retrieved oocytes, mature oocytes (MII), fertilized oocytes, obtained blastocysts, and embryonic developmental timelines between the study and control groups. The chi-square test was employed to assess if there were statistically significant differences between the different stages of embryonic development reached in terms of the total number of embryos. Additionally, a t-test was used to compare blastocyst quality in terms of the degree of expansion and morphology of the Inner Cell Mass (ICM) and Trophectoderm (TE) between the two groups. All statistical evaluations of patient parameters were performed by comparing the study group and controls and conducted using GraphPad Prism software (GraphPad Software , 225 Frankli Street. Fl. 26 Boston MA 02110)  .