Human papillomavirus infection in women undergoing in-vitro fertilization: effects on embryo development kinetics and live birth rate

Study population

A total number of 457 women with couple’s infertility, aged 30–43 years, in the waiting list of IVF program at the Physiopathology of Reproduction and IVF Unit of S. Anna Hospital (University of Turin), were submitted to a cervical swab for HR-HPV test. Among them, 326 underwent their first IVF treatment in the study time period (January – October 2021) and were included in the analysis of IVF outcome. For those who resulted positive for HPV in the cervical swab, HPV genotyping and cervical cytology (PAP test) were performed. Moreover, in case of cervical positivity, the presence of HPV-DNA was also tested in endometrial cells (collected during a mock embryo-transfer before starting IVF procedure) and in granulosa cells (collected from the follicular fluid at the time of oocyte retrieval).

The study was carried out in accordance to the Declaration of Helsinki and was authorized by the local Ethical Committee (Ref. number 0000055). Signed informed consent was obtained from all patients.

Cervical cells sampling, HR-HPV test, PAP test, endometrial cells sampling

Cervical sampling was accomplished approximately one month before starting IVF procedure using a specific sampling set (ThinPrep, Hologic), gently scraping cells from the cervical canal and from the internal and external portion of the cervix. Cervical samples were stored in a 10 ml transport medium (PreservCyt® ,Hologic, ThinPrep), and labelled with an ID number.

Briefly, cervical samples were processed by QIAsymphony®DSP (Qiagen, Hilden, Germany), an automatic system based on magnetic particle technology, for the isolation of human cervical cells from the preserving solution. Subsequently, isolated cervical cells were tested by the Hybrid Capture 2 assay High-Risk HPV DNA test (HC2, Qiagen, Hilden, Germany) by using the Rapid Capture System® (RCS; Qiagen, Hilden, Germany) according to the manufacturer’s instructions. HC2 test is a nucleic acid hybridization assay with signal amplification for qualitative detection of 14 HPV types in cervical cells (HPV16, -18, -31, -33, -35, -18, -39, -45, -51, -52, -56, -58, -59, and − 68). HC2 results were expressed as the ratio between the specimen’s light emission (relative light units [RLU]) and the mean of three concurrently tested positive controls (CO). Samples were considered positive when the ratio (RLU/CO) was ≥ 1 [31].

In case of positivity to HPV test, a cytological smear was set up from the same sample and was stained using Papanicolaou staining (Pap Test) in order to detect morphological features of any eventual cervical lesion. Cytology was classified using the Bethesda 2001 system: data were classified as negative for intraepithelial lesions of malignancy (NILM), low grade (LSIL, ASC-US, and AGC), or high grade (ASC-H, HSIL, adenocarcinoma in situ-AIS-, and cancer).

Endometrial cell sampling was performed using a modified embryo transfer catheter consisting of two components: an outer guide and an inner soft part; first, the guide was inserted into the cervical canal, avoiding contact with the vaginal walls; the inner soft catheter was then gently inserted into the guide and advanced until reaching the endometrium inside the uterine cavity. After rotating the inner part tip in order to collect endometrial cells, it was retracted without exposition the vaginal environment, its distal end (approximately 5–10 mm) was cut using sterile scissors and immediately placed in sterile tubes for HPV infection assessment.

DNA extraction, HPV-DNA detection and genotyping

DNA was extracted from cervical, endometrial and granulosa cells by using the QIAamp DNA Mini Kit® (Qiagen, Hilden, Germany), following the manufacturer’s instruction. Extracted DNA was quantified at the NanoDrop® ND-1000 spectrophotometer and, in order to check DNA quality, the β-globin (152 base pairs length (bp) housekeeping gene was amplified from the clinical samples using iCycleriQ™ Real-Time PCRDetection System (BioRad®, Hercules, CA, USA). PCR reaction contained 2X SYBR Green Supermix (BioRad®), 0.3 µM of β-globin primers [forward (5’GAAGAGCCAAGGACAGGTAC3’); reverse (5’CAACTTCATCCACGTTCACC3’)] [32], and 2 µl of DNA sample. Each sample was analysed in duplicate, and a control without DNA was included in each run.

To test HPV presence, a 150 bp L1 region was amplified with GP5+/GP6 + consensus primers, allowing detection of a broad range of HPV types [33, 34]. Consensus primers GP5+ (5’TTTGTTACTGTGGTAGATACTAC3’) and GP6+ (5’GAAAAATAAACTGTAAATCATATT3’) [35] were used. A positive (SIHA cell lines) and a negative control (without DNA) were added to each PCR run. The presence of PCR amplicon was checked on 2% agarose gel stained with ethidium bromide.

Samples testing positive for GP5+/GP6 + PCR were genotyped by using INNO-LiPA® HPV Genotyping Extra II Amplification test (FUJIREBIO, Belgium). This assay can identify 32 different HPV genotypes at the same time using reverse hybridization, after a preliminary step of amplification. PCR reactions were carried out in thermocycler (GeneAmp PCR System 9700) using SPF10 biotinylated consensus primers.

The biotinylated amplicons were denatured and hybridized with specific oligonucleotide probes fixed on membrane strips. After reverse hybridization, they were incubated with BCIP/NBT chromogen at 20–25° C on a shaker yielding a purple precipitate, and the results were visually interpreted (INNO-LiPA® HPV Genotyping Extra II Ampmanual).

IVF procedure

Controlled Ovarian Stimulation (COS) with gonadotropins was accomplished according to standardized protocols. Follicular growth was monitored by serial transvaginal ultrasound (TV-US) and measurement of circulating estradiol (E2) every second day from day 7. When at least two follicles reached 18 mm mean diameter, with appropriate E2 levels, a single subcutaneous injection of 10.000 IU hCG (Gonasi HP, IBSA, Pambio Noranco, Switzerland) was administered to trigger ovulation.

Transvaginal US-guided oocyte pick-up (OPU) was performed 35–37 h later under local anesthesia (paracervical block). Follicular fluid was aspirated and immediately observed under stereomicroscope. Cumulus-oocytes complexes (COCs) were washed in buffered medium (Follicle Flush Buffer – COOK Medical, Ireland) and incubated in controlled atmosphere (37 °C, 5% O2, 6.5% CO2) using appropriate culture medium (Fertilization Medium - COOK Medical, Ireland).

The same day of OPU, semen samples were examined to assess sperm concentration, motility and morphology according to the World Health Organization guidelines (WHO laboratory manual, 2010); then they were prepared by density gradient centrifugation in order to select normally motile and morphologically normal spermatozoa. A few hours after OPU, oocytes were fertilized by conventional IVF or ICSI (Intra-Cytoplasmic Sperm Injection) according to semen parameters.

Normal fertilization was assessed 16–18 h later (Day 1) by evaluating the presence of two pronuclei (2PN) and the extrusion of the second polar body.

Embryos were cultured in pre-equilibrated cleavage medium (Cleavage Medium - COOK Medical, Ireland) overlain with mineral oil up to day 3 of development; at this stage, a change of medium was performed and a new one (Blastocyst Medium - COOK Medical, Ireland) was kept until the blastocyst stage (day 5–6). Embryo morphological evaluation was first performed on day 2 using the Integrated Morphology Cleavage Score (IMCS) [36], and then on day 5 according to The Istanbul Consensus Workshop [37]. In order to obtain information on the kinetic of embryonic development, embryos were cultured in micro-wells (one zygote/micro-well) using the Geri plus® time-lapse system (Genea Biomed, Germany) with integrated embryo monitoring system. Geri plus ® is a last generation incubator which allows to continuously (24 h/24) observe embryo growth at regular time intervals (every 5 min) without modifying culture conditions. All videos obtained by Geri plus® were analyzed, and the following morphokinetic parameters were considered: time of pronuclear appearance (tPNa), pronuclear fading (tPNf), completion of cleavage to two, three, four and eight cells (t2, t3, t4, and t8 respectively), blastulation start (tSB), full blastocyst development completion (tB), initiation of hatching process (tHN). The following time intervals were also calculated: tPNf-tPNa, t2-tPNf, t3-t2, t4-t3, t4-t2 and t8-t4 [38].

Embryo transfer (ET) in uterus was performed on day 5 using the soft catheter Sydney Guardia (COOK Medical, Ireland) under transvaginal US guidance, as previously reported by our group [39].

Statistical analysis

The primary endpoint for power calculation was the prevalence of HPV infection among infertile women candidate to IVF. Accordingly, the power calculation was made as follows: considering the available epidemiologic data, we hypothesized that HPV infection could be 10% more frequent among women undergoing IVF than in the general population, and 450 subjects would have allowed to reach a statistical power of 80% with 0.05 alpha error.

Analyses were performed subdividing women undergoing IVF according to the presence or absence of HPV infection in the cervical swab. The data normal distribution was assessed using Kolmogorov-Smirnov test. Comparison between HPV-positive and -negative women was performed using the Student t-test (continuous variables) or the χ2 test (categorical variables), and the results were expressed by mean and standard deviation or by percentage. All statistical tests were for unpaired data and two-sided; a p-value < 0.05 was considered significant.

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