1. IntroductionIt is acknowledged that sexually transmitted diseases (STDs) are a major cause of infertility, as 20–60% of cases of infertility in women are related to Chlamydia trachomatis, Ureaplasma urealyticum, and Neisseria gonorrhoeae, which cause cervical, tubal, and mucosal damage to the host [1,2]. Human papillomaviruses (HPV) are double-stranded DNA viruses that constitute the most common sexually transmitted causative agent infecting humans of reproductive age worldwide [3]. Among women of reproductive age, HPV infection is a potential risk factor that predisposes them to subsequent infertility [4], and it infects skin and mucosal and cutaneous epithelial cells [5]. HPV infection is highly correlated with precancerous and cancerous lesions of the cervix uteri, vulva, vagina, penis, and anogenital areas [3,6,7], and some studies indicate that HPV is detectable in cervical endometriotic and ovarian lesion tissues [8,9].HPV infection is primarily self-limiting and can be cleared by self-immunity of the infected individual. However, persistent HPV infection can be carcinogenic and associated with precancerous lesions and cancer of the cervix and uterus in women and of the anogenital mucosa in women as well as men [10]. Persistent HPV infection has been linked to chronic inflammation [11], and infectious virion production may weaken the cells residing in the endometrium in association with infertility and miscarriage [12]. HPV infection increases the risk of spontaneous abortion as well as ectopic pregnancy, and different HPV genotypes may play disparate roles in adverse reproductive outcomes when using assisted reproductive technology (ART) [13].While HPV infection may influence pregnancy outcome, this contention is controversial [14]; thus, the effects of HPV on women’s infertility and subsequent reproductive outcome require further study. Therefore, in the present study, we investigated the prevalence of HPV infection in women from infertile couples treated with IVF/ICSI-ET and assessed their reproductive outcomes. 2. Materials and Methods 2.1. Study DesignA total of 8117 women patients from infertile couples underwent HPV genotype testing; 747 patients were infected with HPV. The HPV (+) group was subsequently sorted into high-risk HPV infection (hrHPV+, 529/747, 70.82%), low-risk HPV infection (lrHPV+, 175/747, 24.42%), and high-risk and low-risk sub-groups (hrHPV+/lrHPV+, 43/747, 5.76%). Of 529 cases of hrHPV positivity and 175 cases lrHPV positivity, patients that did not undergo controlled ovarian hyperstimulation (COH), had no oocyte retrieved, and had no embryo transferred were excluded. Only 130 cases of hrHPV-positive and 94 cases of lrHPV-positive patients underwent IVF/ICSI-ET treatment. One hundred twenty-six HPV (−) patients were selected randomly and designated as negative control (Figure 1). We excluded all patients who showed an abnormal thin-prep cytologic test (TCT). Before the COH cycle, regular vaginal discharge and bacterial vaginitis (BV) were examined to exclude mycosis, trichomoniasis, Gardnerella, and Neisseria gonorrhoeae. Cervical swabs were examined to exclude Chlamydia trachomatis, Ureaplasma urealyticum, and Mycoplasma genitalium. Regular blood tests were also conducted to exclude HIV, HBV, HCV, and TP. All patients underwent a fresh-cycle embryo transfer or a frozen-embryo cycle embryo transfer after the COH cycle. The average transferred embryo number was 1.3 per cycle. We obtained detailed information on infertile patients that included age, years of infertility, body mass index (BMI), cause of infertility, baseline hormonal levels such as follicle-stimulating hormone (FSH) and anti-Müllerian hormone, and antral follicle count (AFC). 2.2. Determination of HPV Genotype

Sexual activities and vaginal medications were restricted prior to HPV analyses. Cervical discharges were swabbed for HPV detection, and genotyping was performed with a BioRad 100 Amplification and Luminex® 200™ System (Thermo, Waltham, MA, USA) that detected 27 genotypes: high-risk genotypes 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, and 82 as well as low-risk genotypes 6, 11, 40, 42, 43, 44, 55, 61, 81, and 83.

2.3. IVF/ICSI-ET ProtocolAll patients underwent a standardized gonadotropin-releasing hormone (GnRH)-agonist long protocol or GnRH-antagonist protocol with oocyte retrieval, fertilization, and embryo transfer. For the GnRH-agonist long protocol, patients who underwent the IVF/ICSI-ET protocol experienced pituitary downregulation with a GnRH agonist administered at the midluteal phase. For the GnRH-antagonist protocol, patients initiated rFSH treatment on the second day of the cycle by once-daily injection. After five days of this treatment, we administered the antagonist cetrorelix acetate (Merck Serono, Darmstadt, Germany) daily, and the rFSH dose was adjusted according to the individual ovarian response as assessed by daily ultrasonographic examination. The antagonist treatment continued until the day of hCG injection. When at least two leading follicles reached 18 mm in diameter in the two COH protocols, ovulation was induced with recombinant α-HCG (5000 to 10,000 IU, Merck Serono, Darmstadt, Germany) and oocytes were collected between 36 and 38 h later. Oocytes were then fertilized by either conventional IVF or intracytoplasmic sperm injection (ICSI) [15], and all embryos were transferred on the third day after oocyte pickup with a standard ET protocol [16]. Vaginal progesterone (8% Crinone vaginal gel, Merck Serono, Darmstadt, Germany) was used daily from the day of embryo transfer (ET) to provide routine luteal support and maintain luteal function until the 10th week of pregnancy [17]. 2.4. Embryonic Development and Reproductive Outcomes

Day-3 high-quality embryo rate, ovum maturation rate, fertilization rate, implantation rate, clinical pregnancy rate, live birth rate, and miscarriage rate were determined. Our rate calculations were as follows: ovum maturation rate = no. of D0 MII oocytes/no. of retrieved oocytes; fertilization rate = no. of Day-1 2PN embryos/no. of D0 MII oocytes; high-quality embryo rate = no. of Day-3 high-quality embryos/no. of Day-1 2PN oocytes; implantation rate = no. of implanted embryos (i.e., pregnancies)/transferred embryos; clinical pregnancy rate = pregnancy cycles/total cycles; and miscarriage rate = miscarriage cycles/total cycles.

2.5. Statistical Analysis

Measurements are presented as means ± standard deviation, and we applied the Statistical Package for the Social Sciences (SPSS, version 23.0, IBM, Armonk, NY, USA) for Windows for all statistical analyses. The Student’s t test and Chi-squared test were used to compare categorical variables, and a p value of <0.05 was considered to be statistically significant. We executed logistic regression analysis on reproductive outcomes, and odds ratios (ORs), 95% confidence intervals (CIs), and p values are reported.

4. DiscussionHPV infection can be spontaneously cleared within one to two years, but repeated infection is associated with multiple malignancies that include cervical, anogenital, and oropharyngeal cancers [18]. Over 200 different HPV genotypes have been identified [19], and the prevalence of HPV differs with respect to geographic location and socioeconomic status [14,20,21,22,23]. Several authors indicated that HPV prevalence was higher in pregnant women than in non-pregnant women and demonstrated overall HPV prevalence rates of 16.82% and 12.25%, respectively [24]. A case-control study suggested that HPV prevalence was 24.2% in pregnant women vs. 14.8% in non-pregnant women, and that HPV prevalence was age and genotype dependent [25]. In our study, HPV prevalence in women from infertile couples was 9.2%, and hrHPV was detected in 78.82% of all HPV-positive women. The predominant hrHPV genotypes were 16, 52, 53, 56, 58, and 59; and the predominant lrHPV genotypes were 6, 43, 44, 55, 61, and 68. Types 16 and 52 were the most common genotypes we observed in the infertile women, which is congruent with a recent report [25]. Genotype 61 is the predominant type in lrHPV, occupying 9.77% of the total HPV infection (73/747), which was higher than genotypes 6 and 11 (25/747). Our results also indicated that there was an elevated infection rate in women of infertile couples who were 26–40 years old (and particularly in women 26–35 years of age), accounting for 70% of total infections and potentially associated with frequent sexual activity in this age group.As HPV DNA has not only been identified in the cervix but also in the placenta, fetal membranes, and amniotic fluid, pregnant women undergo an increased risk of HPV infection [18,24]. However, whether HPV infection exerts adverse effects on pregnancy outcomes remains controversial. While several researchers have suggested a higher HPV prevalence among women who suffered a spontaneous abortion in relation to normal pregnancies [26,27,28], others uncovered no correlation between HPV infection and the risk of spontaneous abortion, miscarriage, or preterm delivery [29,30]. In addition, attenuated HPV infection rates have been observed in patients with recurrent miscarriage, and it has been hypothesized that augmented immunoreactivity may be partially responsible for the recurrent pregnancy loss and that this may be protective against HPV infection [31]. Our results indicated that HPV infection in women of infertile couples did not alter ovum maturation or fertilization rate but reduced the Day-3 high-quality embryo rate (p26,27,28,32,33,34].HPV commonly infects both the male and female partners. Men can be infected with HPV in the penis, anus, and head and neck; and it can be detected in penile swabs and semen. There are reports of a significantly higher HPV infection in infertile couples compared to the general population (20.9% vs. 8.2%) [35], and HPV also affects semen parameters [36]. One study indicated a statistically significant correlation between the rate of pregnancy loss and positivity for HPV DNA in the male partner of infertile couples compared with non-infected couples (66.7% vs. 15%, respectively) [32]. Pregnancy rate was reduced and the miscarriage rate was increased after HPV infection in both women and men [33]. Depuydt et al. reported that the pregnancy rate with intra-uterine insemination (IUI) declined when the sperm DNA fragmentation index (DFI) exceeded 26%; and sperm samples containing HPV exhibited a significantly higher DFI compared with HPV-negative sperm samples (29.8% vs. 20.9%, respectively; p = 0.011) [37]. However, Hana et al. recently reported that men with hrHPV-positive semen samples showed altered seminal parameters that included lower semen volume, sperm concentration, and total sperm count relative to men with HPV-negative samples; but there was no association between seminal hrHPV infection and pregnancy outcomes that included spontaneous abortion [36]. Thus, the impact of HPV on male fertility and associated reproductive outcomes remains debatable.In our study, infertility in 45 couples was caused by single male factors that mainly included oligozoospermia and asthenozoospermia, and this was possibly associated with HPV infection. The single-male-factor group (smHPV group) manifested a lower Day-3 high-quality embryo rate, clinical pregnancy rate, and live birth rate, and it had an increased miscarriage rate compared with the HPV-negative group. Logistic regression analysis indicated that single male factors comprised an independent risk for decreased clinical pregnancy rate, decreased live birth rate, and increased miscarriage rate in infertile couples in which the female partner was infected with HPV. Our results suggested that HPV infection could cause semen parameters to change, which is possibly one of the reasons for oligozoospermia and asthenozoospermia, and that it would contribute to lower high-quality embryo rate [38], reduced clinical pregnancy and live birth rates and increased the miscarriage rate. We did not investigate the prevalence of male-partner HPV infection and changes in semen parameters, so further investigation of male-partner infection status is necessary.Several studies indicated that HPV infection was associated with spontaneous preterm birth (sPTB), defined as delivery between 28 and 37 weeks of gestation [14]. In normal pregnancy, 17.5% of HPV infection occurs at the cervix, which is significantly lower than in sPTB patients, and cervical cytology shows that HPV infection generates placental abnormalities and preterm birth [39,40]. Hr-HPV infection was also associated with a risk of premature rupture of the membranes [41], and persistent HPV-16/18 infection was related to an increased risk of preterm birth independent of cervical treatment [42]. In our study, we observed no rise in the sPTB rate but did observe an attenuation in the live birth rate between the HPV-infected and non-HPV-infected groups.This study also has some limitations. First, we only investigated the prevalence of human papillomavirus infection in the female partner of infertile couples before IVF/ICSI-ET treatment. We have no repeated HPV DNA detection results with intervals three to six months beginning at IVF/ICSI-ET treatment, which could have confirmed ongoing HPV infection. Second, in our study, HPV infection in males were not tested despite the fact that previous reports have shown a correlation between HPV+ male and pregnancy loss [32,33]. Finally, the number of HPV-infected cases undergoing IVF/ICSI-ET was limited, and the influence HPV infection on reproductive outcomes in females or both couples needs to be clarified by further large population-based studies.

In summary, our results indicated that HPV-infected women of infertile couples did not show alterations in ovum maturation, fertilization, implantation, clinical pregnancy, live birth, or miscarriage rates regardless of hrHPV or lrHPV infection, but they did exhibit lower high-quality embryo rates with HPV infection. HPV infection in these women was associated with a reduced miscarriage rate, and single-male-factor-induced infertility influenced reproductive outcomes of couples undergoing IVF/ICSI treatment. However, simultaneous determination of HPV status for both female and male partners of infertile couples is required to further clarify this phenomenon.