The Huhner test (HT) is simple, non-invasive, inexpensive and commonly used in standard fertility investigations but its clinical utility is subject to debate.

The purposes for the HT are numerous: confirm a complete intercourse, identify women with abnormalities in quality or quantity of cervical mucus, determine whether the sperm-mucus interaction is favorable or not to the vitality of the spermatozoa [1], and is the only test to diagnose a cervical factor in idiopathic subfertility [2].

Several studies demonstrated a strong association between the HT and pregnancy rates [3-8]. In a prospective study, Glazener et al., conclude that TH is an effective predictor of conception if duration of infertility is less than 3 years [9]. The use of TH improved the discrimination of two models for the prediction of the chance of treatment-independent pregnancy leading to live birth among subfertile couples [10,11]. Similarly, Van der Steeg et al., got an improved predictive model by adding the HT [12]. More recently, Leushuis et al., showed that subfertile couples with an abnormal HT have a lower probability of a spontaneous pregnancy [13].

On the other hand, Oei et al., concluded, with a substantial number of participating women, that the use of HT would only lead to more interventions without an increase in pregnancy rates [14]. But the study was criticized because it did not assess the HT only [15,16]. Similar findings were published by the team 3 years later [14] Similarly to Oei, Helmerhost et al. explained that the HT has poor diagnostic and prognostic properties and stated that has no benefit on pregnancy rates [17].

The HT identifies a cervical factor in subfertile couples which has important implications for treatment. Three studies clearly indicated a beneficial effect of IUI in couples with an isolated cervical factor [18,19] whereas four others did not report such an effect [20-22]. But Helmerhost et al., in a literature review, revealed numerous biases in these studies [23].

Another limit in HT is the criterion for the positivity of the test. For WHO 1992, more than ten progressive motile spermatozoa per high power microscope field were necessary for positivity [24]. Seven years later, only one progressive motile spermatozoon was needed in six fields [25].

In the meta-analysis of Griffith, the 6 studies used different criteria from 0 (<1 per field) to 5 progressive motile spermatozoa per field. In the retrospective study, Oei et al. concluded that the best definition for a negative HT is <1 progressive motile spermatozoa per field (and specificity and sensitivity compared with different cut off: <1, <5, <10 and <20 progressive motile spermatozoa per field). For Van der Steeg et al., a HT was considered negative if no progressive motile spermatozoa were observed throughout the mucus. In France, most teams consider the HT as positive if more than 5 progressive motile spermatozoa were observed by field.

But even if the positivity criteria varied according to the teams, Glatstein et al. in a study with blinded observers has shown that the test is reproducible on the number and mobility of sperm [26].

The present retrospective study aims at comparing couples with positive HT and couples with negative HT regarding the occurrence of pregnancy, mode of pregnancy obtained and the period of infertility from the realization of HT. Furthermore, the results are expressed according to different criteria of positivity.

Patients

Outcomes of all couples who underwent a Huhner test as part of their routine fertility work-up in 2 sites of Pau’s laboratories from 1st January 2004 to 31^st December 2012, were evaluated. All of the couples had a duration of infertility > 12 months. To avoid bias, couples were excluded if the woman had a FSH >15 IU/l. Similarly, men with an altered sperm (< 20 M/ml, motile spermatozoa < 30%) were excluded from the study. We have not taken into account tubal disease because hysterography has not been performed for every women. Anovulation wasn’t either a criterion of exclusion in our study.

About 854 HT were realized in the laboratory. Time of conception was based on the date of the first TPC.

Huhner test (HT) procedure

Patients were given standardized instructions. The Huhner test was planned 8-12 hours after intercourse. The HT was not performed if a couple failed to have intercourse as recommended, and was reprogrammed.

The HT and slide preparation for each subject were performed by 6 biologists in an identical standardized fashion according to the HAS guidelines.

The patient was placed in the lithotomy and a sterile nonlubrificated speculum was inserted into the vagina and the cervix was exposed. After assessment of the opening of the cervix, endocervical mucus was collected by suction in a standard narrow 1ml disposable syringe. 2 collections were performed, the first from the endocervix and the second into the pouch of Douglas to ensure an effective intercourse.

Evaluation of the cervical factor

The cervical mucus quantity and quality were determined using the Insler score, which is based on evaluation of spinnbarkeit, ferning, cervical mucus volume, and appearance of the external cervical os (condition of the cervix) and ranges from 0–12 (cervical index). Samples with an Insler score of ≥9 were considered ovulatory.

The pH of the cervical mucus was measured by using paper strips (pH Indikatorpapier; Merck, Darmstadt, Germany).

Cellularity

After collection of the test specimens, the biologist examined vaginal pool specimens microscopically for the presence of spermatozoa. A sample of cervical mucus was covered with a glass slip (22×22 mm) and examined at low (x100) and high (x400) power magnification. The number of sperms moving forward per high field was recorded.

4 different cut-off levels were considered varying from one to 10 motile spermatozoa per high power field for positivity.

Cervical mucus inflammation was evaluated with number of leucocytes.

Pregnancy diagnosis

Diagnosis of clinical pregnancy was defined with the presence of embryonic sac visible at sonography. The research regarding women’s potential pregnancies were stopped on 04.30.13.

Semen analysis

Men produced semen samples by masturbation into a specimen cup at the laboratory. Lubricants were not used for masturbation. The men were asked to abstain from ejaculation for 3–5 days before the clinic visit and to report the time of their previous ejaculation.

Samples were processed within 30 minutes of collection. After semen liquefaction, semen analysis was performed according to World Health Organization guidelines [25] for assessing semen volume and sperm concentration. Total sperm count was calculated as concentration x volume. Motility was assessed, classifying the spermatozoa as progressive motile (A+B), total motile (A+B+C), or immotile (D).

Statistical methods

Descriptive statistics were used to assess the similarity of the groups. Cumulative pregnancy rates were calculated as for life table analysis and compared with the Wilcoxon test. Data were assessed by the student test. P<0.05 was considered significant.

Out of the 854 HT performed in the laboratory, 20 were excluded because of FSH>15 IU/ml and 116 were not taken into account (one TPC per couple, if more than one was done for the same couple, we only kept the positive one). Infertility was primary in 95.4% of the women and secondary in 4.6%.

The remaining 718 tests were divided into 2 groups: positive HT or negative HT.

Follow up was known for over 80% of women who underwent the test.

After exclusion of couples with bad semen analysis, 408 couples remained and follow up was known for 88% of them.

In Table 1, HT was considered positive if at least one motile spermatozoon was observed in cervical mucus. The characteristics of the participants did not differ significantly between the 2 groups.

Table 1: Occurrence of pregnancy by result of the post-coital test (PCT) Positive PCT criterion: 1 or more motile spermatozoa in the whole cervix

The cumulative pregnancy rate was 13.5% higher (P<0.01) and occurred quickly (0.80 year vs. 0.99 year) in positive HT group compared with negative HT group.

For spontaneous pregnancy: In the positive HT group, there was more spontaneous pregnancy (+19.8%) than in the negative HT group. The lapse of time to pregnancy was the same (6 months) in the two groups.

For simple stimulation: there were twice as many pregnancies in the positive HT group as in the negative HT group (22.1% vs. 12.2%). But the time to pregnancy was 3 times longer in positive HT group than in negative HT group (0.9 year vs. 0.3 year).

For intrauterine insemination (IUI): there were 50% less pregnancies (P<0.001) in positive HT group than in negative HT group (9.9% vs. 18.7%). But time to pregnancy was longer in the negative HT group than in positive HT group (1.2 year vs. 0.8 year).

For in vitro fertilization (IVF), no significantly difference was found between positive and negative HT groups (10.5% vs. 10.1%).

For intracytoplasmic sperm injection (ICSI): there were three times fewer pregnancies (P<0.001) in the positive HT group than in the negative HT group (7.5% vs. 28.8%). And time to pregnancy was shorter in the negative HT group than in the positive HT group (1.3 year vs. 1.8 year).

In table 2, after exclusion of altered sperm, similar results were obtained: total pregnancy rate was higher in the positive HT group than in the negative HT group (70.5% vs. 57.8%) (P<0.05).

Table 2: Occurrence of pregnancy by result of the post-coital test (PCT) after exclusion of altered sperm (< 20 M/ml, motile spermatozoa < 30%) mPositive PCT criterion: 1 or more motile spermatozoa in the whole cervix.

A negative HT was significantly associated with half chance of natural conception (38.8% vs. 20%) (P<0.05).

For simple stimulation, pregnancy rate was three times higher in the positive HT group than in the negative HT group (17.8% vs. 6.8%) (P<0.05).

For IUI, there were twice as many pregnancies in the negative HT group compared with positive HT group (30.5% vs. 13.2% p<0.01) and three times more in ICSI (27.1% vs. 9.3% p<0.01). For IVF, there was always no significant difference between groups (20.9% vs. 15.2% for positive and negative HT respectively).

In Table 3, we compared 4 different cut-off levels to consider a HT positive or negative, varying from one to 10 motile spermatozoa. We considered a positive result of a HT to be more than 1) 10 progressively motile spermatozoa per field or 2) 5 progressively motile spermatozoa per field or 3) one progressively motile spermatozoon per field or 4) one progressively motile spermatozoon in the whole cervical mucus sample.

Table 3: Occurrence of pregnancy by result of the post-coital test (PCT) after exclusion of altered sperm (< 20 M/ml, motile spermatozoa < 30%) *: p<0.05; **: p<0.01

With the third criterion (1 spermatozoon per field), we could see two significant differences between positive and negative HT with pregnancies by stimulation (19.6% vs. 8.1%) and ICSI (8.8% vs. 22.1%).

Differences were more obvious with the last criterion: at least one spermatozoon seen in the cervix appears to be the best cut off probably because of its simplicity and better reproducibility according to WHO 2010 [27]. We observed significant differences between positive and negative HT with spontaneous pregnancy (38.5% vs. 20.3%), pregnancy by treatment (61.2% vs. 79.7%), pregnancy by stimulation (17.8% vs. 6.8%), pregnancy by insemination (13.2% vs. 30.5%) and ICSI (9.3% vs. 27.1%).

HT was still recommended in WHO and continues to do so in the 2010 edition. In France, for ANAES (Agence Nationale d’Accréditation et d’Evaluation en Santé ), HT is still indicated in the first place contrary to the NICE (National Institute for Clinical Excellence) guideline that decides not to recommend the TH because of “its low predictive value on pregnancy rates”, based on 2 peer-reviewed publications [28,17].

We conducted this retrospective study to assess the usefulness of the controversial HT in investigating subfertility. We aimed to show the interest of the non-invasive and inexpensive HT in terms of a predictive diagnostic and especially of a therapeutic diagnostic.

The diagnostic interest is to identify a cervical factor in the subfertility and avoid classifying these couples in idiopathic subfertility. And it’s essential to understand the type of subfertility in order to be more efficient.

The therapeutic interest is to adapt the strategy according to the positivity or negativity of the test.

We showed predictive interest of the HT for spontaneous pregnancy. Like 3 studies [29,14,7], we have demonstrated that spontaneous pregnancy rate was higher with a positive HT than with a negative HT. Maybe when the HT is positive and the woman young, we must let the chance of natural conception and therefore give more time to the couple before moving on to simple stimulation.

With a negative HT, no clear conclusion can be drawn, but it seems logical to proceed directly to IUI (if all other criteria are met: patent tubes, good ovulation). Similar to our results, Van der Steeg et al., believe that IUI is an effective treatment for cervical factor subfertility [30]. And, if IUI failure, it might be worth considering ICSI rapidly.

Hypothesis that could explain a higher rate of pregnancies with ICSI in case of a negative TH is the presence of anti-sperm antibodies (ASA). Marshburn et al., has demonstrated, that a negative HT could be due to an immune subfertility [31]. Effectively, a poor sperm penetration of cervical mucus (HT negative) could be due to the presence of ASA either in the woman or in the man [32]. These ASA are directed against sperm surface antigens, interfering with sperm motility and their migration through the female reproductive tract. ASA also can inhibit capacitation or acrosome reaction [33-35]. These antibodies in the female can cause sperm agglutinate or become immobilised in the cervical mucus. ASA in the husband prevented the sperm from penetrating the cervical mucus, even though the sperm appear normal on seminal analysis. ASA have been considered as subfertility cause in 10-30 % of subfertile couples [36,37] and prevalence rates of ASA in subfertile men range between 6 and 11% [38].

In conclusion, for a negative HT and IUI failure, it would be judicious to detect ASA in both men and women, and shift rapidly towards ICSI.

Finally, to respond to the lack of a uniform definition of positive test, we have analyzed several cut off. For standardized the HT methodology, we have shown that the best criterion to consider a HT positive is, at least, one motile spermatozoon seen in the cervix, according to WHO 2010 [27].

Whereas some authors may feel uncertain about the method, the clinical importance and the therapeutic approach of HT, we are convinced of the usefulness of this test in diagnostic, predictive and therapeutic interests.