1 Obstetric & Gynecology Department, Poissy-Saint Germain Hospital, 78300 Poissy, Versailles, Saint Quentin en Yveline University and 2 Reproductive Medicine Laboratory, Poissy-Saint Germain Hospital, 78300 Poissy, Versailles Saint Quentin en Yveline University, France
3 To whom correspondence should be addressed at: Obstetric & Gynecology Department, Hôpital de Poissy-Saint Germain, 10 rue du Champ Gaillard, 78300 Poissy, France. Email: bob.wainer{at}wanadoo.fr
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Abstract |
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Key words: intrauterine insemination/number of motile spermatozoa inseminated/sperm morphology/sperm preparation
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Introduction |
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The prognosis value of the initial semen characteristics in assessing the likelihood of successful IUI is still the subject of debate (Horvath et al., 1989; Lelannou, 1994
; Ombelet et al., 1995
; Wainer et al., 1996
; Branigan et al., 1999
; Duran et al., 2002
), and studies on the impact of sperm morphology, before and after preparation, have led to divergent results (Francavilla et al., 1990
; Matorras et al., 1995
; Burr et al., 1996
; Wainer et al., 1996
; Karabinus and Gelety, 1997
; Dickey et al., 1999
; Hauser et al., 2001
).
The minimum recommended number of motile spermatozoa inseminated (NMSI) after preparation varies from 0.8 to 10 x 106, depending on the study (Horvath et al., 1989; Dodson and Haney, 1991
; Wainer et al., 1996
; Berg et al., 1997
; Van Voorhis et al., 2001
; Miller et al., 2002
). At lower values, IVF should be used.
The aim of our study was to assess the likelihood of IUI success as a function of the NMSI and of the percentage of morphologically normal spermatozoa, assessed after sperm preparation.
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Materials and methods |
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Indications for IUI included cervical and male factors, as well as unexplained infertility. Cervical factors were defined by a negative post-coital test with no sperm abnormalities. Normal semen analyses were defined by the threshold values of the WHO (1992) (concentration
20 x 106/ml, total count
40 x 106, progressive motility
50%, typical morphology
30%). The same three observers performed these microscopic analyses throughout the 10 years of the study. All semen samples were collected in the laboratory after 35 days of sexual abstinence. After liquefaction for 30 min at room temperature, volume, pH, sperm count and progressive motility were evaluated according to the WHO standard criteria. Sperm concentration was determined with a haemocytometer on two separate preparations of the semen sample (dilution 1:20 in Ringer solution). Sperm motility was determined by assessing at least five microscopic fields to classify at least 200 spermatozoa (x400 magnification). The motility was graded progressive, non-progressive or immotile.
Motile sperm were selected by: (i) a swim-up procedure from 1991 to 1994; (ii) a two-step Percoll gradient (45% and 90%) from 1995 to 1997; and (iii) a two-step pure sperm gradient from 1998 to 2000. In all cases, the motile sperm fraction was washed twice by centrifugation and the sperm pellet was resuspended in 0.35 ml of a capacitating medium. The following capacitating media were used successively: B2 (CCD, Paris France) from January 1991 to April 1997, Ferticult (Fertipro, Beernem, Belgium) from May 1997 to January 1999, B2 modified (CCD) from February 1999 to March 2000, and P1 (Irvine Scientific, Santa Ana, CA, USA) from April 2000 to December 2000. No medium was supplemented with albumin.
Sperm were then counted and progressive motility assessed to determine the total NMSI.
To analyse sperm morphology, smears were prepared from the whole ejaculated fraction and from the motile selected one. We used Schorr and Harris' haematoxylin staining procedure. The percentages of morphologically normal spermatozoa and of various sperm abnormalities were evaluated on 100 sperm at a final magnification of x1000, according to the method described by David et al. (1975) and modified by Jouannet et al. (1988)
.
All IUI cycles were accompanied by ovarian stimulation with HMG or recombinant FSH. Ovarian response was monitored by plasma E2 and LH concentrations and by ovarian ultrasonography. In the absence of spontaneous LH peaks, ovulation was induced by injection of 5000 IU HCG. The IUI was performed with a Frydman catheter (CCD) 40±4 h after HCG injection. In the presence of spontaneous LH peak the IUI was performed the next day. The patient received 300 mg/day of intravaginal micronized progesterone during the luteal phase until the day of -HCG testing. The principal assessment criterion was the clinical pregnancy rate/cycle, according to semen characteristics. A clinical pregnancy was defined as a pregnancy that was
-HCG-positive and had a gestational sac visible with ultrasonography.
Statistical analysis
A global 2-test was calculated for all population groups according to each set of criteria studied, to determine whether there was a statistically significant difference between the subgroups. Then a 2 x 2 comparative
2-test was carried out. A P-value <0.05 was considered significant. The cumulative rate curves were compared using the log rank test. We used the Cox model to examine the contribution of the independent variables.
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Results |
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The distribution of the different indications for IUI were: male factor subfertility 62.32% (n=554 couples), cervical factor infertility 16.98% (n=151 couples) and unexplained infertility 20.70% (n=184 couples).
The following sperm abnormalities, defined according to the WHO criteria, were observed: isolated oligospermia 5% (n=28), isolated asthenospermia 48% (n=265), isolated teratospermia 5% (n=28), oligoasthenospermia 14% (n=75), oligoteratospermia 2% (n=12), asthenoteratospermia 16% (n=88) and oligoasthenoteratospermia 10% (n=57).
Three hundred and thirty-one clinical pregnancies followed 2564 IUI cycles, for a clinical pregnancy rate/cycle of 12.91% and a clinical pregnancy rate/couple of 37%.
Fifty-three clinical pregnancies ended in spontaneous abortions (abortion rate 16%). There were 55 multiple pregnancies: 42 twins, 10 sets of triplets and three sets of quadruplets (multiple pregnancy rate 16.6%).
Of the couples with cervical factor infertility, 41.7% became pregnant (63 pregnancies for 151 couples). The cumulative pregnancy rate/couple was 38.4% (213 pregnancies for 554 couples) for those with male factor subfertility and 30% (55 pregnancies for 184 couples) for those with unexplained infertility.
Table I shows the clinical pregnancy rate/cycle as a function of the NMSI. When the NMSI was <1 x 106 in our series, the clinical pregnancy rate/cycle was 3.13%, significantly lower than in the subgroups with a NMSI 2 x 106 (P<0.01). The best pregnancy rate/cycle (14.75%) was obtained for NMSI between 5 and 10 x 106, but this rate did not differ significantly from those of any of the subgroups with a NMSI
2 x 106.
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These rates were quite similar, both when the global sperm morphology was excellent (normal sperm rate 70%, pregnancy rate 12.69%) and when it was very poor (normal sperm
20%, pregnancy rate 15.08%). The same is true for the abnormal acrosomal characteristics when considered alone. Accordingly, the clinical pregnancy rate was 10.38% (33 clinical pregnancies in 318 cycles) when the rate of normal acrosomes exceeded 80%, and 9.80% (10 clinical pregnancies in 102 cycles) when the rate of normal acrosomes was below 30% (not significant).
Sperm morphology was studied after preparation only from January 1992 onwards. We thus studied 2238 cycles and found that even when the post-preparation normal sperm rate was <30%, the clinical pregnancy rate/cycle was 11.31%, which was not significantly different from the pregnancy rates observed with higher percentages of normal sperm.
Table V shows the clinical pregnancy rates/cycle according to the NMSI and the percentage of normal spermatozoa after sperm preparation. For a normal morphology sperm rate <30% after preparation, the clinical pregnancy rate was 5.43% (5/92) when the NMSI was <5 x 106 and 18.42% (14/76) when the NMSI was 5 x 106. This difference is statistically significant (P=0.008). On the other hand, when the percentage of normal sperm was
30%, pregnancy rates did not differ significantly, regardless of the NSMI. Similarly, when the NMSI was
5 x 106, the pregnancy rates did not differ regardless of the percentage of normal sperm. In all, 104 IUI cycles were performed with a normal sperm rate after migration of between 20% and 30%, and nine clinical pregnancies occurred (pregnancy rate/cycle 8.65%). Fifty-one cycles with an NMSI <5 x 106 resulted in only one pregnancy (pregnancy rate/cycle 1.96%); 53 cycles were undertaken with a NMSI
5 x 106 and resulted in eight pregnancies (pregnancy rate/cycle 15.09%).
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We studied sperm morphology after preparation for 170 couples in which the man's semen analysis showed a normal sperm rate <30% in the semen analysis immediately before preparation. In these 481 IUI cycles, sperm morphology after preparation improved to a normal rate of 30% for 125 men (73.5%); it did not improve for 45 men (26.5%).
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Discussion |
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With an NMSI <1 x 106, the pregnancy rate/cycle was 3.13%, significantly lower than in the subgroups with an NMSI of 2 x 106. Most authors agree on this minimum threshold of 1 x 106 for the NMSI, and recommend IVF when this value is lower (Horvath et al., 1989
; Dodson and Haney, 1991
; Nulsen et al., 1993
; Brasch et al., 1994
; Lelannou, 1994
; Campana et al., 1996
; Huang et al., 1996
; Wainer et al., 1996
; Berg et al., 1997
). Conversely, Burr et al. (1996)
found no significant difference between pregnancy rates regardless of the NMSI, but only 35 cycles involved <1 x 106 motile spermatozoa.
Other authors have suggested using the total number of motile spermatozoa of the initial sperm count as the criterion for choosing between IUI and IVF, and have recommended threshold values ranging from 5 to 10 x 106 (Campana et al., 1996; Dickey et al., 1999
). The usefulness of this criterion, however, is limited by the variability of quality from one ejaculation to another, as well as the variable results of sperm preparations.
All forms of ART yield very mediocre results for women 40 years old (Van Der Westerlaken et al., 1998
). Our results confirm that IUI is no exception, with a clinical pregnancy rate/cycle of 5.04% for that age group. Many authors therefore suggest that women
38 years old begin directly or very quickly with IVF. Nonetheless, in our series, >200 cycles of IUI in women aged 38 and 39 years led to 32 clinical pregnancies, for a rate/cycle of 15.24%. This should encourage us not to automatically rule out IUI in this age range; for example, we could offer these patients three IUIs over a maximum period of 6 months before resorting to IVF.
Sperm morphology is another factor besides the patient's age and the NMSI that may influence the IUI results. Several IVF studies have shown that the fertilization rate decreases with a low level of normal sperm (Kruger et al., 1986; 1988
; Oehninger et al., 1988
). In a prospective study of 45 couples, Kruger et al. (1988)
assessed sperm morphology before preparation. They used two different morphological criteria: percentage of strictly normal forms and the morphological index, which groups the strictly normal spermatozoa and the slightly amorphous forms. The percentage of fertilized oocytes was 7.6% for men with a strictly normal spermatozoa rate <4% and a morphological index <30%. The fertilization rate was at least 63.9% when the strictly normal spermatozoa rate was >4% and the morphological index >30%.
It is thus logical that sperm morphology modifies the results of IUI. Van Waart et al. (2001) conducted a literature review on this subject: only six of 421 studies could be used. The analysis showed a significant improvement in the pregnancy rate above a 4% threshold for strict criteria. In our study, however, considering sperm morphology only, before or after preparation, did not help to predict IUI results. This was also the case when only the rates of acrosomal abnormalities were considered. Several other retrospective and prospective studies have reached the same conclusions (Matorras et al., 1995
; Karabinus and Gelety, 1997
; Dickey et al., 1999
). Conversely, some authors have observed that likelihood of pregnancy with IUI fell when the prepreparation percentage of normal sperm was low (Francavilla et al., 1990
; Toner et al., 1995
; Burr et al., 1996
; Lindheim et al., 1996
; Hauser et al., 2001
). The threshold for the percentage of spermatozoa with normal morphology below which IVF is recommended thus varies according to team and technique from 4% to 50%. When the strict criteria of sperm normality (Kruger et al., 1988
) were considered, there were fewer than 20 of these couples (i.e. below this threshold) in the population of each series (Toner et al., 1995
; Lindheim et al., 1996
; Hauser et al., 2001
), and no reliable conclusions can be drawn.
These divergent results raise several questions. Which reference criteria should be used to assess sperm morphology (David's, Kruger's, or others)? Should a single criterion be considered (for example a percentage <4% of strictly normal spermatozoa) or several criteria (level of strictly normal spermatozoa + morphological index + abnormal acrosomal characteristics, etc.)? The second issue is that morphological assesments may vary substantially according to the conditions of observation. Semen preparation may modify sperm characteristics considerably, and the number of motile spermatozoa and the morphological criteria should logically be assessed after semen preparation. In our study, morphology improved after preparation in nearly three-quarters of the men (125/170) with a rate of normal sperm before preparation <30%: the rate of normal features exceeded the 30% threshold. This result justifies the use of morphology only after preparation for assessing IUI prognosis.
Our study shows that, when morphologically normal sperm account for <30% of a sample after preparation, the NMSI strongly influences the likelihood of successful IUI. An initial semen preparation that reveals this low level of spermatozoa with normal morphology suggests that IUI will involve 5 x 106 motile spermatozoa.
Ombelet et al. (1997) suggested that the NMSI could compensate for inadequate sperm morphology after observing that morphological scores were significantly lower (<4% of normal forms) in cases of pregnancy failure only in the group with a NMSI <1 x 106. In this study the morphological score was calculated before preparation.
While most teams already offer IVF or even ICSI in cases of very severe teratospermia (<4% normal forms), the initial approach to severe teratospermia (1030% normal forms) is a more difficult question. Our study shows that IUI is not an unreasonable approach when the NMSI is 5 x 106. Our series included 104 cycles when normal forms were between 20% and 30%: there were nine clinical pregnancies (8.65%), with a clinical pregnancy rate ranging from 1.96% for a NMSI <5 x 106 to 15.09% for a NMSI
5 x 106. When normal forms accounted for <20%, there were nonetheless 10 pregnancies in 64 cycles, with a pregnancy rate ranging from 9.76% to 26.09%, depending on the NMSI.
A broader study would improve the assessment of the results obtained for the subgroups with normal sperm rates of 2030%, 1020% and <10%.
It might also be interesting to calculate the threshold value of motile normal spermatozoa necessary to achieve pregnancy. Such a calculation, however, would require assessing the morphology of the motile spermatozoa in the migrated fraction only, which would be technically difficult.
Severe teratospermia may be associated with oligospermia that makes it impossible to obtain an NMSI 5 x 106. A double sperm collection, the day of the IUI, might be realized, since the threshold of 5 x 106 might be reached by combining two semen samples. Future studies should address this question. If this NMSI still cannot be reached, IVF should be recommended.
Overall, our study confirms the efficacy of IUI with gonadotrophin stimulation for a large population. Clinical pregnancy rates are satisfactory, including for patients aged 38 and 39 years old, as long as the NMSI exceeds 1 x 106. Because semen preparation often reduces the level of abnormal sperm, IUI prognosis can best be assessed with the post-preparation morphological characteristics. Our results show that for couples with a rate of normal sperm after preparation <30%, >5 x 106 of motile spermatozoa must be inseminated. If this NMSI threshold cannot be reached, IVF should be recommended to these couples.
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References |
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Submitted on April 17, 2004; accepted on June 2, 2004.
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