1 Assisted Conception Unit, Chelsea & Westminster Hospital, London SW10 9NH and 2 Clinical Director of Surgery, Charring Cross Hospital, London W6 8RF, UK
3 To whom correspondence should be addressed. Email: james.nicopoullos{at}chelwest.nhs.uk
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Abstract |
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Key words: HIV/intrauterine insemination/predictors of outcome/sperm washing
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Introduction |
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Reproductive assistance to HIV-discordant couples can make a significant impact in prevention of viral transmission. In a serodiscordant couple, the female partner has a 0.10.2% risk of acquiring HIV per act of unprotected intercourse (Mastro and Vincenzi, 1996), and attempting to conceive naturally carries a serious risk to the uninfected woman and child (Mandelbrot et al., 1997
). The virus is present in semen as free virus in the seminal plasma and as cell-associated virus in the non-sperm cells, but there is little evidence to support HIV being able to attach to, or infect, sperm. The technique of sperm washing, pioneered in Milan (Semprini et al., 1992
), involves the sperm being washed free of seminal plasma and non-sperm cells before insemination into the woman at the time of ovulation. Following studies confirming the viability of sperm washing as an effective risk reduction option for HIV-discordant couples wishing to conceive (Kim et al., 1999
), several centres in Europe have introduced the technique. The Chelsea and Westminster Assisted Conception Unit has treated HIV+ men with sperm washing as part of a risk reduction programme since 1999, and has performed the largest series of inseminations in the UK (Gilling-Smith, 2000
, 2003
).
Previous reports have assessed the effect of HIV infection on sperm parameters with inconsistent results. Krieger et al. (1991) reported no difference in any parameter and Crittenden et al. (1992)
reported only a decrease in the percentage of motile sperm in HIV+ men. Others, however, have reported a more significant effect of HIV on semen parameters (Dondero et al., 1996
; Muller et al., 1998
; Dulioust et al., 2002
) when compared to controls. The effect of HIV treatment, duration of infection and markers of HIV infection such as CD4 cell count and viral load (VL) has also been reported. CD4 count has been demonstrated to affect semen volume (Politch et al., 1994
; Lasheeb et al., 1997
; Dulioust et al., 2002
), sperm concentration (Politch et al., 1994
; Dondero et al., 1996
; Dulioust et al., 2002
), total count (Dulioust et al., 2002
), motility (Crittenden et al., 1992
; Dondero et al., 1996
; Lasheeb et al., 1997
) and morphology (Politch et al., 1994
; Dondero et al., 1996
). One study has also assessed the effect of VL on sperm parameters with a negative correlation with sperm motility and morphology (Dulioust et al., 2002
).
Several determinants of success have been identified for intrauterine insemination (IUI). IUI outcome is improved by the use of injectable gonadotrophins for ovulation induction in both unexplained infertility (Cohlen et al., 1998) and mild male infertility (Guzick et al., 1999
), and by the use of clomiphene citrate for ovulation induction in only unexplained infertility (Arici et al., 1994
). Similarly, maternal age and number of pre-ovulatory follicles have also been reported to impact on IUI outcome (Duran et al., 2002
). The effect of several sperm parameters has also been investigated. Although sperm motility (Montanaro et al., 2001
), morphology (Van Waart et al., 2001
) and post-preparation motility (Stone et al., 1999
; Hendin et al., 2000
) have been shown to affect IUI outcome, total motile count (TMC) inseminated is the marker most consistently shown to be a determinant of success (Toner et al., 1995
; Campana et al., 1996
; Ombelet et al., 1997
; Van der Westlaken et al., 1998
; Stone et al., 1999
; Khalil et al., 2001
).
The aim of this study was to: (i) present the first UK data assessing the effect of HIV infection on sperm parameters; (ii) assess the effect of markers of HIV infection on sperm parameters in our large cohort of patients; (iii) present the first data to determine whether there are predictors of success in IUI/sperm washing in HIV+ men.
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Materials and methods |
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Table III presents the HIV-related characteristics of the men in group 3, i.e. samples used for sperm washing and IUI. Sixty samples (45.1%) came from men who acquired HIV infection through sexual intercourse, 22 (16.5%) through former intravenous drug abuse (IVDA), 18 through haematological means (contaminated blood or blood products) and in 33 (24.8%) cases the mode of transmission was unknown. The known duration of HIV infection ranged from 0.5 to 17 years, and the majority of patients (55.6%) were on anti-retroviral therapy at the time of semen analysis and IUI. All samples came from men without symptoms of autoimmune deficiency syndrome (AIDS) at the time of collection. CD4 counts and HIV RNA blood plasma concentrations (VL) used to correlate against semen samples and IUI outcome were the most recent routine samples taken as part of the regular HIV monitoring process. The median CD4 count was 410 cells/mm3 (range 1001207), and the majority of patients (59.1%) had an undetectable HIV RNA blood plasma concentration (range, undetectable: 150 000 copies/ml).
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Semen samples for IUI treatment were produced on the morning of insemination (at 08:00), following
3 days of abstinence from ejaculation. After assessment, samples were prepared using a 4590% density gradient to separate progressively motile HIV-free sperm from the infected non-sperm cells (NSC) and seminal plasma found in the supernatant. Puresperm (Nicadon, Sweden) solution was diluted to 45 and 90% using sperm buffer medium (Cook®, Australia) and the solutions warmed to room temperature in centrifuge tubes before use. The ejaculate was layered over the prepared density gradients and centrifuged at 200 g/room temperature for 20 min. Following centrifugation, the supernatant was aspirated, and the sperm pellet removed, resuspended in fresh media and centrifuged at 200 g/room temperature for 10 min. This wash procedure was repeated twice more, before allowing the sample to swim up. The supernatant volume recovered after swim-up was
550 ml.
An aliquot of washed sperm (100 ml) was subsequently tested for detectable HIV RNA using a nucleic acid-based sequence amplification (NASBA) assay according to the manufacturer's instructions (Biomérieux, UK). In seven of the 140 cycles commenced (5%), the NASBA test was positive, necessitating cancellation of the cycle. In the remaining 133, the NASBA test was negative and IUI was carried out (at
16:00). In the lithotomy position, the washed tested sperm (
450 ml) was injected using an insemination catheter (Cook) into the uterine cavity. A pregnancy test was performed 2 weeks later.
Sperm washing programme
Careful preconception counselling, both together and individually, was undertaken prior to treatment in all cases of sperm washing during which several issues were addressed. The couple were advised that sperm washing is a risk reduction technique and does not eliminate risk of horizontal and vertical transmission entirely. The impact of possible treatment failure and of the current and future health of the male partner was addressed. Although a very sensitive area, frank discussion about coping with a child when one parent is HIV+, or as a single parent, is essential. The real possibility that treatment may not be successful was also discussed, enabling them to make an informed decision about whether they wished to begin treatment.
Following counselling, a full sexual health screen and fertility screen of both partners followed. Sexual health screening is mandatory as genital infections or lesions in either partner increase the risk of HIV transmission (Fleming and Wasserheit, 1999). All couples offered IUI required an early follicular phase FSH <12 IU/ml, at least one patent tube and one routine semen analysis showing normal parameters according to criteria set by the WHO Laboratory Manual, 1992
. If there was evidence of spontaneous ovulation (mid-luteal progesterone >30 ng/ml), the couple were initially offered natural cycle IUI. They were scanned from day 8 or 9 of their cycle to ensure correct timing of insemination, and scans performed every 23 days thereafter to track the developing follicle. Once the lead follicle was 17 mm in diameter, either spontaneous ovulation occurred (detected by ovulation predictor kits using early morning urine samples; Assure, Conception Technologies, USA) or hCG (5000 IU) was given by subcutaneous injection if necessary. IUI was subsequently performed 24 h post positive LH surge on ovulation predictor kit, or 40 h post hCG injection.
If the female partner was anovulatory, or following three to six failed natural cycles, insemination was performed in conjunction with ovulation induction using either clomiphene (first line) or gonadotrophins (second line).
Statistical analysis
For comparisons of sperm parameters between groups 1 and 2, the first sample produced for each HIV+ patient was compared to the control samples to minimize potential bias from repeat samples from the same patients. In the analysis of the predictors of IUI success, and in the comparison of groups 3 and 4, all insemination samples were analysed.
For comparisons between groups, age, semen volume, sperm concentration, total count, motility and morphology were considered as continuous variables. The ShapiroWilk W-test of normality was utilized. As none of the factors assessed was normally distributed, the non-parametric MannWhitney U-test was used to compare the groups.
Spearman rank testing was performed to detect any correlation between sperm parameters and CD4 count, VL and years since diagnosis (continuous variables). MannWhitney U-testing was also used to detect an effect of categorical variables VL (detectable versus undetectable), CD4 count (<400 versus >400 mm3) and use of anti-retrovirals on sperm parameters.
For analysis of effect on IUI outcome, pre-preparation total sperm count, motility, morphology, post-preparation total motile count (TMC) inseminated, CD4 count, VL, duration of infection, duration of use of anti-retroviral therapy, maternal age, stimulation regime and the number of follicles were analysed as categorical variables and their effect assessed using Fisher's exact test and 2-tests.
All statistical analysis was performed using Analyse-It statistical software for Microsoft Excel.
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Results |
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There was no significant difference in mean male age between the HIV+ and control men who produced samples (37.3 versus 37.2 years). Ejaculate volume (median, mean of 3.3, 3.1 ml versus 2.9, 2.8 ml), sperm concentration (70.1, 53.1 versus 54.8, 39.7 x 106/ml) and calculated total sperm count (222.4, 159.6 versus 147.3, 104.0 x 106) were all significantly higher in the control group 2 than in the HIV+ men in group 1 (P<0.05, MannWhitney U-tests). Both the estimation of the percentage of total motility (66.1, 68 versus 52.7, 55%) and the assessment of the percentage of progressively motile sperm, i.e. type a (rapid to very progressively motile) + type b (slow or sluggish progressive motility; 54.7, 55% versus 48.5, 54%) were also significantly lower in the HIV+ men. The number of morphologically abnormal sperm was significantly higher in the HIV+ men (76.7, 77% versus 72.0, 73%).
Patient and semen characteristics of the samples used for sperm washing and IUI are presented in Table II. There was no significant difference in male age between the IUI samples from HIV+ men (group 3) and control men (group 4, 38.8 versus 37.6 years). Although ejaculation volume was again significantly lower in HIV+ men (3.5, 3.1 versus 2.9, 2.8 ml), sperm concentration of the samples taken for IUI was significantly higher (66.1, 60.0 versus 57.5, 46.1 x 106/ml). Therefore, there was no significant difference in the calculated total sperm count between groups 3 and 4 (182.6, 157.5 versus 194.9, 140 x 106). The proportion of motile sperm (62.8, 65% versus 55.2, 55%) was significantly lower in the IUI samples from HIV+ men, but the percentage of progressively motile sperm, i.e. type a + type b showed a decrease in the HIV+ group that was only of borderline statistical significance (50.9, 50% versus 47.1, 47%). When analysed separately, the proportion of sperm that were shown to have rapid to very rapid progressive motility (type a) was significantly higher and the proportion shown to have slow or sluggish progressive motility (type b) was significantly lower in the HIV+ samples. The number of morphologically abnormal sperm was again significantly higher in the HIV+ samples (74.4, 75% versus 71.0, 72%).
Relationship between markers of HIV infection and semen characteristics
Table IV shows the relationship between markers of HIV infection and semen characteristics.
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There was no significant correlation between any semen parameter and HIV RNA VL, and no significant difference between any parameter comparing samples from men with undetectable (n=65) and detectable VL (n=45; all P>0.05). There was also no correlation between duration of HIV infection and any semen parameter (Spearman's correlation, P>0.05) and the use of anti-retroviral medication did not significantly affect any semen parameter (MannWhitney U-test, P>0.05).
Outcome of IUI and predictors of success
Of the 140 cycles commenced, 133 led to insemination with 25 positive pregnancy tests. This gave an overall clinical pregnancy rate (CPR) of 17.9% per cycle commenced and 18.8% per insemination.
Table V presents the outcome of the sperm washing/IUI inseminations, analysed according to patient and sperm characteristics. There was no significant difference in outcome according to paternal age. Although there was an obvious trend for worsening outcome with increasing maternal age across the four groups evaluated (CPR of 31.8, 19.5, 15.7 and 10.5% for maternal ages of <30, 3034, 3539 and >40 respectively), this did not reach statistical significance (P>0.05). Similarly, there was also a trend towards improved CPR per insemination as total sperm count of the raw semen sample increased (CPR of 8.1, 18.8 and 27.3% for counts of <100, 100200 and >200 x 106 respectively), but this only reached borderline statistical significance (P=0.08). Pre-preparation sperm motility (using a 50% threshold), sperm morphology (using both a 70 and 85% abnormal threshold) and TMC inseminated had no significant effect on clinical pregnancy outcome.
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Discussion |
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Our comparison demonstrates that every parameter analysed (ejaculate volume, sperm concentration, total count, motility, progressive motility and morphology) was significantly impaired in HIV+ men. Although previous studies have reported sperm parameters to be impaired, this is the first to demonstrate such a consistent effect on all parameters.
The first study to present data on sperm characteristics in HIV+ men found no difference in any parameter between their small (n=24) cohort and a control group of HIV men providing semen for general fertility investigation (Krieger et al., 1991). In a slightly larger group of HIV+ men (n=38), Crittenden et al. (1992)
demonstrated significantly lower sperm motility (% motile, motile sperm density and total motile count per ejaculate) but no difference in ejaculate volume, sperm concentration or total count compared to an HIV control group made up of predominantly haemophiliacs (n=51). Dondero et al. (1996)
compared HIV+ men (n=21) to both a high-risk lifestyle and control group with a significantly lower sperm concentration, motility (determined by linear progression and straight line velocity
25 mm/s) and morphology.
In the largest group of HIV+ men studied (n=250), 6% were found to be azoospermic and 45% had normal concentration and motility according to WHO criteria (Muller et al., 1998). When compared to a small control group of fertile HIV men, they were found to have significantly lower ejaculate volume, sperm concentration and motility, but computer-assisted sperm analysis (CASA) demonstrated no difference in any measurement of motility.
The discrepancies in these reports may be due to the small numbers in some studies, methodological variations, differing levels of HIV disease in the study groups as well as considerable variation in the choice of control group. The most recently published study (Dulioust et al., 2002) of 189 HIV+ men avoided some of these biases. As with our methodology, their study population consisted of a clinically homogeneous group of HIV+ men free of AIDS symptoms and who were therefore well enough to be considered for fertility treatment. As a diagnosis of AIDS (Krieger et al., 1991
; Muller et al., 1998
) and disease progression (by CDC clinical categories, Muller et al., 1998
) is significantly associated with spermatogenesis, this minimizes the possible effect of the immunological and clinical manifestations of advanced disease. Furthermore, the choice of healthy partners of women undergoing IVF for tubal subfertility as the control group avoided any bias from the use of sperm from men of proven fertility. They reported a decrease in ejaculate volume and total sperm count with no difference in sperm concentration or morphology in the HIV+ men. Although they reported no significant difference in progressive motility (a+b) they did suggest a significant shift from type a to b.
In contrast, we reported significant decline in concentration, progressive motility (predominantly due to a decrease in type b motility) and morphology, as well as volume and total sperm count in our comparison of similarly selected groups.
Although this removes several potential areas of bias, there still remains possible bias towards better sperm parameters in the control groups as a consequence of not including men with oligoasthenoteratozoospermia (and likely to require ICSI), who would therefore not have been eligible for IVF. In part, however, this had been addressed in the two early papers that used controls of men undergoing initial fertility investigation (Krieger et al., 1991) and sperm donors (Crittenden et al., 1992
) and still reported significantly impaired sperm characteristics.
Similarly, abstinence delay can also impact on sperm parameters (a minimum of 3 days was stipulated prior to analysis with no stipulated maximum in our cohort). It could be argued that the HIV+ men may potentially have longer abstinence periods due to the effect of illness on libido, fear of transmission and cultural differences in frequency of masturbation. However, although these data were incomplete and therefore not integrated in the analysis, there was no obvious difference in abstinence period between the HIV+ and HIV samples where the data were available. Moreover, Dulioust et al. (2002) found impaired parameters in their cohort before and after matching for abstinence delay.
We further compared samples from men selected on clinical grounds to be suitable for IUI (groups 3 and 4). Although ejaculate volume remained significantly lower, total count was similar as sperm concentration was in fact higher in the HIV+ men. Similarly, although motility remained lower, the difference in progressive motility did not reach statistical significance. The percentage of abnormal sperm remained higher in the HIV+ men in this comparison. Although there may be bias from the inclusion in the control/IUI group of couples where the male partner may have mild male-factor subfertility, this comparison, along with the high IUI success achieved (CPR of 17.9% per cycle commenced and 18.8% per insemination), should allow us confidently to counsel couples undergoing IUI where the male partner is HIV+ that, although sperm parameters are impaired in HIV+ men, once a clinical decision has been made that they are suitable for IUI, the effect of HIV on sperm is less significant.
When sperm characteristics were correlated with markers of HIV infection, we found a significant correlation between CD4 cell count and ejaculate volume, sperm concentration, total sperm count, sperm motility, progressive motility and sperm morphology. Previous reports have again shown variable effects of CD4 counts on sperm characteristics. Dulioust et al. (2002) found only ejaculate volume to be correlated with CD4 count, but sperm concentration and total count were lower in those men with CD4 <200 cells/mm3. Crittenden et al. (1992)
and Lasheeb et al. (1997)
found CD4 count to correlate only with motility, whilst Dondero et al. (1996)
and Muller et al. (1998)
found CD4 count to positively correlate with motility and negatively correlate with abnormal morhology. Unlike Dulioust et al. (2002)
who found VL to correlate with type b motility and sperm morphology, we found that VL had no effect on any parameter. As previously reported in several small series (Krieger et al., 1991
; Crittenden et al., 1992
; Dondero et al., 1996
; Lasheeb et al., 1997
), we found no difference in any parameter in those taking anti-retroviral medicaton. Similarly, the differences demonstrated in sperm parameters between HIV+ men and controls were consistent even after sub-analysis of the positive men according to whether they were on anti-retoviral medication. The effect of anti-retrovirals is difficult to separate from the effect of HIV infection and few studies have prospectively assessed the effect of treatment. Politch et al. (1994)
found that those on AZT treatment, regardless of disease stage, had parameters similar to those of untreated early disease stage patients. One study assessed 26 men about to start treatment for 12 weeks, reporting an overall increase in sperm motility and normal morphology, with no effect on sperm count (Robbins et al., 2001
). Although mitochondrial DNA damage as a consequence of nucleoside analogue toxicity has been reported (Mital et al., 2001
), it may be that any potential deleterious effect of the medication is negated by the effect of improved health on spermatogenesis.
The effect of CD4 count on the post-preparation samples used for IUI was assessed for the first time in this study. There remained a significant effect of CD4 count on sperm concentration, morphology and TMC inseminated.
In the USA in 1990, the Centers for Disease Control recommended against insemination of women with semen from men infected with HIV, following a single report of HIV transmission to a woman who underwent IUI using sperm from her HIV+ husband (Centers for Disease Control, 1990), due to sub-optimal semen processing. Others, predominantly in the USA, have proposed ICSI as the method of choice for assisted reproduction regardless of semen parameters, to minimize exposure from a higher number of sperm required in IUI or IVF (Loutradis et al., 2001
). One series of ICSI cycles in HIV-1+ men reported a fertilization rate, clinical pregnancy rate and ongoing pregnancy rate per transfer of 65, 46 and 31% respectively (Sauer and Chang, 2002
). This series also used ICSI as the method of choice for risk reduction without washing and NASBA testing, with only 57% of men having any semen abnormality of any kind. Despite these reports, we remain of the opinion that in order to safeguard patients from technical errors during washing, viral detection testing of semen samples prior to insemination is the method of choice. Indeed, in animal models, studies have demonstrated contamination of the oocyte and resulting embryos with HIV following the ICSI procedure (Perry et al., 1999
). The sensitivity of NASBA testing also allows us confidently to choose between IUI, IVF and ICSI based solely on the fertility history and results of investigation. This removes the risks of unnecessary stimulation cycles as well as the potential genetic implications of ICSI (Johnson, 1998
). This view is now supported by reports of >300 healthy children born after >2300 cycles of sperm washing (and viral detection testing) and IUI or IVF/ICSI worldwide, with no reported seroconversions in either partner or children (Semprini et al., 1992
, 1998
, 2001
; Marina et al., 1998a
,b
; Tur et al., 1999
; Gilling-Smith, 2003
).
Our analysis of the predictors of IUI success may also allow us to maximize the chances of success for a couple seeking sperm washing as a risk reduction technique. Although there is a trend towards improved CPR with controlled ovarian stimulation, at our present sample size this does not achieve statistical significance. This supports our current practice of initially offering couples natural cycle IUI, unless anovulatory, before proceeding with ovulation induction after several failed cycles. In view of the trend towards impaired outcome with increasing age, however, it may also be reasonable to proceed with controlled ovarian stimulation and IUI immediately at advanced maternal ages. This option may also be sensible in view of the cost involved per cycle, minimal funding available for the fertility treatment of HIV-infected couples and the low incomes of many couples seeking treatment (Gilling-Smith, 2003).
Although sperm parameters have been shown successfully to predict the outcome of IUI (Toner et al., 1995; Campana et al., 1996
; Ombelet et al., 1997
; Van der Westlaken et al., 1998
; Stone et al., 1999
; Hendin et al., 2000
; Khalil et al., 2001
; Montanaro et al., 2001
; Van Waart et al., 2001
), none was shown to have a significant impact on IUI outcome following sperm washing in our cohort of patients. Although there was a trend towards improved outcome as total sperm count increased across the three groups (<100, 100200, >200 x 106), only 6% (n=9) of the samples used had a total count below the WHO threshold of 40 x 106 from which two pregnancies ensued.
Only markers of HIV infection significantly affected IUI outcome in our cohort of cycles. CPR was significantly higher in those cycles from men with low VL (<1000 copies/ml) and in those from men on anti-retroviral therapy. However, CD4 count had no impact on IUI outcome.
The mechanism by which HIV infection alters semen parameters remains unclear. Similarly, why CD4 count (but not VL) correlates with sperm parameters, and why VL and the use of anti-retrovirals (but not CD4 count) predict IUI outcome in HIV+ men also remains unclear.
In the studies that include men with advanced disease, altered parameters have been explained by the effect of severe systemic disease and infection with other microorganisms on spermatogenesis (Krieger et al., 1991). Crittenden et al. (1992)
postulated that the effects they reported of HIV on motility may be a consequence of the lower testosterone levels previously reported in HIV+ men (Dobs et al., 1988
) which may impair acquisition of sperm motility or epididymal sperm maturation. The correlation they reported between motility and CD4 count was explained by either the development of symptomatic disease or more prolonged viral carriage. However, the effect of CD4 on sperm parameters is likely to be a direct effect of advanced disease status on spermatogenesis and sperm maturation (and testosterone production) rather than on viral carriage, as in our study duration of diagnosis had no direct effect on any parameter. This is supported by findings of testicular atrophy in autopsy reports on men with AIDS (Chabon et al., 1987
). Dondero et al. (1996)
reported a significant increase in cytoplasmic droplet forms and immature germ cells in HIV+ compared to the control groups, which may suggest a stress condition after HIV infection affecting spermatogenesis and epidydmal function.
In summary, although the majority of HIV+ men have sperm parameters that are within the defined WHO normal range, we found all parameters to be significantly impaired compared to HIV controls and to correlate with CD4 cell count as a likely consequence of the deleterious effects of chronic disease on the male reproductive system. As more couples seek fertility advice, the sperm washing programme allows HIV+ men to father children whilst minimizing the risk of viral transmission. We have found the use of anti-retroviral medication and low HIV RNA serum VL measurements to be factors that significantly improve IUI outcome regardless of CD4 count, sperm parameters and stimulation regime. It may therefore be sensible to advise HIV+ men to commence anti-retroviral medication to minimize VL at the time of treatment. The mechanism by which this occurs requires further investigation.
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Acknowledgements |
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Submitted on April 13, 2004; accepted on June 26, 2004.