Reproductive BioMedicine Online (2016) 32, 584590

w w w. s c i e n c e d i r e c t . c o m
w w w. r b m o n l i n e . c o m

ARTICLE

Semen residual viral load and reproductive

outcomes in HIV-infected men undergoing ICSI
after extended semen preparation
Maria Jose Zamora a, Albert Obradors a, Bryan Woodward b,
Valerie Vernaeve a, Rita Vassena a,*

a
Clnica EUGIN, Barcelona 08029, Spain; b IVF Consultancy Services, 144a New Walk, Leicester, LE1 7JA, UK
* Corresponding author. E-mail address: rvassena@eugin.es (R Vassena).

Maria Jos Zamora is a Senior Embryologist (ESHRE Regular Track 2008) with extensive experience in andrology
and embryology laboratory techniques as well as PGD techniques. She obtained her Bachelor of Science degree
in Biology in 1999 and her Masters in Human Reproductive Biology and Assisted Reproduction Techniques in 2001.
In 2005, she joined Clnica EUGIN in Barcelona, where she developed reproductive clinical care. Her main in-
terest is improving the results and efcacy of laboratory outcomes in terms of embryo culture, oocyte and embryo
vitrication and pregnancy rates.

Abstract The aim of this study was to evaluate the residual presence of the human immunodeciency virus (HIV) following a triple
gradient extended semen wash from ejaculates of serodiscordant couples, and analyse their reproductive outcomes after intracy-
toplasmic sperm injection (ICSI). For this purpose, a retrospective analysis of our database was performed in serodiscordant couples,
with HIV-infected men and non-infected women, using fresh or frozen sperm with ICSI in oocytes from either the patients or donors
from January 2006 to September 2013. Overall, the rate of positive HIV test after semen washing was 1.86%. The positive beta human
chorionic gonadotrophin, clinical and ongoing pregnancy rates in patients with their own oocytes were 47.1%, 37.5% and 30.8%, re-
spectively, and 58.6%, 50.8% and 39.1%, respectively, in oocyte donation cycles. To summarize, the described method of sperm washing
based on triple gradient sperm selection coupled with extensive centrifugations is a highly reliable technique for HIV removal, as it
provides lower than reported post-wash positive tests while maintaining high pregnancy rates in assisted reproduction cycles. Despite
extensive personnel training and effectiveness of the washing protocol, post-wash HIV test on semen is recommended to identify
residual positive samples.
2016 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

KEYWORDS: assisted reproductive treatment, HIV, ICSI, sperm wash

http://dx.doi.org/10.1016/j.rbmo.2016.02.014
1472-6483/ 2016 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
Semen wash in HIV-positive men
Introduction
Until the development of highly active antiretroviral therapy
(HAART), women infected with the human immunode-
ciency virus (HIV) had a high risk of transmitting HIV to their
child during pregnancy and perinatally, and couples with one
HIV-infected partner had a high risk of horizontal transmis-
sion of the virus to the uninfected partner. Patients were
advised not to get pregnant, and couples in which the man
was HIV-positive were not considered eligible for assisted re-
productive treatment (Gilling-Smith et al., 2001).
With the introduction of HAART, both life expectancy and
quality of life for HIV-infected people improved signi-
cantly (Scandlyn, 2000). Nowadays, couples with an HIV-
infected partner consider having children and are willing to
take transmission risks in an assisted reproductive technol-
ogy programme. (van Leeuwen et al., 2008) HIV-positive pa-
tients have reproductive desires and reproductive needs, as
rates of infertility are higher in HIV-infected people; in men,
for instance, semen parameters can decrease due to the HIV
infection or HAART treatment-associated hypogonadism occurs
(Bujan et al., 2007; Nicopoullos et al., 2004; van Leeuwen
et al., 2004). The availability of assisted reproduction for HIV-
discordant couples has had a signicant impact in the pre-
vention of viral transmission (Semprini et al., 1992). Since HIV
receptors (CD4, CCR5, CXCR4) are not expressed by sperma-
tozoa, a combination of density gradient centrifugation fol-
lowed by swim-up has been proposed as a way to separate
HIV-free, motile spermatozoa from seminal plasma and non-
seminal cells, thus preventing infection of female partners
(Marina et al., 1998; Semprini et al., 1998).
Sperm washing coupled with intrauterine insemination (IUI),
IVF, or intracytoplasmic sperm injection (ICSI) is safely used
in serodiscordant couples without any case of seroconversion
reported (Barnes et al., 2014), although theoretical risks of
viral transmission cannot be completely ruled out (Peckham
and Newell, 2000). Prepared samples should be screened for
HIV presence before use, and only HIV-free samples should
be used for assisted reproductive treatment (WHO, 2010).
Despite the availability of very sensitive polymerase chain
reaction (PCR)-based HIV detection assays, many assisted
reproduction centres do not offer treatment for HIV
serodiscordant couples, who might resort to unprotected in-
tercourse in order to achieve a pregnancy.
The aim of this study was to evaluate the residual pres-
ence of HIV following a triple gradient extended semen wash
from ejaculates of serodiscordant couples, and analyse their
reproductive outcomes after ICSI cycles using either oocytes
from the female patient or a donor.
Materials and methods
Study population
This study included data from 269 sperm wash procedures (cor-
responding to 218 men/couples) carried out from January 2006
to September 2013 at a large private assisted reproductive
treatment clinic in Spain. So far, 183 couples have under-
gone assisted reproductive treatment, resulting in 234 com-
pleted cycles. Of these, 105 cycles were performed using the
womans own oocytes and 129 using donor oocytes.
585
All female patients were aware of their partners HIV status,
and informed consent was obtained after extensive counsel-
ling on the marginal risks of HIV transmission involved in the
procedure. In order to be accepted in the IVF programme, male
partners were required to be under the care of an infec-
tious disease specialist, who certied the time elapsed since
diagnosis, route of transmission, ongoing antiretroviral therapy,
viral load and CD4 counts, and suitability for treatment. There
were no specically set threshold limits for HIV viral load
(copies/ml) and CD4 counts (cells/mm3) in blood for enrol-
ment in the IVF programme.
A standard fertility evaluation was performed in both part-
ners. Both men and women (patients and donors) were also
tested for hepatitis C virus, hepatitis B virus, gonorrhoea, chla-
mydia, and syphilis. A determination of HIV viral load and HIV
antibody was performed in women before treatment and again
15 days after embryo transfer, in order to conrm the lack
of infection (GESIDA, 2013).
Semen wash procedure
Semen samples were obtained by masturbation after 3 to 5
days of abstinence. After liquefaction at room tempera-
ture, ejaculates were analysed with a computer-assisted sperm
analyser (Sperm Class Analyser, SCA Human Edition, Mi-
croptic S.L., Barcelona, Spain) and classied (WHO, 2010).
Semen samples were transferred into 15 ml conical tubes
(Falcon) and diluted 1/1 (vol/vol) with sperm medium (SM;
Sperm Rinse, Vitrolife), with a maximal volume of 6 ml (3/3)
in each tube. The diluted samples were then centrifuged at
400g for 20 min and the supernatants were discarded using
a disposable sterile Pasteur pipette for each tube. Pellets were
then re-suspended by adding 1 ml of fresh sperm medium per
100 150 million spermatozoa. One conical tube of triple-
density gradient (90%70%45%; Sperm Grad, Vitrolife/ Sperm
Rinse, Vitrolife) with 1 ml volume of 45% fraction, 1 ml of
70% fraction, and 2 ml of 90% fraction was used for each 1 ml
of re-suspended sample. Severely oligospermic samples, with
two million or fewer total motile spermatozoa, were pro-
cessed using smaller volume gradients. For all samples, 1 ml
was gently layered on top of the triple-density gradient and
centrifuged at 300g for 20 min. Supernatants were dis-
carded with extreme care using 1000 l micropipette tips.
Pellets were unied in a new conical tube, re-suspended in
5 ml of sperm medium and centrifuged at 250g for 10 min.
The supernatant was discarded and the pellet was trans-
ferred into a new conical tube, re-suspended with 2.5 ml of
sperm medium and centrifuged again at 250g for 10 min
(Figure 1). After centrifugation, the supernatant was dis-
carded using a disposable sterile Pasteur pipette and 1 1.2 ml
of sperm medium was gently added for swim-up. The tube was
placed at a 45 angle and incubated for 1 h at room tem-
perature. After incubation, the upper 1 ml of medium was re-
covered and divided into two parts. One part was used for
post-wash HIV testing and the other half was used for ICSI
treatment, once PCR results were available. Sperm concen-
tration and motility were assessed microscopically.
Until the end of December 2010, the viral assessment was
based on a double assay: a real-time PCR (Smart Cycler II,
Cepheid) for the loci of GAG, POL, ENV of the HIV proviral
DNA (set up by the external laboratory), and RNA detection
586
Figure 1 Semen wash procedure.
by means of AMPLICOR HIV-1 MONITOR (Roche). The rst one,
DNA test, was the diagnostic method of choice from the labo-
ratory supplying the test in cases with expected risk of low
amount present (such as in newborn). For this reason, it was
applied to semen, suspecting a low amount of HIV post-
wash. The false negative risk is only theoretical, given the
assay of three different genes at the same time. The false posi-
tive is also theoretical and related to environmental con-
tamination. The AMPLICOR HIV-1 MONITOR test has a sensitivity
of 100% for the detection of 7.5 copies of HIV-1 RNA/reaction
(400 copies/ml) and specicity of 100%, as validated by the
supplier.
From January 2011 on, the testing laboratory substituted
the two previous ones with the kit RealTime HIV-1 (Abbott
Laboratories), accepted by international guidelines as a pos-
sible diagnostic tool for HIV. The sensitivity is >99.5% and the
specicity is >99.5% for the detection of >40 copies/ml of HIV-1
RNA.
Ovarian stimulation
When a patient was using her own oocytes, the stimulation
protocol included Follitropin alfa (Gonal, Merck-Serono,
Spain) or highly puried urinary human menopausal gonado-
trophin (Menopur, Ferring, Spain) with daily injections from
150 to 300 IU. Pituitary suppression was obtained with a long
protocol of gonadotrophin-releasing hormone (GnRH) agonist
(0.1 mg of Triptorelin, Decapeptyl, Ipsen Pharma Biotech,
France) from the 21st day of the previous menstrual cycle and
0.05 mg when the stimulation was started), or with a GnRH
antagonist (GnRHan) protocol (Cetrorelix [Cetrotide, Merck,
Spain] 0.25 mg or 3 mg initiated on the sixth day of
stimulation).
Final oocyte maturation was triggered with 250 g of
Ovitrelle (Merck Serono, Spain) when three or more fol-
licles of 18 mm diameter were observed.
MJ Zamora et al.
All oocyte donors were stimulated with exogenous go-
nadotrophins, while the pituitary suppression was obtained
with two possible protocols: GnRH agonists starting in the
second phase of the preceding menstrual cycle or GnRH an-
tagonists xedly from the sixth day of ovarian stimulation.
Ovulation was triggered when three or more follicles 18 mm
diameter were present, regardless of the stimulation proto-
col. Ovulation trigger was performed with either 250 g of
human chorionic gonadotrophin (HCG) (Ovitrelle, Merck,
Germany) or 0.2 mg of the GnRH agonist triptorelin
(Decapeptyl, Ipsen Pharma Biotech, France) depending on
the stimulation protocol.
In all cases, oocyte collection was performed 36 h after
triggering, by means of ultrasound-guided transvaginal fol-
licular aspiration.
Before 2011, when the post-wash PCR test could be per-
formed in a few hours, semen was also collected, pro-
cessed, and tested before ICSI on the same day of oocyte
retrieval; since 2011 the PCR testing has taken longer, so sper-
matozoa were collected and tested beforehand, and frozen
until the day of fertilization.
Embryo transfer was performed at day 2 or 3 of embryo
development, but also at day 5, usually in patients with mul-
tiple previously failed attempts. The number of embryos trans-
ferred was one to two in all cases. Positive HCG test was
dened as a positive HCG blood test 14 days after embryo
transfer; clinical pregnancy was dened by the presence of
a gestational sac with a heartbeat at 7 weeks of pregnancy,
and ongoing pregnancy was determined by a normally devel-
oping pregnancy at 12 weeks of gestation.
Endometrial preparation
Endometrial preparation differed within embryo transfer cat-
egories. In case of embryo transfer with embryos originat-
ing from a patients own oocytes, the only treatment was
Semen wash in HIV-positive men 587

progesterone (400 mg/12 h) (Utrogestan, SEID, Spain or Table 1 Demographic characteristics of the patients included
Progefk, Efk Laboratory, Spain) starting on the day of in the study overall and by treatment group.
oocyte retrieval. In case of embryo transfer with embryos origi-
Overall Own oocytes Donor oocytes
nating from donated oocytes, two scenarios were possible:
in case of residual ovarian function in the oocyte recipient,
the hypophysis was suppressed with GnRH agonists (triptorelin, n 234 105 129
3.75 mg); moreover, the endometrium was prepared with oes- Womans age, 39.5 (4.90) 36.5 (4.52) 42.05 (3.68)
tradiol valerate orally in an increasing dose, from 2 mg to 6 mg mean (SD)
per day (Progynova, Bayer Health Care, Germany or Mens age, 42.8 (6.43) 41.85 (7.12) 43.72 (5.74)
Provames, Sano-Aventis) or oestradiol hemihydrate mean (SD)
transdermally from 75 g to 150 g (Estradot, Novartis Number of 1.95 (0.44) 1.99 (0.53) 1.92 (0.35)
Pharma or Vivelledot, Novartis Pharma). In case of no re- transferred
sidual ovarian function, only endometrial preparation was per- embryos, n (SD)
formed. Oestrogen and progesterone treatment was continued Day of transfer, 2.68 (0.79) 2.66 (0.76) 2.71 (0.81)
until the pregnancy test 14 days after the embryo transfer n (SD)
and, in case of a positive result, until week 12 of pregnancy.

Table 2 Number of ICSI cycles using spermatozoa from fresh or

Statistical analysis frozen washed semen samples and HIV test results.
Fresh Frozen Overall
Descriptive statistics were performed for demographic and semen semen
outcome variables. Differences in pregnancy outcomes (posi-
tive HCG, clinical and ongoing pregnancies) between using IVF cycles, n 154 80 234
fresh sperm (until December 2010) or frozen sperm (from 2011 PCR, n 154 115 269
onwards) were tested for statistical signicance using the chi- HIV + , n (%) 5 (3.2) 0 (0) 5 (1.86)
squared test. A P-value <0.05 was set as statistically signicant.
ICSI = intracytoplasmic sperm injection; PCR = polymerase chain
reaction.

Ethical approval
This is a retrospective analysis of anonymized data managed
as a cohort; it therefore does not require the approval of an
ethical committee for clinical investigation (CEIC) under
Spanish law (Orden SAS/3470/2009).
Results
Population characteristics
The average age of women was 39.5 (SD 4.90) years. For
women using their own oocytes, the average age was 36.5 (SD
4.52) years, and for women using donor oocytes the average
was 42.05 (SD 3.68) years. The average age of HIV-infected
men was 42.8 (SD 6.43) years. In the group using their own
oocytes, the average male age was 41.85 (SD 7.12), and 43.72
(SD 5.74) years in the group using donor oocytes (Table 1).
The average number of embryos transferred was 1.95 (SD
0.44), being slightly higher in the group using their own oocytes
(1.99 (SD 0.53)) compared with the group using donor oocytes
(1.92 (SD 0.35)).
Extended sperm wash
In total, 269 semen samples were processed using the de-
scribed protocol of triple-density gradient coupled with ex-
tensive centrifugation and swim-up. Following WHO (WHO,
2010) and national recommendations (GESIDA, 2013), all
washed semen samples were tested for HIV presence before
performing ICSI (WHO, 2010). Five out of 269 (1.86%) samples
washed with the presented protocol remained positive for HIV
at the post-wash test. All ve of the positive post-wash tests
occurred before January 2011; in four of these cases a new
semen sample was obtained, washed, and in all four cases it
tested negative at the post-wash test, while in one case ICSI
was performed with donor sperm as a replacement. This case
was therefore excluded from the total number of IVF cycles
analysed in the study (Table 2).
Reproductive outcomes
Of the 234 ICSI treatments initiated, 233 reached embryo
transfer (99.6%) and 232 had reproductive results, 104 using
their own oocytes and 128 using donor oocytes. There was one
case of a cancelled embryo transfer, resulting from donor
oocytes, where embryo quality was considered too poor for
transfer.
Of the 104 embryo transfer with their own oocytes, 49 posi-
tive HCG tests (47.1%), 39 clinical pregnancies (37.5%) and
32 ongoing pregnancies (30.8%) resulted. Of the 128 embryo
transfer with embryos from donor eggs, there were 75 posi-
tive HCG tests (58.6%), 65 clinical (50.8%) and 50 ongoing
pregnancies (39.1%) (Table 3). Positive outcomes with embryos
originating from donor oocytes were higher, but no signi-
cant differences were found in pregnancy rates between the
two groups.
Moreover, no statistically signicant differences were found
between using fresh or frozen sperm (Table 4), neither in the
own oocytes group (Table 5) nor in the donor oocytes group
(Table 6).
588 MJ Zamora et al.

Table 3 Pregnancy results after ICSI according to oocyte origin. 2003). Depending on the availability of alternatives, 1040%
of them would be potentially exposed to HIV transmission
Overall Patient Donor
(Klein et al., 2003). Up to 10% of HIV-infected men with un-
n = 232 oocytes oocytes
n = 104 n = 128
detectable viral load in blood have been reported to have a
detectable viral load in semen (Vernazza et al., 1997), pos-
Positive HCG test, n (%) 124 (53.4) 49 (47.1) 75 (58.6) sibly increasing the population of at risk women trying to
Clinical pregnancy, n (%) 101 (43.5) 39 (37.5) 65 (50.8) conceive. The rate of HIV transmission in serodiscordant
Ongoing pregnancy, n (%) 80 (34.5) 32 (30.8) 50 (39.1) couples has been reported to be approximately 1 for 500
1000 unprotected intercourses (Mandelbrot et al., 1997).
HCG = beta human chorionic gonadotrophin. We describe here an extended sperm washing with triple
There were no statistically signicant differences. gradient, coupled with post-wash PCR test. All cycles were
performed by ICSI to minimize the contact between semen
and oocyte, in agreement with previous reports (Garrido et al.,
Table 4 Overall pregnancy results after ICSI using spermato-
2004; Pena et al., 2003). Moreover, the use of ICSI may also
zoa from fresh or frozen washed semen samples.
be considered a technical necessity due to the loss of viabil-
Fresh sperm Frozen sperm ity intrinsic to the extended sperm wash procedure. Fresh
Overall
n = 152 n = 80 semen samples were originally used, but this was changed to
n = 232
semen post-wash cryopreservation to allow for an easier work-
ow and better coordination with the testing laboratory,
Positive HCG test, n (%) 84 (55.3) 40 (50.0)
without loss in ongoing pregnancy rates, as shown (Tables 5
Clinical pregnancy, n (%) 74 (48.7) 30 (37.5)
and 6). Regardless of availability of the result of the PCR test
Ongoing pregnancy, n (%) 57 (37.5) 25 (31.3)
the same day as semen preparation, freezing semen samples
HCG = beta human chorionic gonadotrophin; ICSI = intracytoplas- is a useful choice for patients residing far away from the clinic,
mic sperm injection. and especially as a backup in case of unexpected difculties
There were no statistically signicant differences. in collecting the sperm sample the day of insemination; more-
over, semen post-wash cryopreservation avoids cancelling of
the cycle in case a positive PCR test is returned.
Table 5 Pregnancy results after ICSI using sperm from fresh or Using the described extended wash, the rate of HIV-
frozen washed semen samples with patients oocytes. positive post-wash test was 1.86%, or ve out of 269 samples
Fresh sperm Frozen sperm washed. The rst two positive cases occurred in 2006, when
n = 77 n = 27 the procedure was recently introduced in the laboratory,
whereas the subsequent three cases all happened in 2010 and
Positive HCG test, n (%) 36 (46.8) 13 (48.1) were performed in the same week by the same operator. After
Clinical pregnancy, n (%) 30 (39.0) 9 (33.3) re-training of the operator, no more post-wash positive results
Ongoing pregnancy, n (%) 23 (29.9) 9 (33.3) were reported, for the same operator as well as others.
Since 2011, even with a more sensitive PCR method, with
HCG = beta human chorionic gonadotrophin.
a threshold of 40 copies/ml, no positive post-wash test has
There were no statistically signicant differences.
been returned. Comparison with the literature is sometimes
difcult because, unfortunately, either post-wash tests were
Table 6 Pregnancy results after ICSI using spermatozoa from not performed (Mencaglia et al., 2005; Pena et al., 2003; Sauer
fresh or frozen washed semen samples with donor oocytes. et al., 2009), or when the post-wash test was performed and
reported, there were different sensitivities reported among
Fresh sperm Frozen sperm tests. In a study using a post-wash reverse transcription PCR
n = 75 n = 53 (RT-PCR) with a threshold of 500 copies/ml, the positive rate
was 4% (Savasi et al., 2007); the authors speculate that this
Positive HCG test, n (%) 48 (64.0) 27 (50.9)
relatively high rate of post-test positivity could have been due
Clinical pregnancy, n (%) 44 (58.7) 21 (39.6)
to accidental contamination during RT-PCR. However, when
Ongoing pregnancy, n (%) 34 (45.3) 16 (30.2)
using a highly sensitive nested PCR test, a positive rate of 10%
HCG = beta human chorionic gonadotrophin. was reported (Garrido et al., 2004). The two studies using a
There were no statistically signicant differences. post-wash test with a 200 copies/ml threshold reported 5.6%
(Marina et al., 1998) and 7.7% positive rates (Veiga et al.,
1999), respectively.
Heterogeneity of thresholds notwithstanding, the rate of
Discussion 0/115 positive cases since the introduction of the 40 copies/
ml threshold tests indicates that the success of the proce-
The reproductive wishes and needs of serodiscordant couples dure is linked to the use of the triple density gradients coupled
often lead to an increased risk of both horizontal and verti- with a longer swim-up, and by the thorough application of the
cal HIV transmission, even in economically developed coun- protocol by highly trained personnel. It is likely that each one
tries. In fact, 20% of serodiscordant couples report having had of these factors, taken independently, would not achieve the
unprotected intercourse in the past in order to achieve a preg- same level of success, since the triple density gradient alone
nancy, and 12% of couples report the wish to continue un- has been used previously (Marina et al., 1998; Melo et al.,
protected intercourse in order to have children (Klein et al., 2008; Veiga et al., 1999). Our protocol included a longer
Semen wash in HIV-positive men
swim-up and a second wash and centrifugation step after se-
lection, probably further avoiding the carryover of HIV par-
ticles from the gradient; to the best of our knowledge, a
washing protocol that encompasses all these measures has not
been reported so far in the literature.
Although the current statistical analysis does not address
the presence of patient-specic variables that might impact
reproductive outcomes, the clinical pregnancy rates per trans-
fer described in this study for patients using their own oocytes
and using donor oocytes were 37.5% and 50.8%, respec-
tively. These rates are comparatively higher than the rates
described for ICSI for the same type of cycles (32.0% and 47.4%)
by the European Society of Human Reproduction and Embry-
ology (Kupka et al., 2014). In part, this may be due to the
fact that patients using their own oocytes were not infer-
tile; rather, they were assumed as fertile as the general popu-
lation of the same age. Our results were similar to the
pregnancy rates reported by Melo et al. (Melo et al., 2008),
which compared 30 serodiscordant couples to 79 couples with
tubal factor infertility as controls. The serodiscordant couples
had a pregnancy rate of 46.7% versus 40.5% in the control
group; however, unlike our report, cases with severe male
factor and women older than 37 years were excluded from
the study. Other studies reported comparable pregnancy rates
between serodiscordant and control seronegative couples un-
dergoing IVF/ICSI for infertility reasons; in the study of Prisant
et al., the clinical pregnancy rate per embryo transfer in
serodiscordant couples was 22.2% versus 10.8% in the control
group (Prisant et al., 2010), while Santulli et al. reported a
clinical pregnancy rate per oocyte retrieval of 23% and 32%
in an age-matched control group (Santulli et al., 2011).
Savasi et al. (Savasi et al., 2007) described a pregnancy
rate of 24% per embryo transfer in IVF/ICSI when the woman
was infertile, both partners had subfertility conditions or when
fewer than one million motile spermatozoa were recovered
after washing. Results were lower when compared with cases
of fertile serodiscordant couples who underwent ICSI (Garrido
et al., 2005). Our results were higher even though there was
no exclusion threshold by sperm concentration post sperm
washing, in agreement with a report of assisted reproduc-
tive treatment outcomes not altered in couples with male HIV
infection, despite poorer sperm quality (Sauer et al., 2009);
however, while a clinical pregnancy rate per embryo trans-
fer in fresh non-donor cycles was 45%, this study reported a
multiple birth rate of 41% with 5% triplets.
There are few comparative data about donor oocyte cycles
in the literature. One study presented a pregnancy rate of 33%,
with a cohort of just 12 cycles (Sauer et al., 2009), and another
one reported a pregnancy rate of 40% in 23 cycles from 18
patients (Garrido et al., 2004). We report the largest cohort
so far, with 128 cycles using donor oocytes. We found a clini-
cal pregnancy rate of 50.8% when using donor oocytes, al-
though this was not signicantly higher than the pregnancy
rate when using a patients own oocytes. This difference could
be due to the fact that oocyte donors in Spain must be by law
younger than 35, and were 27.4 6.5 years old in our cohort.
In conclusion, we propose the use of the described ex-
tended wash for semen of HIV-positive men, which offers lower
than reported post-wash viral positivity, and all but elimi-
nates the detection of HIV, while maintaining high preg-
nancy rates, even when using frozen semen. As a consequence,
and in the light of the difference in viral load between blood
589
and semen, we see no reason not to offer extended semen
wash to all serodiscordant couples, regardless of the sero-
logical viral load.
Acknowledgements
The authors wish to thank Desiree Garcia for statistical
support, Sarai Brazal for administrative assistance, and Dr Raul
Santamaria for virological support.
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Declaration: The authors report no nancial or commercial con-
icts of interest.
Received 2 November 2015; refereed 17 February 2016; accepted 24
February 2016.