Document Type : Original Article
Authors
1 Human Reproduction Unit, Clinical Management Unit for Obstetrics and Gynaecology, Virgen de las Nieves University Hospital, Granada Institute for Healthcare Research, Granada, Spain
2 Infectious Disease Unit, Internal Medicine Service, Virgen de las Nieves University Hospital, Granada, Spain
3 4Microbiology Service, Virgen de las Nieves University Hospital, Granada, Spain
Abstract
Keywords
Assisted reproductive technology (ART) first
came into use to address problems of infertility, and
was subsequently applied to fertile couples with the
aim of reducing the risk of transmission of genetic
and infectious diseases. With this latter objective,
ART has been applied to couples in which one or both
partners are infected by human immunodeficiency virus
(HIV), hepatitis C virus (HCV) or hepatitis B virus
(HBV). Semprini et al. (
To date, many studies have described the safety
of the semen wash-intracytoplasmic sperm injection
(ICSI) technique for couples that are serodiscordant
for HIV (
A similar situation occurs with studies analysing the
use of ART for couples in which the male partner is
seropositive for HCV or HBV (
Since late 2005, the Human Reproduction Unit at
Hospital Universitario Virgen de las Nieves, Granada,
Spain has been the only one in the public health system
of Andalucia (8.2 million inhabitants) providing
fertility care for couples with an HIV, HCV or HBV
infection. We follow the recommendations of the ethics
taskforce of the european society of human reproduction
and embryology (ESHRE) about suitable
treatment compliance, avoidance of other risk factors
such as drug abuse, treatment in reference centres with
established protocols, a separate adapted laboratory,
as well as separate tanks for storage of infected material,
and appropriate multidisciplinary support (
The aim of this retrospective study was to determine, for couples in which the male was seropositive for HIV, HCV or HBV: i. the efficiency of sperm wash in terms of viral load; ii. the results of ART-ICSI; iii. the seroconversion rates after the treatment; and iv. the obstetric and neonatal outcome for such couples at a public hospital.
A retrospective review was conducted of men who were seropositive for HIV, HCV or HBV and underwent assisted reproduction treatment between November 2005 and December 2009.
To be enrolled, all the couples were required to sign informed consent, and to attest to safe sex practices since four months before beginning the treatment and not to have sex from one month before until one month after the end of the treatment. Male and seropositive female partners were requested to be under the care of an infectious disease specialist and to provide a full report of their disease including current serological study, CD4 counts in blood (only for HIVseropositive), blood viral load with a maximum age of 4 months and treatment received and evolution of the disease for the past 12 months. To enrol an HIV positive female, both undetectable viral load and CD4 counts >200 cells/mm3 were required. Seronegative female partners were required to provide a current serological study and a blood viral load. HBV negative females with HBV positive male partners were vaccinated, and if immunity was not achieved, the partner’s semen was washed and viral load was determined. The couples had to wait an average of two years for ICSI treatment, as our hospital is a public one and it has a waiting list. Every couple was allowed a maximum of two attempts (two cycles with embryo transfer) to achieve a pregnancy.
A standard evaluation was performed on both partners,
consisting of anamnesis and physical examination.
Female fertility was also assessed by gynaecological
examination, a vaginal ultrasound examination
of the uterus and ovaries, a vaginal sample for bacteriological
testing and a smear test. Basal hormonal
study was determined from blood samples on day 3
of the menstrual cycle. The men were also subjected
to seminal analysis, following world health organization
(WHO) criteria (
Semen samples were obtained after sexual abstinence
of 3 days, and subsequently kept and manipulated in a class II biological safety cabinet. After
liquefaction, semen parameters were evaluated as
outlined by the WHO (
Quantification of HIV RNA, HCV RNA and HBV DNA in final processed semen was performed by real-time PCR COBAS TaqMan™ 96 instrument (COBAS Ampliprep™ analyser; Roche Diagnostics GmbH, Manheim, Germany).
Ovarian stimulation protocols were selected according
to clinical data, patient’s age and hormonal
profile and the result of any previous stimulation.
Normally, the long protocol was adopted for the first
cycle, and then, for the second one, the long or the
short protocol was chosen depending on the results
of the first cycle. The cycles were monitored by serial
transvaginal ultrasound examination and serum
estradiol (E2) levels. When the follicles had reached
a mean diameter of 18-22 mm, human chorionic gonadotropin
(hCG) was administered, and 34-36 hours
later, oocyte retrieval was performed with ultrasound
guidance. The oocytes collected were incubated in GIVF
Plus supplemented with human serum albumin
(HSA, Vitrolife, Göteborg, Sweden) and the mature
oocytes with extrusion of the first polar body were
then microinjected with sperm selected from the motile
fraction. The procedure was performed as previously
described (
Complications such as ovarian hyperstimulation
syndrome (OHSS) were noted. Clinical pregnancy
was assessed by ultrasonographic visualization of
one or more gestational sacs or definitive clinical
signs of pregnancy as described by the International
Committee for Monitoring Assisted Reproductive
Technology. Delivery was defined as the expulsion or
extraction of one or more foetuses from the mother
after 20 completed weeks of gestational age, and live
birth was considered to be the complete expulsion or
extraction from its mother of a product of fertilization,
irrespective of the duration of the pregnancy, which,
after such separation, breathes or shows any other evidence
of life such as heart beat, umbilical cord pulsation,
or definite movement of voluntary muscles, irrespective
of whether the umbilical cord has been cut
or the placenta remains attached (
Comparisons of rates between groups were performed using chi-squared tests. All the tests were twosided, with a p value of 5% considered as significant.
The blood viral load of the female partner was tested using RNA polymerase chain reaction (PCR) at 3 weeks, 3 months and 6 months after the ART. If the woman became pregnant, the blood viral load was tested every 2 months, and the serological study performed every 3 months. The infants were also tested at birth and at age 3 months.
After considering a total of 105 couples, 93 couples were included in this study and twelve were rejected as they did not meet the inclusion criteria; two men had active drug consumption, three men did not present adequate adherence to highly active antiretroviral therapy (HAART), another two men had active opportunist infection and five women were aged older than 40 years.
In 33 of the couples treated, the male was HIV seropositive (23 men were also HCV positive, one was HBV positive and another was both HCV and HBV positive). In another 23 couples, the male was HCV seropositive (one man was also HBV positive), and in the remaining 37 couples, the male was HBV seropositive. The men were infected with different genotypes of HCV: 43.5% (20 men) with genotype 1, 6.5% (n=3) with genotype 2, 26.1% (n=12) with genotype 3 and 23.9% (n=11) with genotype 4. The characteristic profile of the couples who underwent ICSI is shown in table 1.
Characteristic profile of couples undergoing IVF-ICSI
Reference | HIV n = 33 | HCV n= 23 | HBV n= 37 | |||
---|---|---|---|---|---|---|
mean±SD | Range | mean±SD | Range | mean±SD | Range | |
34.6±4.1 | 25-40 | 34.2±2.8 | 29-39 | 34.0±3.8 | 26-40 | |
2(6.1) | ||||||
1(3) | 1(4.4) | |||||
0(0) | 0(0) | |||||
6.5±2.1 | 5-8 | 5±0 | ||||
2(100) | ||||||
0(0) | 0(0) | |||||
376000 | ||||||
2(100) | ||||||
0(0) | 0(0) | |||||
0 | ||||||
666±5.7 | 662-670 | |||||
34(91.9) | ||||||
40.1±5.7 | 28-55 | 39.4±6.1 | 32-50 | 36.4±3.8 | 26-44 | |
2(6.1) | 1(4.4) | |||||
11±4.8 | 2-19 | 17±6.1 | 10-21 | 10.3±8.5 | 4-20 | |
28(84.9) | ||||||
3(23.1) | 3(23.1) | |||||
31631.5±47110.4* | 68-123000 | 3367025.6±4075433.2 | 1071-15937448 | 1837.5±2577.3 | 17-10481.1 | |
27(81.8) | ||||||
7(30.4) | 3(13) | |||||
1(50) | 1(100) | 7(18.9) | ||||
569.3±296.6 | 109-1654 | |||||
* ; Viral load (VIH??HIV) in copies/ml
A total of 62 sperm washes from 59 couples were performed, and none were positive for the detection of viral molecules. The semen samples from the 34 HBV-positive males were not washed since the female partner had immunity to hepatitis B.
The results of our ICSI programme, with respect to viral infection, are summarized in table 2. A total of 173 cycles were performed for 93 couples, whereas 25 cycles (14.5%) were cancelled before oocyte retrieval: 2 cycles (8%) due to the presence of an ovarian cyst, 19 cycles (76%) because of low response, 2 cycles (8%) due to hyper-response of the woman, one cycle (4%) due to failure of down-regulation, and one couple failed to attend for oocyte retrieval.
IVF performance and outcome of IVF-ICSI cycles
HIV(n=33) | HIV(n=23) | HIV(n=37) | ||||
---|---|---|---|---|---|---|
Mean ± SD | Range | Mean ± SD | Range | Mean ± SD | Range | |
61 | 48 | 64 | ||||
2409.6 ± 1153.8 | 1013-5550 | 2305 ± 964.3 | 1275-4725 | 2315.5 ± 845.8 | 825-4875 | |
11.2 ± 2.7 | 7-22 | 10.7 ± 2.2 | 7-16 | 10.6 ± 1.9 | 8-18 | |
5.3 ± 4.1 | 0-15 | 5.7 ± 3.4 | 0-13 | 5.6 ± 3.1 | 1-14 | |
2760.6 ± 1408.2 | 672-6816 | 2536.4 ± 1417.1 | 759.8-6230 | 2214.6 ± 1302.6 | 263-5240 | |
2.4 ± 1.7 | 0.30-8 | 2.8 ± 1.9 | 0.21-9 | 2.4 ± 1.6 | 0.1-6.1 | |
5 (8.2) | 1 (2.1) | |||||
1.8 ± 1.3 | 1-4 | 5 ± 0 | 0 ± 0 | |||
48 | 43 | 56 | ||||
13 (21.3) | 4 (8.3) | 8(12.5) | ||||
10.6 ± 6.1 | 1-23 | 9.4 ± 4.6 | 0-20 | 9.5 ± 5.2 | 0-19 | |
8.5 ± 5.4 | 1-20 | 7.6 ± 4.1 | 0-15 | 7.5 ± 4.4 | 1-18 | |
4.6 ± 3.6 | 0-17 | 4.1 ± 2.9 | 0-12 | 4.5 ± 3.4 | 0-15 | |
54.6 ± 29.1 | 0-100 | 52.3 ± 21.1 | 0-100 | 60.5 ± 28.4 | 0-100 | |
6 (18.2) | 5 (11.6) | 3 (5.4) | ||||
1.9 ± 0.4 | 1-3 | 2.1 ± 0.5 | 1-3 | 2.0 ± 0.5 | 1-3 | |
13.2 | 16.9 | 16.8 | ||||
22.2 | 0 | 45.5 | ||||
7 (14.6) | 5 (11.6) | 12 (21.4) | ||||
2 (4.2) | 0 (0) | 0 (0) | ||||
1.9 ± 0.4 | 1-3 | 1.4 ± 0.9 | 1-3 | 2.8 ± 2 | 1-8 | |
19 (59.4) | 5 (71.4) | 16 (48.5) | ||||
9 (47.4) | 2 (0) | 11 (68.8) | ||||
1 (1.6) | 0 (0) | 0 (0) | ||||
With respect to the different infection diseases, there were no significant differences in the number of oocytes retrieved, the number of mature oocytes, the fertilization rate, the number of embryos transferred or the number of embryos cryopreserved per retrieval. In the case of two women, no transfer was performed, due to ovarian hyper stimulation syndrome (OHSS) and cystitis.
The mean fertilization rate achieved was 56.2%,
with a mean implantation rate of 15.7% for fresh
transfers and 31.8% for thawed embryos. In total,
38 clinical pregnancies (22% per cycle and
40.9% per couple) took place, with 28 live births
delivered (16.2% per cycle and 30.1% per couple).
The miscarriage rate was 26.3% and that of
multiple pregnancies, 21.4% (
No seroconversion was detected in any of the 34 newborns (tested at birth and at age 3 months) or in the 62 women treated with washed sperm during assisted reproduction programmes.
Pregnancy data
HIV | HCV | HBV | |||||
---|---|---|---|---|---|---|---|
n | (%) | n | (%) | n | (%) | ||
0 | 0 | 0 | |||||
8 | 10 | 14 | |||||
(13.1) | (20.8) | (21.9) | |||||
(16.7) | (23.3) | (25) | |||||
(20) | (27.0) | (27.5) | |||||
(24.2) | (43.5) | (37.8) | |||||
2 | 0 | 4 | |||||
(33.3) | (0) | (44.4) | |||||
(40) | (0) | (66.7) | |||||
10 | 10 | 18 | |||||
(16.4) | (20.8) | (28.1) | |||||
(20.8) | (23.3) | (32.1) | |||||
(22.2) | (25.6) | (31.6) | |||||
(30.3) | (43.5) | (48.6) | |||||
2 | (20) | 2 | (20) | 6 | (33.3) | ||
0 | (0) | 0 | (0) | 0 | (0) | ||
7 | 8 | 9 | |||||
(11.5) | (16.7) | (14.1) | |||||
(14.6) | (18.6) | (16.1) | |||||
(17.5) | (21.6) | (17.6) | |||||
(21.2) | (34.8) | (24.3) | |||||
1 | 0 | 3 | |||||
(16.7) | (0) | (33.3) | |||||
(20) | (0) | (50) | |||||
8 | 8 | 12 | |||||
(13.1) | (16.7) | (18.8) | |||||
(16.7) | (18.6) | (21.4) | |||||
(17.8) | (20.5) | (21.1) | |||||
(24.2) | (34.8) | (32.4) | |||||
aa
HIV | HCV | HBV | ||||
---|---|---|---|---|---|---|
mean±SD | Range | mean±SD | Range | mean±SD | Range | |
8 | 8 | 12 | ||||
10 | 10 | 14 | ||||
6(75) | 6(75) | 10(83.3) | ||||
2(25) | 2(25) | 2(16.7) | ||||
0(0) | 0(0) | 0(0) | ||||
2(25) | 2(25) | 2(16.7) | ||||
6(75) | 3(37.5) | 9(75) | ||||
2(25) | 5(62.5) | 3(25) | ||||
6 | 7 | 9 | ||||
8(80) | 8(80) | 9(64.3) | ||||
39±1.5 | 38-42 | 39±1.5 | 37-41 | 38±1 | 37-40 | |
2925.6±2630 | 2630-3315 | 2809±470.2 | 2150-3700 | 3376±353.7 | 2760-3890 | |
1 | 1 | 3 | ||||
1(10) | 2(20) | 5 | ||||
36±0 | 36-36 | 34±0 | 36±0.6 | 35-36 | ||
2880±0 | 2880-2880 | 2342.5±343 | 2100-2585 | 2286±531.6 | 1840-3200 | |
0(0) | 0(0) | 0(0) | ||||
0(0) | 0(0) | 0(0) | ||||
0(0) | 0(0) | 0(0) | ||||
1 | 0 | 0 | ||||
1(10) | 0(0) | 00 | ||||
0(0) | 2(20) | 4(28.6) | ||||
- | 2125±35.4 | 2100-2150 | 2057.5±169.2 | 1840-2215 | ||
1 | 0 | |||||
1(10) | 0(0) | 0(0) | ||||
750±0 | ||||||
Our data show that sperm wash testing for detectable viral load of final processed semen and ICSI seems to be a safe and effective option for serodiscordant couples to conceive and to avoid transmitting the virus to the mother and child.
It is currently estimated that following recent advances
in antiretroviral treatment, when total suppression
of the viral load is achieved, the risk of sexual
transmission is 1:100000 (
To date, no seroconversions have been reported
following ICSI treatment in which semen samples
are processed by density gradient centrifugation
of sperm, with or without routine testing of final
semen processes (
Some assisted reproduction centres perform ICSI
for all couples, rather than Intrauterine insemination
(IUI), justifying this on the greater success rate, and
therefore, less exposure to possibly contaminated
sperm, or on the theory that ICSI requires only in
vitro contact between a single sperm and egg, which
should dramatically reduce the risk of transferring the
viral particles that are often present in the semen or
seminal cellular compartment (
Other authors consider this theory to be unproven,
and believe the process could involve the introduction
of viral particles directly into the oocyte, with as yet
unknown effects (
Considerable differences have been reported in
the percentage of positive results for RNA-HIV
of final processed semen, ranging from 0% (
Our results for clinical and ongoing pregnancies
achieved for couples in which the male partner is
HIV positive are similar to those described by other
authors (
With respect to clinical and ongoing pregnancies
for couples with HCV and HBV-positive male
partners, our results are similar to those published
by other authors (
Although we found no statistically significant
differences in the pregnancy rates among the three
types of couples studied, the trend for rates to be
lower among the HIV-infected couples could be
related to reduce fertility rate among HIV-infected
men as a result of treatment or infection (
Although not expressly described, we found no
significant differences between embryology laboratory
results for males who were only HIV seropositive,
and those who were positive for both HIV
and HCV. This has also been reported by other authors,
in studies of these two groups of patients,
or comparing them with the results obtained from
healthy couples (
Although the rate of caesarean delivery was
higher among the group in which the male partner
was HCV infected, these differences were
not statistically significant. Moreover, the overall
rate of caesareans in our study (35.7%) was
lower than that described by other authors for
this type of couple (
We showed sperm washing and ICSI is a safe and effective option for reducing the risk of transmission or super infection in serodiscordant or concordant couples who wish to have a child. The pregnancies obtained by ART among couples in which the male partner has CVD produce good obstetric and neonatal results.