A new method to rescue embryos

contaminated by bacteria
Ruiqi Li, Ph.D.,a,b Fengjiao Du, B.D.,a,c Songbang Ou, B.D.,a,c Nengyong Ouyang, Ph.D.,a,c
and Wenjun Wang, Ph.D.a,c
a
Reproductive Medicine Center, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, People’s Republic of
China; b Reproductive Medicine Center, The First People’s Hospital of Kashgar, Kashgar, People’s Republic of China; and
c
Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-sen Memorial
Hospital, Guangzhou, People’s Republic of China

Objective: To report a case of successful pregnancy involving embryos that wereaffected by bacterial contamination.
Design: A case report.
Setting: Academic assisted reproductive center.
Patient(s): A 31-year–old infertile patient with obstructed fallopian tubes facing bacterial contamination in her embryos during
in vitro fertilization.
Intervention(s): The zona pellucida (ZP) of the embryos that was contaminated by bacteria was removed by acidic Tyrode’s solution.
The ZP-free embryos were then cultured in a time-lapse culture dish with 1 zygote per well until day 5 when a single ZP-free blastocyst
was selected for transfer.
Main Outcome Measure(s): The rate of obtaining embryos without recurrence of bacterial contamination and the developmental po-
tential of the embryos.
Result(s): Twenty oocytes were retrieved and were coincubated with sperm in vitro overnight. A total of 9 zygotes with 2 pronuclei and
3 zygotes with 1 pronucleus were obtained. Unfortunately, all zygotes were contaminated by the Klebsiella pneumoniae bacteria. The ZP
of 7 zygotes were removed using acidic Tyrode’s solution (ZP-free group), whereas the remaining 5 zygotes and 3 metaphase II (MII)
stage oocytes were washed with G-1 PLUS medium multiple times (washing treatment group). In the washing treatment group, all em-
bryos experienced recontamination on day 2 and were dead by day 3. In the ZP-free group, 2 embryos were found to be recontaminated
on day 2. The remaining 5 embryos that stayed uncontaminated were selected for blastocyst culture. On day 5, 2 of the cultured embryos
developed into blastocysts. One blastocyst was transferred during the fresh cycle, and the other was vitrified. A single intrauterine
gestation was confirmed 4 weeks after the transfer. At the time of writing this article, the patient was 30 weeks pregnant without
any occurrence of intrauterine infection during pregnancy.
Conclusion(s): Zona pellucida removal is a safe and effective method to rescue embryos contaminated with bacteria. (Fertil Steril RepÒ
2022;3:168–71. Ó2022 by American Society for Reproductive Medicine.)
Key Words: Bacterial contamination, zona pellucida, in vitro fertilization, pregnancy
Discuss: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/posts/xfre-d-21-00186

INTRODUCTION Although the frequency of conta into financial and psychological

Bacterial contamination during em- mination is <1%, it is still a serious burdens for patients (6). With the
bryo culture is a serious problem faced problem when it occurs, resulting in sheer number of IVF cycles performed
in all in vitro fertilization (IVF) labora- no embryos being available for worldwide each year, microbial
tories. According to previous reports, transfer (1, 4, 5). The damage caused contamination should not be
the frequency of microbial contamina- by microbial contamination in IVF underestimated as a complication in
tion is between 0.35% and 0.86% (1–3). procedures can be directly translated an assisted reproductive technology
treatment cycle. Therefore, it is of
Received November 1, 2021; revised and accepted May 2, 2022. utmost importance for any IVF
R.L. has nothing to disclose. F.D. has nothing to disclose. S.O. has nothing to disclose. N.O. has nothing laboratory to prevent bacterial
to disclose. W.W. has nothing to disclose.
Supported by Guangdong Basic and Applied Basic Research Foundation (2018A030313911,
contamination. Likewise, the method
2021A1515010308), Natural Science Foundation of Xinjiang Autonomous Region, China to rescue contaminated embryos is
(2020D01C011), and China Pakistan Science Foundation (2020M673645XB). equally important.
Reprint requests: Wenjun Wang, Ph.D., Reproductive Medicine Centre, Department of Obstetrics and
Gynecology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, No. 107 Yanjiang West At present, the most commonly
Road, Guangzhou 510120, People’s Republic of China (E-mail: [email protected]). used method when bacterial contami-
Fertil Steril Rep® Vol. 3, No. 2, June 2022 2666-3341
nation occurs involves washing the
© 2022 Published by Elsevier Inc. on behalf of American Society for Reproductive Medicine. This is an embryos thoroughly with a medium
open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc- containing antibiotics as an attempt
nd/4.0/).
https://doi.org/10.1016/j.xfre.2022.05.002 to remove microorganisms from their

168 VOL. 3 NO. 2 / JUNE 2022

 Fertil Steril Rep®

surfaces. However, because of the unique porous network In the first 3 days, the embryos were cultured in the G-1
structure of the zona pellucida (ZP) that surrounds the oocyte PLUS medium (Vitrolife, 10128). Next, the embryos were
(7, 8), it is often difficult to remove microorganisms from it moved into a G-2 PLUS medium (Vitrolife, 10132) for blasto-
completely. In most cases, bacterial contamination may cyst culture.
happen again even after the embryos have been washed. In The embryo culture medium used in this study contains
this study, the recontamination with bacteria was successfully gentamicin as the antibiotic unless otherwise stated.
prevented by the removal of the ZP from embryos affected by On the first day after fertilization, cloudy particles were
bacterial contamination. To our knowledge, this is a new observed in the fertilization dishes, and a large number of
method of rescuing contaminated embryos, eventually lead- sperms were noticed to be immotile (Fig. 1A). Thus, we sus-
ing to a successful pregnancy. pected that bacteria contaminated the fertilization dishes.
The semen suspensions used for the insemination of the oo-
cytes, follicular fluid, and the IVF medium were collected
CASE REPORT and sent to the Department of Medical Microbiology for
The key patient in this study is a 31-year–old woman with a 3- microbiological examination.
year history of secondary infertility and regular periods. Her Test results showed that the contaminating microor-
right fallopian tube was obstructed (distal obstruction), and ganism was the Klebsiella pneumoniae bacteria. It is, howev-
her left fallopian tube suffered partial blockage. Her baseline er, noteworthy that the follicular fluid samples were found to
follicle-stimulating hormone, luteinizing hormone, estradiol, contain Enterococcus faecalis, whereas no bacteria were de-
and antim€ ullerian hormone levels as well as her husband’s tected in the semen samples. Each zygote was then washed
semen analysis were normal. Controlled ovarian stimulation several times in the G-IVF medium and then cultured in the
was performed using the long gonadotropin-releasing hor- G-1 PLUS medium with a ratio of 1 embryo to 1 drop of cul-
mone agonist protocol. ture medium. Nonetheless, bacteria were still present in all
Sperm preparation and insemination procedures were as embryo culture droplets by the afternoon of day 1. Consid-
described previously (9). Briefly, the man washed his hands ering that washing the zygotes again may have little effect,
and penis with soap and disinfected his hands with alcohol the ZP of some zygotes was removed instead after obtaining
before semen collection. The semen sample was collected the patient’s informed consent.
through masturbation, and the sperms were selected via The ZP of 6 2PN zygotes and 1 1PN zygote was removed
gradient centrifugation by collecting the sperm pellet and by acidic Tyrode’s solution (Sigma, T1788) after washing
washing it twice. The final concentration of sperm used in several times with the G-1 PLUS medium (ZP-free group).
IVF was 1.0
105/mL. The semen preparation media used Then, these ZP-free zygotes were cultured in a time-lapse cul-
in this study were G-IVF PLUS (Vitrolife, 10136) and Sperm- ture dish (CultureCoin, ESCO,1821072, Singapore) with a G-1
Grad (Vitrolife, 10099). SpermGrad is antibiotic-free, whereas PLUS medium. The culture dish was subsequently incubated
the media used for dilution and sperm wash, such as G-IVF with the following conditions: 1 zygote per well in a time-
PLUS, contain gentamicin as an antibacterial agent. lapse incubator (Miri, ESCO, Singapore) at 37 C with 6%
Twenty oocytes were obtained and exposed to sperm CO2, 5% O2, and 89% N2. This culture dish has a narrow bot-
overnight for 16–20 hours. The fertilization result on day 1 tom that restricts blastomere movement and limits external
is shown in Table 1. A total of 9 zygotes with 2 pronuclei disturbances. The rest of the zygotes, including 3 2PN zygotes,
(2PN) and 3 zygotes with 1 pronucleus (1PN) were obtained. 2 1PN zygotes, and 3 MII stage oocytes, were washed several

TABLE 1

Baseline information of the patient.

Reference ranges for semen
Maternal Values Paternal Values analysisb
Baseline FSH (U/L)a 1.86 Sperm concentration 62
106/mL R15
106/mL
Baseline LH (U/L)a 5.66 Motility 47% R40%
Baseline E2 (pg/mL)a 132 Normal morphology 10% R4%
AMH (ng/mL) 5.04 Sperm DNA fragmentation index 14% <25%
Total Gn (IU) 1,775
Oocyte number 20
2PN 9
1PN 3
Multiple PN 2
MII 3
MI 1
Gv 2
Note: 1PN ¼ 1 pronucleus; 2PN ¼ 2 pronuclei; MII = metaphase II stage; MI = metaphase I stage; GV = germinal vesicle stage; AMH ¼ antim€
ullerian hormone; DNA ¼ deoxyribonucleic acid;
E2 ¼ estradiol; FSH ¼ follicle-stimulating hormone; LH ¼ luteinizing hormone; Gn = Gonadotropin.
a
Levels of FSH, LH, and E2 on day 2 during the patient’s menstruation.
b
According to the World Health Organization laboratory manual for the examination and processing of human semen, 5th edition.
Li. Saving embryos contaminated by bacteria. Fertil Steril Rep 2022.

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ORIGINAL ARTICLE: CASE REPORT

FIGURE 1

Bacterial contamination of the fertilization dish on day 1 and the embryonic morphology on day 5. (A) Bacterial contamination in the fertilization
dish on day 1. (B) The embryo selected for transfer on day 5. (C) The embryo selected for cryopreservation on day 5.
Li. Saving embryos contaminated by bacteria. Fertil Steril Rep 2022.

times using the G-1 PLUS medium and then cultured in 35 pregnancy. To our knowledge, this is a new method of
mm culture dishes containing G-1 PLUS medium, 1 zygote rescuing contaminated embryos, which ultimately leads to a
per drop (washing treatment group). The embryos in the successful pregnancy. In 1 other report, Shu et al. (10)
washing treatment group were incubated in an incubator at removed the ZP of contaminated frozen blastocysts, which
37 C, with saturated humidity containing 5% CO2, 5% O2, led to successful clinical pregnancies after the transfer of
and 89% N2. In the washing treatment group, all embryos ZP-free blastocysts that were previously contaminated during
experienced recontamination on day 2 and were dead by IVF culture. However, the removal of the ZP in that study was
day 3. As for the ZP-free group, 2 of 7 zygotes experienced re- not done on the day the contamination was discovered but
contamination on day 2 and were dead by day 3 because of right before embryo transfer.
disrupted development. The remaining 5 embryos remained In our case, the microorganism that had contaminated the
uncontaminated and were transferred to the G-2 PLUS me- embryo culture medium was K. pneumoniae. However, E. fae-
dium on day 3. Two cultured embryos developed into blasto- calis was found in the follicular fluid samples instead, and no
cysts by day 5; hence, 1 blastocyst was transferred on day 5 of bacteria were detected in the semen samples. Some studies
the fresh cycle (Fig. 1B) and the other was cryopreserved suggest that the work environment and improper operation
(Fig. 1C). No antibiotic was prescribed for the patient after em- may be the possible causes of contamination, besides the
bryo transfer. Luteal support was administered twice a day us- reproductive tract microbiota of donors (1, 11). Therefore,
ing vaginal capsules at 400 mg each time. Luteal support was the microbes may have originated from ambient air in the
continued until 12 weeks into pregnancy. workplace or the inadvertent introduction during in vitro pro-
A single intrauterine gestation was confirmed by vaginal cedures performed in this case.
ultrasound examination 4 weeks after embryo transfer. At the Bacterial endotoxin, which is produced in the process
time of writing this article, this patient was in the 30th week of of proliferation, has a serious impact on embryonic
pregnancy, and no intrauterine infection had occurred development (3). Apart from causing fragmentation and
throughout this period of time. blebbing to embryos, it also reduces the pregnancy rate
To verify the effectiveness of this method, some discarded (4, 5). Therefore, washing bacteria-contaminated embryos
embryos were infected with Escherichia coli. A total of 14 with a medium containing antibiotics is a method of
triploid embryos were cultured with a medium containing rescuing the embryos by attempting to remove microor-
E. coli and were noticed to be contaminated by E. coli the ganisms from the embryo surface. Studies have shown
next day. These embryos were then randomly divided into 2 that the growth of bacteria can be inhibited by adding
groups of 7 embryos each. Each group was cultured for 72 exogenous penicillin (31.5 IU/mL) and streptomycin (10
hours, and the bacterial growth in the embryo culture droplet mg/mL) or gentamicin (0.15 g/L) into the washing me-
was observed every day. All 7 embryos in the washing treat- dium (12, 13). However, this might not be the most
ment group suffered from E. coli contamination again on the effective method for decontaminating embryos. Our pre-
next day after treatment. In contrast, the ZP-free group ex- vious study showed that only 7 of 42 cases of contami-
hibited no bacterial growth after 3 days of culture, and the nation had embryos available for transfer after treatment
embryo morphology was maintained without blastomere lysis with this method (14). There are two possible explana-
and separation. tions for this outcome: there was a high bacterial load
in the culture medium or inaccuracy in the applied anti-
biotic susceptibility test. Because bacterial susceptibility
DISCUSSION testing usually requires several days before results can
In this study, the recontamination of embryos by bacteria was be obtained, the antibiotic choice to reverse contamina-
prevented through the removal of the ZP of the contaminated tions in embryos can therefore only be selected
embryos, which in turn enabled the patient to achieve subjectively.

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