Assisted reproductive technology (ART), first introduced in the 19th century, is used to treat infertility. The ART used in present times has developed in many aspects that include obtaining gametes (stimulation with gonadotropins and retrievals),
in vitro maturation (IVM) of immature oocytes, in vitro fertilization (IVF), insemination by intra-cytoplasmic sperm injection (ICSI), embryo transfer, freezing of gametes and embryos, biopsy of embryos for pre-implantation genetic diagnosis, and cytoplasmic transfer. As the process of becoming pregnant to giving birth is complex, ARTs have helped millions of infertile women worldwide to overcome childlessness till date. Multiple factors can affect the success of pregnancy in infertile women. For ARTs, the age of the woman is typically considered to be the key point related to the success rate of the treatment. The quality of oocytes produced by women degrades after a certain age (usually considered after 35 years of age) [ 1 ]. Therefore, oocyte quality can be considered the most important factor for the success of the treatment of infertility with ARTs.
In addition to the active role of sperm during fertilization, the sperm is essential for the diploid genetic constitution of the embryo, thus important for further development
[ 2 , 3 ]. The sperm interacts with the oocyte cytoskeletal apparatus to form a functional pair of centrosomes that regulate the pronuclear position in preparation for the first embryonic cleavage [ 4 ]. The sperm centrioles are essential for cell division during the embryonic development. Therefore, sperm DNA integrity is essential for embryonic development and health. In the presence of high level of sperm DNA damage, the DNA repair machinery is activated in oocytes to ensure optimal DNA transfer to the zygote, resulting in a long gap before the first embryonic cell division occurs [ 5 ]. Increased sperm DNA damage adversely affects embryo quality starting on the day 2 of early embryonic development and continuing after embryo transfer, resulting in reduced implantation rates and pregnancy outcomes [ 6 ]. The contribution of the sperm to embryonic development goes far beyond its well-established role in fertilization, highlighting the importance of good-quality sperm [ 7–9 ].
There are different methods of preparing sperm for infertility treatment. Sperm samples prepared by gradient centrifugation may be more stable in terms of DNA fragmentation than those prepared by swim-up (SU)
[ 10 ]. Other than sperm DNA fragmentation, variations in sperm telomere length (STL) may affect the development of zygotes; notably critically short telomeres in sperm may adversely affect the development of zygotes [ 11 ]. Both gradient centrifugation and SU methods can recover a sperm population with long STL and generate good DNA integrity for ART [ 12 ]. However, the use of selected STL measurements to evaluate the reproductive potential of male patients or to predict the success rates of ART treatment could not be substantiated, since the total population of the selected STLs was not verified against sperm quality and clinical outcomes [ 13 ]. Recently, microfluidic chip-based sperm sorting has been introduced as an alternative to conventional centrifugation-based techniques [ 14–16 ]. Interestingly, microfluidic sorting of unprocessed semen allows for the selection of clinically usable and highly motile sperm with nearly undetectable levels of DNA fragmentation [ 17 ]. Nevertheless, density gradient centrifugation (DGC) in combination with SU is still widely used worldwide for IVF treatment [ 18 ].
Given the efficiency of IVF and improvements in the culture system, natural-cycle IVF or mild stimulation may be suitable for women undergoing IVF treatment
[ 19 ]. In contrast to that of standard stimulation IVF treatment, the aim of mild stimulation is to develop safer and patient-friendlier protocols. Importantly, it is recommended that a successful IVF cycle should be redefined as the birth of a healthy singleton baby at term without compromising the health and safety of both the woman and baby and that it is achieved at the lowest possible cost [ 20 , 21 ]. Recovery of immature eggs following IVM is a potential useful treatment for infertile women. As the scientific community gains more experience and accumulates more outcome data from natural-cycle IVF and mild stimulation IVF, there is a growing interest in IVM treatments as they may prove to be potential alternatives to standard stimulation treatments, as well as first-line treatment choices [ 22 ].
In the development of IVM treatment, one attractive possibility for promoting successful outcome is to combine natural-cycle IVF treatment with immature oocyte retrieval followed by IVM of the retrieved immature oocytes
[ 23 ]. The use of IVM technology can be increased to treat women suffering from all types of infertility with acceptable pregnancy and live birth rates [ 24–28 ]. Notably, it is estimated that thousands of healthy babies are delivered by employing IVM oocytes [ 28 , 29 ], and evidence from molecular analysis indicates that there are no differences between in vivo and in vitro matured human eggs [ 30 , 31 ].
During natural-cycle IVF treatment in combination with immature oocytes,
in vivo and in vitro mature oocytes may also be retrieved. As per the protocol, only one sperm sample is required on the day of oocyte retrieval, and in vitro matured oocytes are then inseminated by ICSI with the collected sperm on the second day [ 23 ]. Although there are many healthy live births by employing sperm samples prepared a day before the IVM of oocytes, it is necessary to confirm whether these sperms are suitable on the second day for insemination by ICSI. Therefore, the objective of this study was to verify the suitability of the selected sperm on the second day for insemination of in vitro matured oocytes by ICSI by evaluating the sperm quality in terms of motility and DNA fragmentation index (DFI). Materials and methods
Semen samples were collected from 30 outpatients who visited the Center for Reproductive Medicine for semen analyses. All participants included in the study signed the written informed consent form. The procedures performed in this study were approved by the ethics committee of Shanghai Tenth People’s Hospital. The inclusion criteria were healthy males under 35 years of age, and semen parameters that met the fifth edition of the World Health Organization (WHO) criteria. Males with genetic diseases were excluded from this study. Semen samples were collected in a sterile semen cup by ejaculation after 2–3 days of sexual abstinence. The collected semen cup was placed on a rotary shaker at room temperature (RT) to allow liquefaction. After liquefaction, routine semen analysis was performed according to WHO guidelines, and the remaining semen samples were used for DGC and SU procedures.
Sperm preparation procedures
DGC procedure: Motile spermatozoa were separated from seminal plasma using the DGC technique. In brief, semen samples were added to a 14-mL centrifuge tube previously prepared with 1.0 mL 80% and 40% gradients (COOK, USA). The tubes were then centrifuged for 20 min at 300
×g. After centrifugation, the bottom of the tube precipitate was aspirated and transferred to another tube and washed twice with 3.0 mL of Gamete Buffer (COOK, USA) by centrifugation at 200 ×g for 10 minutes.
SU procedure: The supernatant was discarded and sperm pellet was added to 1.0 mL of Sperm Medium (COOK, USA, equilibrated previously) in tri-gas incubator, 90% N
2, 5% O 2, and 5% CO 2 for 30 minutes of incubation. After SU incubation, the upper supernatant (0.5 mL) of the sperm medium was transferred to a new tube for sperm motility and DFI assessments. Examination of sperm motility
Sperm motility refers to the percentage of sperm with progressive or non-progressive movement. A computer-aided sperm analysis system (CASA, Model CX43RF, Spain) was used to evaluate sperm motility, according to the manufacturer’s instructions. Briefly, 2 µL of the semen samples were pipetted into a glass slide counting chamber and then assessed using the CASA system. A minimum of 200 sperms were examined in each assay. Sperm motility was calculated according to the following formulae: Motility (%) = (number of progressive + non-progressive movement sperms)/ (number of all motile + non-motile sperms)] × 100.
Determination of sperm DFI
Sperm DFI was examined using a DFI Kit (Cellpro Biotech, Zhejiang, China;
) according to the manufacturer’s instructions. First, the sperm concentration was adjusted to 1–2 × 10 http://www.cellpro.com.cn 6/mL with Buffer A, followed by the addition of 200 µL of Buffer B and mixed well. The mixture was incubated for 30 seconds at RT. Subsequently, 600 µL of the prepared staining Buffer C was added. After staining for 1 minutes, the sperm mixture was analyzed using a cell flow cytometer (Beckman Coulter, Model DxFLEX, USA). Experimental design
The sperm motility and DFI were compared before and after DGC and SU procedures. Following DGC and SU procedures, the sperm samples were incubated at 4, 25, and 37°C after tightly capping the tubes. The sperm motility and DFI were assessed following incubation for 24, 48, and 72 hours.
The data were analyzed using SPSS version 20.0 (SPSS Inc., Chicago, CA, USA), and the percentages are expressed as mean ± standard deviation. Statistical significance was determined using the Student’s
t-test ( P <0.05). Results
As shown in
Fig. 1, the sperm motility (91.8% ± 8.6%) significantly increased ( P <0.01) following the combination of DGC and SU procedures in comparison to that prior treatment (50.8% ± 13.1%). Moreover, the sperm DFI (5.1% ± 7.9%) significantly decreased ( P <0.01) following the combination of DGC and SU procedures in comparison to that prior treatment (13.0% ± 11.6%) ( Fig. 2). Fig. 1.:
The changes in sperm motility following the combination of density gradient centrifugation (DGC) and swim-up (SU) procedures. *Significant difference among groups (
P <0.01). Fig. 2.:
The changes in sperm DNA fragmentation index (DFI) following the combination of density gradient centrifugation (DGC) and swim-up (SU) procedures. *Significant difference among groups (
The changes in sperm motility following incubation at 4, 25, and 37°C for 24, 48, and 72 hours, respectively, are shown in
Fig. 3. After incubation for 24, 48, and 72 hours, sperm motility gradually declined ( P <0.05) at 4, 25, and 37°C, respectively, compared to sperm motility before incubation (0 hours). However, the sperm motility was significantly higher ( P <0.05) when incubated at 25°C (76.9% ± 10.4%) than that at 4°C (53.5% ± 11.0%) and 37°C (47.6% ± 10.2%) after incubation for 24 hours. Fig. 3.:
The changes in sperm motility following incubation at 4, 25, and 37°C for 24, 48, and 72 h, respectively. *Significant difference among groups (
The changes in sperm DFI following incubation at 4, 25, and 37°C for 24, 48, and 72 hours, respectively, are shown in
Figure 4. After incubation for 24, 48, and 72 hours, at 37°C, the sperm DFI significantly increased ( P <0.05) compared to the sperm DFI prior to incubation (0 hours). However, the sperm DFI did not significantly increase when they were incubated at 4°C (5.7% ± 5.9%) and 25°C (6.8% ± 5.6%) following incubation for 24 hours compared to that before incubation (5.1% ± 7.9%). Following incubation at 25°C for 48 hours, the sperm DFI (10.9% ± 8.2%) was significantly lower ( P <0.05) than that at 37°C for 48 hours (16.9% ± 11.4%). However, the sperm DFI at 25°C for 48 hours of incubation did not differ from the sperm DFI at 4°C (7.2% ± 7.6%) for 48 hours of incubation ( Fig. 4). Fig. 4.:
The changes in sperm DNA fragmentation index (DFI) following incubation at 4, 25, and 37°C for 24, 48, and 72 h, respectively. *Significant difference among groups (
P <0.05). Discussion
This study demonstrated that the sperm motility and DFI can be improved significantly following the combination of DGC and SU procedures for sperm selection. Post the selection procedures, the sperm samples can be incubated at RT for 24 hours without increasing the sperm DFI. Therefore, these sperm samples may be used for insemination of
in vitro matured oocytes by ICSI.
While investigating the effect of different sperm preparation methods on the sperm DNA fragmentation, it was observed that sperm samples prepared by DGC may be more stable in terms of DNA fragmentation than those prepared by SU
[ 10 ]. However, the efficiency of the DGC procedure alone for sperm selection may be relatively low [ 18 ]. Thus, sperm preparation in IVF treatments using DGC in combination with SU has been widely adopted worldwide. The results of this study confirm that sperm motility increased and the level of sperm DFI improved significantly following the combination of DGC and SU procedures ( Figs. 1 and 2).
Sperm quality is an important factor in ART treatment and affects the success rate of the treatment
[ 32 ]. It is commonly believed that one of the causes of ART treatment failure is sperm DNA fragmentation, which may be associated with the incubation period of the sperm sample following its preparation. There is a positive correlation between the incubation time and sperm DNA damage. Prolonged incubation of prepared sperm samples at 37°C was associated with high rates of sperm DNA fragmentation, indicating that the sperm samples intended for ART procedures should be used within 2 hours of incubation at 37°C [ 33 , 34 ]. Interestingly, it has been shown that following 2 and 4 hours of incubation at RT, the sperm progressive motility and viability decreased significantly [ 35 ]. Sperm DNA fragmentation increased significantly after both 2 and 4 hours of incubation at RT and 37°C, respectively, and no significant differences were found between the sperm parameters and DNA fragmentation after the different incubation periods at RT and 37°C, suggesting that IVF, ICSI, and intrauterine insemination procedures should be performed at the earliest after sperm preparation [ 35 ]. However, our results indicated that although following the combination of DGC and SU procedures the sperm motility significantly decreased at the 3 temperatures for 24, 48, and 72 hours of incubation, the sperm motility was significantly higher at 25°C than that at 4 and 37°C for 24, 48, and 72 hours of incubation, respectively ( Fig. 3). More importantly, our results showed that following sperm selection treatment with the combination of DGC and SU procedures, the level of sperm DFI did not significantly increase at 4 and 25°C compared to that at 37°C for 24 hours of incubation ( Fig. 4). These results suggest that sperm samples may be used for insemination of in vitro matured oocytes following IVM by ICSI.
The recovery of immature oocytes following IVM is a potentially useful treatment for infertile women
[ 36 ]. IVM treatments seems particularly effective for women with polycystic ovaries or polycystic ovarian syndrome-related infertility [ 37 ] because of the numerous antral follicles within the ovaries in these two groups of patients [ 38 , 39 ]. Although many healthy babies are delivered by employing in vitro matured oocytes, its efficiency is comparable to that of conventional ovarian stimulation during IVF cycle treatment [ 19 , 29 ]. Furthermore, the Practice Committees of the American Society for Reproductive Medicine and Society for Assisted Reproductive Technology indicated that IVM should be offered by experienced personnel with expertise gained by specific training, accompanied with appropriate counseling about the expected results, and require informed consent. Therefore, this technology is no longer considered experimental [ 40 ], and IVM provides an alternate treatment protocol for infertile women [ 29 , 41 ].
Natural-cycle IVF, mild stimulation IVF, and IVM treatment may be alternatives to standard stimulation treatments as well as a potential first-line treatment
[ 22 ]. An attractive possibility for enhancing successful outcome is the combination of natural-cycle IVF treatment with immature oocyte retrieval followed by IVM of these immature oocytes [ 23 ]. The use of IVM technology can treat women suffering from all types of infertility, with acceptable pregnancy and live birth rates [ 24–28 ]. However, although many healthy babies were delivered by employing in vitro mature oocytes [ 28 , 29 ], there is still a practical question as to whether the sperm samples collected and treated on the day of oocyte retrieval can be used on the second day for insemination of in vitro matured oocytes by ICSI, as it is not always convenient to obtain a second sperm sample from the male donor. The results of this study demonstrated that sperm samples can be incubated at 25°C for 24 hours following the combination of DGC and SU procedures without leading to an increase in the level of sperm DFI. Therefore, these sperm samples may be used on the second day for insemination of in vitro matured oocytes by ICSI. Conclusions
These results demonstrate that sperm quality, in terms of motility and DFI, can be improved efficiently by using DGC in combination with SU for sperm selection. After the selection procedures, the selected sperms can be incubated at 25°C (RT) and be used on the second day for insemination of
in vitro matured oocytes by ICSI. Acknowledgments
Y.N.Y. and L.W.: experimental execution, data analysis, and critical discussion; Y.B.L., Y.J.X., C.C.L., F.S., and X.D.: experimental execution and manuscript comment; R.C.C.: study design, manuscript writing, and critical discussion. All the authors have read and approved the final version of the manuscript.
This research was supported by the Ministry of Science and Technology of China, National Key R&D Program of China (No. 2017YFC1002003 and No. 2017YFC1001601)
Conflicts of interest
All authors declare no conflict of interest.
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