|Year : 2015 | Volume
| Issue : 1 | Page : 41-46
Impact of sperm cryopreservation on child sex after intracytoplasmic sperm injection
Ahmed M Omar MD 1, Mahmoud F Abdel Hamid1, Amr H Abbassy2
1 Department of Dermatology and Venerology, National Research Centre, Cairo, Egypt
2 Department of Obstetrics and Gynecology, National Research Centre, Cairo, Egypt
|Date of Submission||27-Nov-2014|
|Date of Acceptance||28-Jan-2015|
|Date of Web Publication||25-Jun-2015|
Ahmed M Omar
Department of Dermatology and Venerology, National Research Centre, Dokki, PO Box 12622, Cairo
Source of Support: None, Conflict of Interest: None
Sperm cryopreservation causes extensive damage to sperm membranes and its ultrastructural morphology, affecting the fertilization ability by decreasing the percentage of normal intact acrosomes and consequently the acrosine activity. This retrospective study aims at detecting the effect of sperm cryopreservation on the baby's sex after intracytoplasmic sperm injection (ICSI) in terms of the susceptibility of X versus Y chromosome baring spermatozoa to cryopreservation.
Patients and methods
This retrospective study included 87 ICSI cycles performed with post-thawed spermatozoa. The patients were classified into two groups (I and II) according to the total sperm count before freezing.
This study included 87 ICSI cycles performed with post-thawed spermatozoa. Patients were classified into two groups (I and II) according to the total sperm count before freezing. Group I included 43 patients with a sperm count less than 0.1 × 10 6 /sample (countable samples). Group II included 44 patients with a sperm count more than 0.1 × 10 6 /sample (uncountable samples). The numbers of fertilized M II, good embryos, clinical pregnancy, and male babies were significantly higher in group I compared with group II.
ICSI using post-thawed spermatozoa of countable samples yielded a higher male sex ratio (80.8%) compared with uncountable samples (28.6%). Thus, spermatozoa that successfully survived the freeze-thaw procedure exhibited an improved chromatin structure and nuclear maturity. These data suggest that sperm cryopreservation may improve the fertilization rate, enhance early embryo development parameters, as well as pregnancy outcome after ICSI.
Keywords: baby sex, intracytoplasmic sperm injection, sperm cryopreservation
|How to cite this article:|
Omar AM, Abdel Hamid MF, Abbassy AH. Impact of sperm cryopreservation on child sex after intracytoplasmic sperm injection. J Arab Soc Med Res 2015;10:41-6
|How to cite this URL:|
Omar AM, Abdel Hamid MF, Abbassy AH. Impact of sperm cryopreservation on child sex after intracytoplasmic sperm injection. J Arab Soc Med Res [serial online] 2015 [cited 2017 Jun 28];10:41-6. Available from: http://www.new.asmr.eg.net/text.asp?2015/10/1/41/159376
| Introduction|| |
Cryopreservation of human spermatozoa is routinely used in many assisted insemination and fertilization programmes  . Cryopreservation causes extensive damage to the sperm membranes and decreases the percentage of motile spermatozoa and the velocity of their movement ,, , decreasing the fertilization ability by decreasing the percentage of normal intact acrosomes and consequently the acrosine activity ,, .
As early as 1971, Pedersen and Lebech  described severe impairment of sperms in terms of ultrastructural morphology. Several investigators have confirmed this damage at the level of the membranes and acrosomes after freezing ,,, , which appear swollen and ruptured  ; however, a comparison of the ultrastructural morphology of fresh and frozen-thawed testicular retrieved spermatozoa showed intactness of the nuclear membranes and chromatin in frozen-thawed samples.
Cryopreservation increases sperm DNA fragmentation, as concluded by Li et al.  and Pérez-Cerezalesin et al.  . Further, Steele et al.  using the comet assay showed that control ejaculated sperm DNA was significantly more damaged than testicular sperm DNA from control men; they also showed that the percentage of undamaged DNA in testicular sperm from fertile men was significantly greater than the percentage of undamaged DNA in ejaculated sperm from the same men; this may explain the higher percentage of recovered spermatozoa in testicular samples compared with ejaculated samples and may also explain the higher pregnancy rates that were observed in frozen-thawed surgically retrieved spermatozoa used for intracytoplasmic sperm injection (ICSI)  .
Despite the fact that sperm cryopreservation reduces sperm quality, samples from fertile men are significantly better than those from infertile individuals , . An explanation for this discrepancy may be the higher rate of reactive oxygen species production in low-quality samples than in normal-quality samples [20-24] .
As ejaculate contains several cellular components other than spermatozoa, their sublethal damage results from the combined effects of cell dehydration/rehydration, membrane lipid phase transition, alteration in the enzyme activity or energy metabolism, and activation of lipid peroxidation cascade with generation of reactive oxygen species ,, . This may explain the lower results of ejaculated spermatozoa compared with other sperm sources. Several authors reported that the clinical pregnancy rate is similar when frozen-thawed testicular spermatozoa or fresh gametes are used for ICSI ,,,,, . Severe impairment of sperm motility depends largely on the initial quality of the sample, the cryoprotectant, or the method of freezing ,,,,, .
The present study aims at detecting the effect of sperm cryopreservation on the baby's sex after ICSI in terms of the susceptibility of X versus Y chromosome baring spermatozoa to cryopreservation.
| Patients and methods|| |
This is a retrospective study of patients who underwent cryopreservation of motile ejaculated (26 cases) and testicular (61 cases) spermatozoa using sperm freeze (FertiPro NV, Beernem, Belgium) as a cryoprotectant, programmed freezing protocol, and sperm storage in liquid nitrogen as the refrigerant (−196°C). One of the authors was the working embryologist.
Clinical history (full history and genital examination), laboratory data (hormonal profile if needed, including follicular stimulating hormone, leutenizing hormone, testosterone, and prolactin), prethawing and post-thawing sperm count, motility, and vitality were determined.
Sample analysis was performed according to the guidelines of the WHO , under a light microscope (Olympus CH 30 RF 200; Olympus Company Limited, Tokyo, Japan).
Testicular tissue samples were collected in a HEPES buffered Earle's balanced salt solution; the biopsy samples were shredded with two sterile microscopic glass slides. Fine pincers were used for further mincing  , and the sample was examined under an inverted microscope (Olympus 1 × 70 S8F2; Olympus Company Limited). If no spermatozoa were detected the tissue fluid was treated with erythrocyte lysing buffer and the sample was centrifuged  . A dish with microdrops was then prepared (2 mm each drop) from the pellet, covered with sigma oil, and examined under an inverted microscope , .
The sample was diluted 1 : 1 (v/v) with the sperm freeze medium, which was added dropwise from the side of a 15-ml falcon tube (Falcon 2095; Becton Dickinson, New Jersey, USA) over 10 min to minimize hyperosmotic stress, while continuously shaking the tube. The diluted semen was loaded into straws using an automatic pipette. The straws were sealed at one end with a cotton plug. After aspiration each straw was sealed on the other side. The straws were labeled individually with the name of the patient, file number, date of cryopreservation, and nature of the sample (Brady, New Jersey, USA), and placed in the chamber of a computer-controlled biological freezer (Nicool LM 10; Air Liquide, Paris, France) and cooled with the freezing program described by Yavetz et al.  . The rate of freezing was as follows: from room temperature to 10°C at a rate of -1.6°C/min for 6 min (program 5), and from 10 to -120°C at a rate of -5.5°C/min for 20 min (program 10). The sample was then removed from the controlled-rate freezer and plunged directly into the liquid nitrogen storage tank at -196°C  .
When a straw had to be thawed, it was removed from the liquid nitrogen tank according to the patient identification number, file number, registration data, and cryopreservation tank map and thawed at 37°C for 10 min. One end of the straw was cut and the straw was placed near the tip of a conical falcon tube and the other end was cut to let the sample fall into the tube. The cryoprotectant was removed by placing the sample in a tube to which 10 ml of Earle's balanced salt solution containing 0.5% human serum albumin was added slowly dropwise. Equilibration at 37°C for 10 min and then centrifugation at 1500 rpm for 10 min were carried out. The supernatant was removed and the pellet was resuspended in a fixed volume of 200 ml. The sample was then evaluated using the guidelines of WHO , and the results were recorded.
The ICSI procedures involved sperm and oocyte preparation , . The ICSI procedure was performed as described by Van Steirteghem et al.  , Al-Hassani et al.  , Merchant et al.  . A duration of 16-18 h after injection, the oocytes were examined for the presence of pronuclei and polar bodies , , and at 25 h after injection the oocytes were monitored for early cleavage  . The embryos were monitored at exactly 48 h for four-cell stage (day 2 transfer), or at exactly 72 h for 7-9-cell stage (day 3 transfer). The embryos were then transferred to the uterine cavity according to the protocol followed by Merchant et al.  , Racowsky et al.  , Alikani et al. , , Ziebe et al.  . Luteal support was provided with either human chorionic gonadotropins (hCG) or natural micronized progesterone (600 mg/day).
| Results|| |
This study included 87 ICSI cycles performed with post-thawed spermatozoa. The patients were classified into two groups (I and II) according to the total sperm count before freezing. Group I included 43 patients with a sperm count less than 0.1 × 10 6 /sample (countable samples). Group II included 44 patients with a sperm count more than 0.1 × 10 6 /sample (uncountable samples).
There was no statistically significant difference in male clinical parameters, sperm freezing and thawing data, and female clinical parameters between pregnant and nonpregnant ladies in groups I and II, as shown in [Table 1] [Table 2] [Table 3], respectively.
|Table 1 The difference in male clinical and endocrinal parameters between pregnant and nonpregnant women|
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|Table 2 The difference in prefreezing and post-thawing data between pregnant and nonpregnant women|
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|Table 3 The difference in female clinical, hormonal, and biological parameters between pregnant and nonpregnant women|
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There was a statistically significant difference in the numbers of fertilized M II and good-quality embryos between pregnant and nonpregnant ladies in groups I and II, whereas other ICSI parameters were statistically not significant, as shown in [Table 4].
|Table 4 The difference in intracytoplasmic sperm injection parameters between pregnant and nonpregnant women|
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All patients underwent a pregnancy test 2 weeks after embryo transfer; 28 of 87 (32.2%) cases showed a positive pregnancy test as detected by the b-hCG in their serum. Gestational sac and presence of pulsating fetal heartbeat were detected by means of transabdominal ultrasound in 27 of 87 (31%) cases 2 weeks after the pregnancy test. Only one of the ejaculated samples resulted in a positive pregnancy (1/26, 3.8%), compared with 26 of 40 (65%) from retrieved spermatozoa in groups I and II, as shown in [Table 5]. Twelve of the 27 positive pregnancies resulted in successful deliveries and the sex of the babies is shown in [Table 6]; seven of 27 cases are still pregnant and eight of 27 aborted. This study showed that the take baby home was 11 males and seven females in group I and II as shown in [Table 6].
|Table 5 The percentage of frozen ejaculated sperm samples and testicularly retrieved spermatozoa among pregnancy test results in groups I and II|
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|Table 6 The number and percentage of delivered cases and their baby's sex in groups I and II|
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The percentage of good embryos to fertilized M II and the number of clinical pregnancies were significantly higher in group I compared with group II. In contrast, no significant difference between groups I and II was detected in other parameters, as shown in [Table 7].
|Table 7 A comparison between groups I and II regarding embryological records|
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To detect a possible explanation to these findings, we compared the female clinical parameters and ICSI data in groups I and II, as shown in [Table 8]. The results showed that the number of good and fair embryos and the number of embryos transferred was significantly higher in group I, which may explain the higher pregnancy rate [Table 7].
|Table 8 The differences in female clinical parameters and intracytoplasmic sperm injection data between groups I and II|
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A comparison between the two groups regarding the sex of delivered babies showed that the number of male babies was significantly higher in group I when compared with group II, as described in [Table 9].
| Discussion|| |
Cryopreservation is associated with extensive damage to cell membranes, and results in alteration of the functional and metabolic status of the cells and mitochondria. Evidence suggests an increase in DNA single-strand breaks, and degree of DNA condensation or fragmentation in sperm after cryopreservation , . Pregnancy rate was significantly higher in group I compared with group II [18/43 (41.9%) vs. 9/44 (20.4%)]. The difference is attributed to the high percentage of good embryos/fertilized M II (115/275, 41.8%) in group I versus group II (67/257, 26%) (P < 0.05) and to the mean numbers of embryos transferred, which was statistically higher in group I (4.16 ± 1.65) compared with group II (3.23 ± 1.87) (P < 0.05). This difference could not be explained by female factors as the differences in mean age, serum follicular stimulating hormone level, number of cumulus masses, mature oocytes, number of injected oocytes, fertilized oocytes, and embryos were statistically insignificant between groups I and II. This may point to the effect of sperm factor on the ICSI outcome as some changes caused by cryopreservation resemble the changes occurring during normal capacitation that contribute to more efficient oocyte activation and/or pronuclear formation after ICSI , .
The mean number of fertilized M II and the number of embryos were significantly higher in pregnant ladies. No significant differences between pregnant and nonpregnant ladies were detected in other parameters.
ICSI using frozen-thawed samples in group I yielded a higher male sex ratio (80.8%) compared with countable samples (28.6%). This study is contradictory to a previous work that showed no significant difference in sex ratio when frozen-thawed spermatozoa were used for artificial insemination  , as artificial insemination donor  or as IVF donor  . In contrast, Sidhu et al.  had reported that more male children (101 male versus 83 female children) were born after IUI using cryopreserved thawed spermatozoa . However, to our knowledge, no studies have reported differences in sex ratio when ICSI was performed using cryopreserved thawed spermatozoa.
During spermatogenesis the chromatin becomes highly condensed within a protamine matrix  . The DNA is organized into loops, attached at their bases to the nuclear matrix, anchored to the base of the sperm tail by the nuclear annulus, and stabilized by disulfide bonds , . This tight packing of the DNA reduces exposure to free radical attack. It has been proven that hidden anomaly such as higher levels of loosely packaged chromatin and damaged DNA can be present in sperm nuclei from men with deficient spermatozoa. The most frequent visible changes are related to the protamine-deficient, nicked, and partially denatured DNA , .
A properly performed cryopreservation may selectively affect defective rather than normal spermatozoa  . Furthermore, spermatozoa that successfully survived the freeze-thaw procedure also exhibited an improved chromatin structure and nuclear maturity. These data suggest that sperm cryopreservation when performed correctly may not only improve the fertilization rate but may also enhance early embryo development parameters as well as pregnancy outcome after ICSI  .
As most of the cases had few uncountable sperms, limited number of spermatozoa have been retrieved and used for oocyte injection. This finding may be explained by the fact that cryopreservation acts as an artificial selecting process with minimal effect on the Y chromosome (60 megabase) due to its smaller size and lower molecular weight compared with the larger X chromosome (160 megabase)  . We think that the flow cytometric sorting used to separate spermatozoa baring Y chromosome based on sex chromosome content might be of added value for use as a preconception method of influencing baby sex  to avoid having children affected by sex-linked disease. There are over 1100 X-linked diseases and ~60 Y-linked diseases. The embryonic sex data (as determined by PGD) show that the proportions of XX embryos after X-sort and XY embryos after Y-sort were consistent with the post-sort FISH results.
If sperm cryopreservation sorting did adversely affect the sperm function, one would expect lower rates of fertilization, cleavage, and pregnancy, which was not the case in this study.
In our study seven cases are still pregnant and the baby's sex has not been determined yet; the sex of these unborn children may or may not confirm the results of this study.
In conclusion, ICSI using post-thawed spermatozoa of countable samples yielded a higher male sex ratio (80.8%) compared with uncountable samples (28.6%). Thus, it is suggested that sperm cryopreservation might affect the pregnancy outcome after ICSI, which needs further controlled studies on a larger scale to be validated.
| Acknowledgements|| |
Conflicts of interest
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9]