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Sperm DNA Damage Linked To Increased Risk of Pregnancy Loss after IVF and ICSI

Earlier studies have indicated that spermatozoa of infertile men possess considerably more sperm DNA damage than that of fertile men, which could adversely affect both natural reproduction and assisted reproduction technology (ART) outcomes. Since the use of ART to treat infertility has increased substantially from its introduction in the US, in 1981, there is a huge concern regarding the safety of using DNA-damaged spermatozoa. Now, a recent study published online in the journal, Human Reproduction, demonstrates that sperm DNA damage is associated with a significantly enhanced risk of pregnancy loss following in vitro fertilization (IVF) and intra cytoplasmic sperm injection (ICSI).
Armand Zini from the St Mary’s Hospital Center, Canada and coworkers conducted a systematic review and meta-analysis of studies involving sperm DNA damage and pregnancy loss after IVF and/or ICSI treatment to determine the relationship between the two. The researchers analyzed 11 studies, which included 808 IVF and 741 ICSI cycles of treatment, resulting in 640 pregnancies (345 with IVF and 295 with ICSI) and 122 pregnancy losses. From the estimates of pregnancy loss, two by two tables were constructed and odds ratios (ORs) were calculated to examine the association. It was found that the combined odds ratio was 2.48, suggesting that damage to spermatozoa DNA raises the risk of pregnancy loss in IVF and ICSI cycles. The data indicates that sperm DNA damage has to be evaluated prior to ARTs to achieve better outcomes and also forms a basis for further investigations to validate the findings.
Recently, Bhattacharya SM (International Urology and Nephrology, 2008) conducted a study to examine the link between different sperm parameters and repeated unexplained early pregnancy loss. Semen samples obtained from male partners of 74 couples with a history of repeated pregnancy loss were assessed according to WHO criteria and the DNA integrity in each case was evaluated using Acridine Orange staining test. A comparison of the results was also drawn by studying semen samples obtained from 65 husbands of proven fertility. It was noted that there were a lack of statistically significant differences between the two groups in the following criteria: age of husbands, total count per ejaculate, sperm concentration, and rapid progressive motility of sperms. However, DNA integrity value, percentage of motile sperm and total motile sperms per ejaculate, were different in the two groups. Based on the findings, it was concluded that repeated embryonic or early fetal loss is associated with sperm DNA-integrity damage, implying that sperm DNA damage may be a key paternal factor for predicting pregnancy outcomes.
To determine the relationship between sperm DNA fragmentation in IVF/ICSI patients, ART outcome, and sperm parameters, Borini and colleagues (Human Reproduction, 2006) conducted a study on 132 men undergoing ART. The scientists found that the embryo post-implantation development in ICSI procedures is affected by sperm DNA fragmentation; with a high fragmentation rate compromising embryo viability, and thereby leading to pregnancy loss.
Sperm DNA damage is attributed to various intra-or extratesticular factors such as chemotherapy, radiation therapy, genital tract inflammation, testicular hyperthermia, varicoceles, cigarette smoking and environmental toxins. Although previous studies have found no consistent relation between sperm DNA damage and fertilization rates during IVF or ICSI, the recent research provides evidence that sperm DNA damage may be associated with an increased risk of significant spontaneous abortion. Substantiating the current research with further larger trials may help in the development of stringent processes for selecting sperms and embryos during ART to alleviate the adverse effects related to sperm DNA damage.
References
1. Zini A, Boman JM, Belzile E, Ciampi A. Sperm DNA damage is associated with an increased risk of pregnancy loss after IVF and ICSI: systematic review and meta-analysis. Hum Reprod. 2008 Dec;23(12):2663-8. Epub 2008 Aug 29.
2. Bhattacharya SM. Association of various sperm parameters with unexplained repeated early pregnancy loss–which is most important? Int Urol Nephrol. 2008;40(2):391-5.
3. Borini A, Tarozzi N, Bizzaro D, et al. Sperm DNA fragmentation: paternal effect on early post-implantation embryo development in ART. Hum Reprod. 2006 Nov;21(11):2876-81. Epub 2006 Jun 22.

Dominant Follicle Diameter Helps Select Optimal Day for Oocyte Retrieval in IVM Cycles

In vitro maturation (IVM), a novel assisted reproduction technique, reduces risks associated with in vitro fertilization (IVF) as the eggs are retrieved, matured and fertilized in vitro prior to implantation, thereby eliminating ovarian stimulation. However, the factors predisposing the success or failure of IVM cycles are unclear. Now, a recent study published in the December issue of the journal, Human Reproduction suggests that dominant follicle (DF) size of ≤14mm at oocyte retrieval following human chorionic gonadotropin (hCG) priming improves pregnancy outcomes in cycles programmed for IVM treatment.
Weon-Young Son from the McGill University, Montreal, and coworkers conducted a study on 160 women with polycystic ovaries (171 cycles) to compare the DF size at oocyte retrieval after hCG priming with IVM outcome. When the endometrial thickness reached a minimal of 6 mm, the researchers subcutaneously administered 10,000 IU hCG, 35 to 38 hours prior to oocyte collection. The retrospective analysis was performed in 3 study groups based on the DF diameter: group 1, with a diameter of ≤10 mm; group 2, between 10 and 14 mm; and group 3 of >14mm. In the corresponding 3 groups, 6.9%, 10.6%, and 15.1% of the in vivo matured oocytes were collected, suggesting a positive correlation between the size and number of oocytes.
Results showed that among the sibling immature oocytes extracted in the 3 groups, the rates of IVM, fertilization and embryo development were similar. It was found that group 3 exhibited a lower clinical pregnancy rate (17.1%) compared to group 2 (40.3%). Furthermore, groups 1 (13.6%) and 2 (14.3%) had higher implantation rates than group 3 (4.9%). Based on the study findings, the researchers proposed DF ≤14mm as the optimal oocyte retrieval time for IVM cycles, as DF >14 mm may detrimentally affect the sibling immature oocytes.
Earlier, the same group of researchers conducted a retrospective study (Human Reproduction, 2008) to investigate if an extension in the time interval between hCG priming and immature oocyte retrieval enhances the oocyte maturation rate after IVM. The assisted reproduction technique was performed on 113 polycystic ovary syndrome patients (120 cycles) and the oocytes were collected at either 35 hours (group 1=76) or 38 hours (group 2 = 44) following 10,000 IU of hCG priming. The oocyte maturity was analyzed after the retrieval and the culture of the immature oocytes was performed till day 2 using IVM medium. It was found that the number of in vivo matured oocytes was considerably lower in group 1 (13.6%) compared to group 2 (7.3%). Also, group 2 exhibited a higher oocyte maturation rate after day 1 (46.3 vs. 36.0%), clinical pregnancy (40.9 vs. 25%) and implantation rates (15.6 vs. 9.6%) than group 1. Based on the findings, the scientists suggested that extending the time of hCG priming from 35 hours to 38 hours for oocyte retrieval could improve the pregnancy outcome of IVM cycles.
In vitro maturation of immature oocytes collected from unstimulated ovaries is an assisted reproduction technology that is extensively being studied. Some of the advantages of IVM over IVF are that it is less expensive, has shorter treatment regimen, and does not require the use of hormonal fertility drugs for ovarian stimulation. It may thereby eliminate the risk of developing ovarian hyperstimulation syndrome and multiple pregnancies.
Several previous studies have indicated that controlled ovarian stimulation in combination with in vitro fertilization cycles provide better results compared to in vitro maturation techniques. Now, the identification of the optimal hCG priming time and dominant follicle size for oocyte retrieval may help in enhancing the success rates of the novel IVM technique with fewer adverse effects compared to IVF.
References
1.Son WY, Chung JT, Herrero B, et al. Selection of the optimal day for oocyte retrieval based on the diameter of the dominant follicle in hCG-primed in vitro maturation cycles. Hum Reprod. 2008 Dec;23(12):2680-5. Epub 2008 Sep 4.
2.Son WY, Chung JT, Chian RC, et al. A 38 h interval between hCG priming and oocyte retrieval increases in vivo and in vitro oocyte maturation rate in programmed IVM cycles. Hum Reprod. 2008 Sep;23(9):2010-6. Epub 2008 Jun 12.

Pretreatment with Transdermal Testosterone may Enhance Ovarian Response to FSH

The synergistic effects of androgens and follicle stimulating hormone (FSH) on folliculogenesis, a process critical for assisted reproduction techniques, have been previously established in primates. Now, a recent randomized clinical trial published in the journal Human Reproduction demonstrates that pretreatment with transdermal testosterone enhances the follicular response in poor IVF responders compared to the high-dose gonadotropin and minidose GnRH agonist protocol.
Francisco Jose Fabregues Gasol, from the Institut Clínic of Gynecology, Obstetrics and Neonatology, University of Barcelona, Spain, and colleagues, conducted a randomized trial to explore the efficacy of transdermal testosterone pretreatment in poor IVF responders. Sixty-two infertile women with a history of poor follicular response to IVF cycle were equally divided into two groups for their second IVF treatment. The first group received transdermal testosterone prior to standard ovarian stimulation, along with gonadotropin under pituitary suppression. On the other hand, the second group was administered with high-dose gonadotropin, along with a low-dose gonadotropin releasing hormone (GnRH) agonist protocol for ovarian stimulation.
Investigators observed that the first group had 32.2% cycles with low response, as compared to 71% in the other group. Also, ovum retrieval was more in the first group of patients (80.6%) in contrast to the second group (58.1%), with statistically significant difference (81.2% vs. 41.1%) in patients with normal basal FSH levels. The study results showed that the ovarian sensitivity to FSH and the response of follicles to gonadotropin treatment could be enhanced through transdermal testosterone pretreatment in poor IVF responders.
Earlier, Balasch and colleagues (Human Reproduction, 2006) conducted a self-controlled, therapeutic clinical trial on 25 infertile women with a history of poor follicular response, leading to cancellation of first and second IVF cycles. It was observed that pretreatment with transdermal testosterone could be an effective approach for poor IVF responders with normal basal FSH concentration but poor controlled ovarian stimulation response.
In a contradictory study, Massin, et al. (Human Reproduction, 2006) observed that testosterone administration does not significantly affect the ovarian response to FSH. The researchers conducted a double-blind study on women with a background of low response to controlled ovarian stimulation and a low hormonal ovarian reserve, to evaluate the effects of androgen application. The subjects were randomized to receive either transdermal testosterone or placebo gel for 15 days prior to gonadotropin administration for a second IVF cycle. Both cycles involved identical GnRH analogue and equal FSH daily doses. Plasma testosterone levels were substantially enhanced, with no similar effects on the antral follicular count in the test group. There was no significant difference in both the groups with regard to the main ovarian response parameters such as pre-ovulatory follicular number, and the count of total and mature oocytes and embryos. Based on their findings the investigators emphasized on further clinical trials to study the effects of optimal dose and duration for testosterone administration.
Conditions, such as high body mass index, pelvic adhesions, prior ovarian surgery, or progressive age, could be related with low ovarian response to gonadotropin treatment. The outcome of an IVF treatment is directly dependent on the number of embryos available. Poor ovarian response could lead to low embryo count, resulting in decreased pregnancy rates. Kailasam and colleagues (Human Reproduction, 2004) defined poor response in women aged <40 years, as cancellation of the cycle in patients on ≥300 IU FSH/day, (<3 pre-ovulatory follicle development), or the requirement for the total administration of ≥3000 IU FSH for sufficient follicular recruitment, substantiating oocyte retrieval.
Poor ovarian response to standard FSH treatment is a major concern in assisted reproduction processes. The current study, which demonstrates the enhanced ovarian sensitivity to gonadotropin in poor IVF responders, by pretreatment with transdermal testosterone, could serve as a novel approach to achieve enhanced success of IVF treatment.
References
1. Fábregues F, Peñarrubia J, Creus M, et al. Transdermal testosterone may improve ovarian response to gonadotrophins in low-responder IVF patients: a randomized, clinical trial. Hum Reprod. 2008 Dec 3. [Epub ahead of print]
2. Balasch J, Fábregues F, Peñarrubia J, et al. Pretreatment with transdermal testosterone may improve ovarian response to gonadotrophins in poor-responder IVF patients with normal basal concentrations of FSH. Hum Reprod. 2006 Jul;21(7):1884-93.
3. Massin N, Cedrin-Durnerin I, Coussieu C, Galey-Fontaine J, Wolf JP, Hugues JN. Effects of transdermal testosterone application on the ovarian response to FSH in poor responders undergoing assisted reproduction technique–a prospective, randomized, double-blind study. Hum Reprod. 2006 May;21(5):1204-11.
4. Kailasam C, Keay SD, Wilson P, Ford WC, Jenkins JM. Defining poor ovarian response during IVF cycles, in women aged <40 years, and its relationship with treatment outcome. Hum Reprod. 2004 Jul;19(7):1544-7.

Cryptorchidism May be Associated with Genetic Mutations

Cryptorchidism, a common congenital defect of the male genitalia that affects 3-4% of full-term infants and 30% of those born prematurely, poses a risk for infertility and testicular cancer. However, the exact etiology and pathogenesis of cryptorchidism are not clearly known. Now, a recent study published in the Journal of the American Medical Association has identified a potential link between cryptorchidism and genetic alterations such as insulin-like factor 3 (INSL3) receptor gene mutations and Klinefelter syndrome.
Alberto Ferlin from the Department of Histology, Microbiology, and Medical Biotechnologies, University of Padova, Italy, and coworkers, conducted the case-control study on 600 male infants in Italy during 2003 to 2005, to investigate the rate of genetic alterations in cryptorchidism. Three hundred noncryptorchid boys between 1 to 4 years were selected as controls. Follow-up of the subjects was done for 2 to 3 years and those with persistent cryptorchidism were treated with orchidopexy.
Study results showed that mutations ranged from low in boys with cryptorchidism (2.8%), to high in those with persistent cryptorchidism (5.3%) and bilateral cryptorchidism (8.3%), compared to controls (0.3%). The calculated odds for having genetic alteration in boys with persistent cryptorchidism were more than 17 times; however, there was lack of mutations in boys with low gestational age or low birth weight and those who had spontaneous descent of the testes. The researchers found that Klinefelter syndrome and INSL3 receptor gene mutations were most frequently associated with cryptorchidism, suggesting a significant relationship between the disease and genetic alterations.
Early studies have indicated that INSL3 plays a key role in testicular descent, with mutations in the gene or its G protein-coupled receptor (leucine-rich repeat, containing G protein-coupled receptor (LGR8)) likely to be associated with cryptorchidism.
Ferlin, et al. (Molecular Human Reproduction, 2006) conducted a study to investigate the hypothesis pertaining to the relationship between INSL3 mutations and the signs of testicular dysgenesis syndrome (TDS), characterized by undescended testis, poor semen quality, testis cancer, and hypospadias. The analysis was performed on 967 subjects with maldescended testes and/or infertility and/or testicular cancer and 450 controls. The researchers also conducted in vitro functional analysis of genetic alterations (R4H, W69R, and R72K) by observing the increase of INSL3-dependent cAMP (cyclic adenosine monophosphate) in cells that express LGR8. It was found that 1.9% of the subjects had 6 INSL3 mutations, with no similar finding in the controls. Based on the findings, it was suggested that a significant link may exist between INSL3 gene alterations and TDS syndrome signs; although, a definite causative role was not established.
Although cryptorchidism or undescended testicle may occur bilaterally, it commonly affects the right testes. A combination of factors such as maternal health, genetics, and environment may play an important role in its development, but the exact cause is unknown. Generally, the condition resolves by itself but sometimes may require orchidopexy to relocate the testis into the scrotum.
Substantiating the current research, which identifies the association between undescended testicles and genetic mutations in INSL3 receptor gene and Klinefelter syndrome, with further larger studies, may help to elucidate the underlying disease mechanism. Further, this may facilitate in developing novel therapeutic strategies for patients with persistent and bilateral cryptorchidism, thereby reducing the burden associated with male infertility.
References
1. Ferlin A, Zuccarello D, Zuccarello B, Chirico MR, Zanon GF, Foresta C. Genetic alterations associated with cryptorchidism. JAMA. 2008 Nov 19;300(19):2271-6.
2. Ferlin A, Bogatcheva NV, Gianesello L, et al. Insulin-like factor 3 gene mutations in testicular dysgenesis syndrome: clinical and functional characterization. Mol Hum Reprod. 2006 Jun;12(6):401-6. Epub 2006 May 10.