Hum. Reprod. Advance Access originally published online on November 29, 2006
Human Reproduction 2007 22(3):751-755; doi:10.1093/humrep/del449
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Twin pregnancy possibly associated with high semen quality
1 University Department of Growth and Reproduction 2 The Ultrasound Clinic, University Hospital of Copenhagen, Denmark
3 To whom correspondence should be addressed at: University Department of Growth and Reproduction, GR-5064, Rigshospitalet, Blegdamsvej 9, 2100 Copenhagen, Denmark. E-mail: camilla.asklund{at}rh.hosp.dk
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Abstract |
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BACKGROUND: Recent studies found an association between a long waiting time to pregnancy (TTP) and reduced probability of twinning and a reduced dizygotic (DZ) twinning rate in subfertile men. However, it remains unsolved whether semen quality is associated with twin offspring. We therefore studied the semen quality in a group of fathers of naturally conceived twins.
METHODS: In this study, 37 fathers of DZ twins and 15 fathers of monozygotic (MZ) twins participated, and 349 normal fertile men served as a reference group. All men delivered a semen sample, underwent a physical examination and completed a questionnaire.
RESULTS: After adjustment, fathers of DZ and MZ twins had 3.6 (95% CI 1.7; 5.4) and 4.6 (95% CI 2.0; 7.2) percentage points higher percentage of sperm cells with normal morphologic features and percentages of motile sperm cells were 11.5 (95% CI 7.2; 15.9) and 12.5 (95% CI 6.3; 18.6) percentage points higher than the reference group (P < 0.01). Fathers of DZ twins and MZ twins had 24.7 (95% CI; –9.1; 71.3) and 17.0% (–25.2%; 83.0%) higher sperm concentration than the reference group.
CONCLUSIONS: Fathers of DZ twins had a better semen quality than the reference group, which supports the assumption that spontaneous DZ twinning rate can be used as a sensor of male fecundity of a population.
Key words: dizygotic twinning rates/semen quality/fecundity/twinning
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Background |
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Trends in twinning rates may be a marker of changing fecundity of both women and men (Lazar et al., 1978
; James, 1982; James, 1997; Tong and Short, 1998). Previous studies have shown that spontaneous dizygotic (DZ) twinning were influenced by inheritance, race, maternal age, body mass index and parity, whereas the causes of monozygotic (MZ) twinning remain unclear (Hall, 2003; Basso et al., 2004; Herskind et al., 2005). There has been a widespread decline in the DZ twinning rates in the 1960s and 1970s, whereas the MZ twinning rates have been reported to be relatively constant (Rachootin and Olsen, 1980; James, 1982; James, 1986). With the increased use of fertility drugs and in vitro fertilization, there has been a marked rise in twinning rates since the 1980s (James, 1982; James, 1986; Hall, 2003). Therefore, recent trends cannot be used as fecundity measures.
The hypothesis that DZ twinning rate may be a marker of fecundity (Lazar et al., 1978) has only been tested in two studies, one of which reported that long waiting time to pregnancy (TTP) reduced the chance of twinning (Basso et al., 2004). The other found reduced chance of DZ twinning in subfertile men (Richiardi et al., 2004), suggesting that decreasing trends in semen quality could contribute to the low twinning rate. However, studies on semen quality in fathers of twins are lacking. We therefore studied the semen quality in a group of fathers of naturally conceived twins.
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Materials and methods |
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Study population
Fathers of ‘naturally conceived’ twins (Group A = 85) were identified from an ongoing cohort study among twin couples recruited in pregnancy (Sperling et al., 2006
). Men residing in the Copenhagen area were invited to participate. In order to include a higher number of participants, another group of fathers of twins (Group B = 57) were recruited by a gynaecologist from files from the Hvidovre University Hospital in the Copenhagen area. A total of 52 fathers of twins participated (overall participation rate 37%, Group A = 42% and Group B = 28%). At the time of examination, no information about the zygosity of the twins was available. At the time when all other information was obtained, DNA typings were performed. Among the 52 fathers of twins, 37 were fathers of DZ twins and 15 were fathers of MZ twins. Two of the 37 fathers of DZ twins had been vasectomized and were excluded for the semen analysis. As a reference group, we included a cohort of 349 normal fertile men investigated previously (Jørgensen et al., 2001), who had been recruited when their partners showed up for their first antenatal visit at the hospital (participation rate 44%).
For all men, the eligibility criteria was that he and his mother had to be born in Denmark and the pregnancies had to be achieved by normal sexual relations, and not as a result of any treatment for subfertility or infertility (hormonal treatment, insemination, IVF or intracytoplasmic sperm injection, etc.). Participation in the study was accepted even if the man had a past history of cryptorchidism, orchitis, epididymitis, surgery of the genital tract (including varicocelectomy), chemotherapy, radiotherapy or other diseases, which may affect reproduction. Chronic illness, previous treatment for infertility or subfertility, unwanted pregnancy or prolonged TTP was not an exclusion criteria. Additionally, being a father of twins was not an exclusion criteria in the reference group, but since the spontaneous twinning rate in the Danish population is expected to be 1.24% (Herskind et al., 2005), we would only suspect few fathers of twins to be in this group. The zygosity of the twins was determined by DNA typing after the father had been examined. The inclusion period was June 2003–January 2005 in the study of fathers of twins and September 1996–October 1997 in the reference group. All examinations took place at the University Department of Growth and Reproduction, Rigshospitalet, Copenhagen.
Questionnaires
On the day of attendance, the men returned a completed standardized questionnaire, which they had received in advance. The questionnaire included information on age, health, occupational factors, TTP, lifestyle factors and previous and/or current diseases and genital diseases such as cryptorchidism, epididymitis and operation for torsio testis. Furthermore, the men answered whether they were twins themselves. In addition, fathers of twins reported all previous pregnancies (including spontaneous abortions), whereas the reference group only reported previous births. From fathers of twins, information was obtained on whether they ever had been diagnosed with high blood pressure, hepatitis, diabetes, metabolic disorders, cancer, rheumatic diseases, asthma/cold or other diseases. The reference fathers only answered whether they ever have had any long lasting or chronic diseases.
Physical examination
Physical examination of each participant was performed on the day of the delivery of his semen sample. The same physician performed all examinations in the group of fathers of twins, whereas another physician previously had conducted the examination of the reference group. Evaluations of testes disposition, varicocele and Tanner stages of pubic hair were performed with the men in standing position. A Prader orchidometer as well as ultrasound was used to determine the volume of the testis.
Semen samples
The participants delivered one semen sample obtained by masturbation and ejaculated into a clean collection tube in a room close to the semen laboratory. The period of abstinence was recorded and the semen sample was analysed according to the World Health Organization 1999 guidelines (WHO, 1999) modified in accordance with Jørgensen et al. (1997, 2001, 2002).
Semen smears were stained by the Papanicolaou method at the time of semen delivery. The slides from the control group were stained 10 years ago and stored in darkness to avoid fading. The same experienced technician assessed the sperm morphology according to strict criteria (WHO, 1999) within 21 consecutive working days. All slides were analysed randomly and without knowledge of the identity of the groups. A set of slides scored by one of the technicians, which contributed to the study of Guzick et al. (2001), was used as the standard for purposes of quality control. Each of the 21 working days, our technician scored two of these slides and compared the results with the standard value, and, if any disagreement, the slides were reanalysed until agreement. The semen smears of the reference group have previously been assessed in France as part of a multicenter study using David criteria (Jørgensen et al., 2001). Some slides were accidentally damaged during transportation. Thus, only 226 slides were available for reassessment from the reference group. We do, however, believe that they are a random sample of the 349 original slides.
Statistical analysis
Outcome variables were semen volume, sperm concentration, total sperm count and the percentage of motile and morphologically normal spermatozoa. The semen quality among all fathers of twins was compared with that of the reference group. First, the distribution of potential confounders was compared among fathers of twins and the reference group. Potential confounders were then entered in multiple linear regression analysis. Maternal age and parity, paternal age, birth cohort effect and season of year were evaluated as possible confounders for the semen parameters and the percentages of motile sperm, the duration from ejaculation to assessment of motility was evaluated as an additional confounder. Only duration of abstinence was found to have an effect, and thus the other variables were excluded in the final model. Diseases in the reproductive organs found at the physical examination (varicocele, hydrocele, dispositon or consistency of the testis (soft or normal)) or reported in questionnaires (cryptorchidism, epididymitis, veneric diseases and operation for torsio testis) found to affect any of the outcome variables separately were transformed into one variable for each outcome (present or not present). The variables hydrocele, disposition of the testis, cryptorchidism, operation for torsio testis, epididymitis and veneric diseases were excluded since they showed no effect.
Normally distributed outcome variables were entered directly as continuous variables in the linear multiple regression analysis, whereas sperm concentration and total sperm count were transformed by the use of the natural logarithm to obtain the normality of residuals. Confounders were excluded stepwise if they were not statistically significant at the 10% level. The results are presented with 95% confidence intervals (95% CI). The fit of the regression models was evaluated by testing the residuals for normality and by inspecting the residual plots.
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Results |
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Table I summarizes the results of the unadjusted semen parameters. Fathers of both DZ twins and MZ twins had a better semen quality (sperm concentration, sperm counts, sperm motility and percentage of sperm with normal morphology) than the reference group, although only significant for sperm morphology and motility (Table I). Information on TTP was provided by 34 (92%) fathers of DZ twins and 13 (87%) fathers of MZ twins (Table I). The median TTP was 2 (range 0;120) and 1 (range 0;12) months among fathers of DZ and MZ twins, respectively. In the reference group the overall TTP was 2 months (range 0–72) (Table I).
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The majority of fathers of twins were singletons themselves; one twin father reported to be a twin himself. Fathers of DZ twins were significantly older (mean age 37 years) at the time of examination than fathers of MZ twins (mean age 34 years) and the reference group (mean age 31 years) (P < 0.01) (Table I). No significant difference between the groups was found concerning the mean size of testis measured by orchidometer or by ultrasound (data not shown). Fathers of DZ twins more often reported genital diseases, and a higher frequency was found at the clinical examination (37.8%) (Table II). This was mainly due to the fact that 16.2% of fathers of DZ twins had a left varicocele in contrast to 5.9% of fathers of MZ twins and 2.6% in the reference group. Four (11%) of fathers of twins had been diagnosed with a chronic disease (asthma or articular rheumatism) when compared with three (1%) of the controls (epilepsy, depression or thyroid disorder) (Table II).
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After adjustment, the percentages of motile sperms in fathers of DZ and MZ twins were 11.5 (95% CI; 7.2; 15.9) and 12.5 (95% CI; 6.3; 8.6) percentage points higher than the reference group (P < 0.01). The percentages of morphologically normal sperms were 3.6 (95% CI; 1.7; 5.4) and 4.6 (95% CI; 2.0; 7.2) percentage points higher than the reference group (P < 0.01). In addition, fathers of DZ twins had a 24.7% (95% CI –9.1; 71.3) higher sperm concentration and a 21.8% (95% CI; 11.7; 169.0) higher total sperm count, whereas fathers of MZ twins had 17% (95% CI; –25.2; 83.0) higher sperm concentration and a 16.4% (95% CI; –26.0; 183.0) higher total sperm count (Table III).
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We repeated the analyses among fathers with a TTP
12 months and the levels of the semen parameters were unchanged.
Discussion
We found that fathers of DZ twins had more normal and more motile sperms than the reference group of fertile men. Their sperm counts were also higher, although not significantly. All semen analyses including morphology assessment were carried out blindly, and our laboratory participated in an external quality control program. Differences in participation rates between fathers of twins and controls could not explain our findings since the participation rates in the groups were quite similar. The two groups of fathers of twins were recruited and examined identically, and the zygosity was unknown to the physician performing the physical examination of the father. We, therefore, believe that our results represent a true difference in the semen quality between fathers of twins and the reference group.
Our findings in the group of fathers of DZ twins are in accordance with the hypothesis that natural DZ twinning is a marker of high fecundity, as it reflects the probability of fertilization of two ovas in the same cycle (Lazar et al., 1978; James, 1997). Furthermore, our findings are also in line with a recent study that found reduced DZ twinning rate in subfertile men, although no data on semen quality were reported (Richiardi et al., 2004).
The percentage of sperms with normal morphological features is probably the sperm parameter with the greatest discriminatory power regarding male fecundity (Guzick et al., 2001). The finding of 3.6 percentage points higher morphologically normal cells among fathers of DZ twins compared with the reference may at a glance appear small. However, Guzick et al. found that the discriminatory range with regard to percentage normal forms between fertile men and subfertile men was relatively small (>2% normal sperm cells and <9% normal sperm cells, respectively) (Guzick et al., 2001). Thus, our results concerning morphology may represent a significant difference in fertility potential between the groups. Also, the motility was significantly better in the group of fathers of DZ twins, but this finding should be interpreted with some caution as evaluation of sperm motility is considered to be highly subjective (Jørgensen et al., 1997). Also, the sperm concentration among fathers of DZ twins was higher, although not significantly, than in the reference group. Taken together, our findings strongly indicate a possible correlation between the high semen quality and the chance of being a father of twins.
The percentage of fathers of DZ twins who had diseases in reproductive organs and chronic diseases (37.8 and 10.8%) was similar to that of the reference group (30 and 1%). We did not have information about the birth outcome among the fertile men who served as the reference group and theoretically a few of the men may have given birth to twins in earlier pregnancies. This would, however, bias our findings towards the null hypothesis.
Artificial reproductive technologies are known to increase the risk of DZ twinning and accounts for >95% of these pregnancies (Sperling et al., 2006). When validating DZ twinning as a marker of semen quality, it is therefore important to include only fathers of naturally conceived twins. All participants were questioned about the conception. In addition, the gynaecologists who recruited fathers of twins in group A were informed only to include fathers of naturally conceived twins, and fathers of twins in group B were only included from files that could confirm that no assisted reproduction had been used to conceive the twins. However, analysing TTP, we found that some of fathers of DZ twins had a surprisingly long TTP (one even reported to have waited for 10 years), and we suspect some men from this group to have received assisted reproduction despite the information from the files. This may have biased our findings towards the null hypothesis. Repeating our analyses among participants with a TTP 12 months did, however, not change our results.
The physical examinations of fathers of twins and the reference group were performed by two different physicians, which may cause inter-observer variation, for example, in testis size (data not included). The frequency of a present varicocele was higher among fathers of MZ twins (6.7%) and fathers of DZ twins (14.7%), compared with the reference group (2.6%). However, other studies have found incidences between 8 and 20.5% (Lewis, 1950; Horner, 1960; Clarke, 1966; Øster, 1971), which point to that the frequency of varicoceles at the clinical examination of the reference group may have been under-diagnosed. We performed no quality control between the physicians since the examinations were conducted during different time periods, and it was not possible for the two investigators to meet to assess the inter-observer variation. Therefore, the differences found in testis size and varicocele should be interpreted with caution.
Maternal age and parity influence the DZ twinning rate (Tong and Short, 1998). The mean age and the frequency of multiparous mothers were higher among DZ twin mothers than among the reference mothers (Table I). In the multiple regression analysis, we controlled for maternal age and parity, which however did not change the estimates of differences in semen parameters. Thus, high maternal age and parity among the twin couples do not seem to influence the results.
Spontaneous DZ twinning is considered a marker of high fecundity, as it reflects the frequency of double ovulation, the probability of fertilization and maintenance of a multiple pregnancy (Lazar et al., 1978). It is, as discussed earlier, well documented that DZ twinning increases with maternal age, as the probability of releasing two ovas in one cycle rises with maternal age. If maternal factors, however, are considered equal among fathers of twins and singletons, differences in the fertilization of the two released ovas may be related to semen quality. Other studies have suggested that paternal factors may be important for twinning, although they did not study the semen quality. Basso et al. (2004) found a correlation between increasing TTP and decreased chance of conceiving twins after adjustment for maternal age and parity, and Richiardi et al. (2004) reported fewer twins among subfertile men after adjustment for maternal age. We found a better semen quality among fathers of twins even after adjustment for maternal age. In addition, it can be argued that the higher parity among partners of fathers of twins may be attributed to their semen quality.
The semen quality has been reported to decline with increasing year of birth (Auger et al., 1995; Irvine et al., 1996; Bonde et al., 1998). Since the study of normal fertile men was conducted in 1996, they belonged to an earlier birth cohort (1965) than fathers of twins (1970). We controlled for a possible birth cohort effect, which did not change the results in the semen quality.
It was a surprise that fathers of MZ twins also had a better semen quality than controls. Furthermore, only 20% of fathers of MZ twins had genital diseases (versus 37.8% among fathers of DZ twins and 30% in the reference group). In the analyses, we, however, adjusted for differences in genital diseases, which did not change the results in semen quality, and therefore, we do not believe that differences in these can explain our findings. The causes of MZ twinning are unclear (Hall, 2003). This high semen quality in the group of fathers of MZ twins was an unexpected finding that remains unexplained. However, the result should be considered with some caution, as only 15 fathers of MZ twins participated in the study.
Decreased fecundity of the couples has been hypothesized as one of the causes of the reported decline in twin rates in the 1960s and 1970s. The exact mechanisms behind these secular changes have never been explained, although both female and male factors may be responsible. Our results, however, support the idea that changes in the semen quality may influence the DZ twinning rates.
In conclusion, fathers of DZ twins had a higher semen quality than the reference group of fathers of singletons. Our results lend support to the assumption that spontaneous DZ twinning rate can be used as a sensor of male fecundity in a population.
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Acknowledgements |
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Anne-Grethe Andersen is acknowledged for her examinations of the reference group and biotechnician Maiken Simonsen for the assessment of the sperm morphology. The study was supported by contact BMH4-CT96-0314 from the European Union, the Danish Research Council, grant no. 2107-05-0006 and 9700833, and the Svend Andersen Fond.
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Submitted on August 11, 2006; resubmitted on October 9, 2006; accepted on October 16, 2006.
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