Document Detail

Predictive value of sperm motility characteristics assessed by computer-assisted sperm analysis in intrauterine insemination with superovulation in couples with unexplained infertility.
Jump to Full Text
MedLine Citation:
PMID:  22384418     Owner:  NLM     Status:  PubMed-not-MEDLINE    
Abstract/OtherAbstract:
OBJECTIVE: To determine whether characteristics of sperm motility obtained by computer-assisted sperm analysis (CASA) could predict pregnancy after intrauterine insemination (IUI) in couples with unexplained infertility.
METHODS: Three hundred eighty-three cycles of intrauterine insemination with superovulation were retrospectively analyzed. Semen analysis was performed with CASA before and after swim-up and the parameters were compared between pregnant and non-pregnant women.
RESULTS: The pregnancy rate per cycle was 14.1%. Pregnant and non-pregnant women were comparable in terms of age, infertility duration, the number of dominant follicles. While sperm concentration, motility, and parameters such as average path velocity (VAP) and percentage rapid (RAPID) before semen preparation were significantly different between the pregnancy and non-pregnancy groups, there were no differences in sperm parameters when comparing the two groups after preparation. Using a receiver operating characteristic curve to measure sensitivity and specificity, the optimal threshold value for the predictors of pregnancy was revealed to be a concentration of ≥111×10(6)/mL, a motility of ≥51.4%, and RAPID ≥30.1% before preparation for IUI.
CONCLUSION: Sperm parameters including concentration, motility, and RAPID before sperm preparation could have predictive value for pregnancy outcome after intrauterine insemination with superovulation in couples with unexplained infertility, and would be helpful when counseling patients before they make the decision to proceed with IVF/ICSI-ET.
Authors:
Joung Sub Youn; Sun Hwa Cha; Chan Woo Park; Kwang Moon Yang; Jin Yeong Kim; Mi Kyoung Koong; Inn Soo Kang; In Ok Song; Sang Chul Han
Related Documents :
12213848 - Discordance of monozygotic twins for thyroid dysgenesis: implications for screening and...
8771488 - Maintaining the safe position in a palmar splint. the "double-t" plaster splint.
25307528 - Limk1 regulates human trophoblast invasion/differentiation and is down-regulated in pre...
22281078 - Human spermatozoa vitrified in the absence of permeable cryoprotectants: birth of two h...
20228378 - Activins and related proteins in the establishment of pregnancy.
1917648 - Types of colic and frequency of postcolic abortion in pregnant mares: 105 cases (1984-1...
Publication Detail:
Type:  Journal Article     Date:  2011-03-31
Journal Detail:
Title:  Clinical and experimental reproductive medicine     Volume:  38     ISSN:  2233-8241     ISO Abbreviation:  Clin Exp Reprod Med     Publication Date:  2011 Mar 
Date Detail:
Created Date:  2012-03-02     Completed Date:  2012-08-23     Revised Date:  2013-05-29    
Medline Journal Info:
Nlm Unique ID:  101563916     Medline TA:  Clin Exp Reprod Med     Country:  Korea (South)    
Other Details:
Languages:  eng     Pagination:  47-52     Citation Subset:  -    
Affiliation:
Department of Obstetrics and Gynecology, Clinic of Dr. Min, Cheongju, Korea.
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms
Descriptor/Qualifier:

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

Full Text
Journal Information
Journal ID (nlm-ta): Clin Exp Reprod Med
Journal ID (publisher-id): CERM
ISSN: 2233-8233
ISSN: 2233-8241
Publisher: The Korean Society for Reproductive Medicine
Article Information
Download PDF
Copyright © 2011. The Korean Society for Reproductive Medicine
open-access:
Received Day: 23 Month: 12 Year: 2010
Revision Received Day: 11 Month: 2 Year: 2011
Accepted Day: 18 Month: 2 Year: 2011
Print publication date: Month: 3 Year: 2011
Electronic publication date: Day: 31 Month: 3 Year: 2011
Volume: 38 Issue: 1
First Page: 47 Last Page: 52
ID: 3283051
PubMed Id: 22384418
DOI: 10.5653/cerm.2011.38.1.47

Predictive value of sperm motility characteristics assessed by computer-assisted sperm analysis in intrauterine insemination with superovulation in couples with unexplained infertility
Joung Sub Youn1
Sun Hwa Cha2
Chan Woo Park2
Kwang Moon Yang2
Jin Yeong Kim2
Mi Kyoung Koong2
Inn Soo Kang2
In Ok Song2
Sang Chul Han3
1Department of Obstetrics and Gynecology, Clinic of Dr. Min, Cheongju, Korea.
2Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Cheil General Hospital and Women's Healthcare Center, Kwandong University College of Medicine, Seoul, Korea.
3Laboratory of Reproductive Biology and Infertility, Cheil General Hospital and Women's Healthcare Center, Kwandong University College of Medicine, Seoul, Korea.
Correspondence: Corresponding author: Sun Hwa Cha. Department of Obstetrics and Gynecology, Cheil General Hospital and Women's Healthcare Center, 1-19 Mukjeong-dong, Jung-gu, Seoul 100-380, Korea. Tel: +82-2-2000-4738, Fax: +82-2-2264-1490, dr-baram@hanmail.net

Introduction

Although assisted reproductive technologies (ARTs) including in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) have been developed, intrauterine insemination (IUI) still remains a reliable method that can be applied to many infertile couples. Compared to IVF or ICSI, IUI is less invasive and associated with lower costs and a low incidence of complications, making it a relatively cost-effective treatment for many forms of infertility [1]. Indications for IUI have included mild male factor infertility (oligozoospermia, asthenozoospermia, pyospermia), cervical factor infertility, and cases of unexplained infertility.

In cases of unexplained infertility, the pregnancy rate with IUI has been reported to be 9-20% [2-5]. Moreover, a meta-analysis revealed that the live birth rate of IUI increased twofold when it was undergone with ovarian stimulation rather than the natural cycle [6]. The success rates of IUI with superovulation depend on the woman's age, duration of infertility, sperm quality, and tubal patency on either side [3,7]. Among sperm parameters, total motile sperm count and morphology are known to be closely associated with the success of IUI [2,8-10].

Standard semen quality assessment describes the volume, viscosity, concentration, normal and abnormal morphology, and motility of spermatozoa when conducted according to guidelines by the World Health Organization (WHO) [11]. However, it is a rather subjective method which has been associated with large inter-laboratory variation in estimating the respective percentages of progressive and non-progressive motile and immotile spermatozoa, which makes it difficult to predict fertility [12,13]. In an attempt to make assessments of sperm movements more objective and detailed, tools for computer-assisted sperm analysis (CASA) have been developed. CASA is an automated method used to visualize sperm that can assess several specific motility parameters describing the movements of spermatozoa. Several studies have addressed CASA parameters as predictors of male fertility. Larsen et al. [14] reported that CASA parameters were useful in predicting the possibility of natural pregnancy in the general male population. In studies of male infertility, some CASA parameters were reported to be useful in predicting pregnancy after IUI with partner or donor sperm [15-18], and in the assessment of sperm fertilizing ability in IVF [19].

On the other hand, some studies have failed to show the association of CASA parameters with the success of IUI [20,21], and there have been few studies about its usefulness in predicting pregnancy after IUI in cases of unexplained infertility.

The aim of this study was to determine whether assessing characteristics of sperm motility using CASA could predict pregnancy after IUI with superovulation in couples with unexplained infertility.


Methods
1. Subjects

This is a retrospective case-control study that was performed in our infertility clinic between October 2006 and September 2008. Three hundred and eighty-three patients with unexplained infertility that underwent IUI with superovulation were included in this study. Patients were divided into two groups, the pregnant group and non-pregnant group.

The inclusion criteria were a follicular stimulating hormone (FSH) level no greater than 12 mIU/mL, at least unilateral tubal patency demonstrated in hysterosalpingogram, and semen analysis resulting in normal sperm counts, morphology, and motility according to the WHO classification. Exclusion criteria were a woman's age of more than 40 years, ovarian cyst detected in the ultrasound examination, uterine lesions such as submucosal leiomyoma, and a previous diagnosis of moderate to severe pelvic endometriosis.

Successful pregnancy after intrauterine insemination was confirmed by ultrasound as one or more visible gestational sacs in the uterine cavity.

2. Superovulation and intrauterine insemination

Superovulation was conducted with either 150 IU of intramuscularly injected uhMG (IVF-M; LG Life Science, Seoul, Korea) or 100 mg of oral clomiphene citrate (Clomifene; Young Poong Pharmaceutical, Incheon, Korea) for five days starting on day 3 of the menstrual cycle, followed by daily injection of 150 IU of hMG. When more than two dominant follicles reached a diameter of 17 mm or the urinary specimen showed positive results for LH surge, 5,000 IU of hCG (Pregnyl; Organon, Oss, the Netherlands) was injected intramuscularly, and after 24 to 36 hours, IUI was performed only once.

Semen was collected from subjects via ejaculation after at least 48 hours of abstinence. The semen was then analyzed after at least 30 minutes of liquefaction. For semen analysis, 10 µL of semen was analyzed by computer assisted sperm analysis (CASA; Sperm Analysis Imaging System [SAIS] ver. 10.1, Medical Supply, Wonju, Korea), and concentration, sperm motility, total motile sperm count, hyperactivity percent (HYP), curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP) , linearity (LIN), straightness (STR), beat cross frequency (BCF), and amplitude of lateral head displacement (ALH), percentage rapid (RAPID) were reported. Semen preparation was performed using the conventional swim-up method, and the same parameters were reanalyzed via a computerized process.

3. Statistical analysis

Statistical analysis was carried out with the SPSS ver. 16.0 (SPSS Inc., Chicago, IL, USA). Calculations of statistical differences between the groups were carried out by a Student's t-test or a chi-square test. Receiver operating characteristic (ROC) curves were constructed for the calculation of the clinically acceptable cut-off value of each individual parameter in predicting pregnancy by IUI. A p-value of <0.05 was defined as representing a significant difference.


Results

A total of 383 cycles of intrauterine insemination with superovulation were conducted. There were 54 cases of successful intrauterine pregnancy and the pregnancy rate was estimated to be 14.1%. There were no significant differences between the pregnant group and the non-pregnant group in terms of female age, duration of infertility, basal hormone levels, number of dominant follicles, or endometrial thickness at the time of hCG administration (Table 1).

Statistically significant differences were found in some variables associated with semen analysis before swim-up: sperm concentration (152.9±13.7×106/mL vs. 118.8±4.3×106/mL in the pregnant and non-pregnant group, respectively; p=0.021); motility (56.8±1.8% vs. 51.2±0.9%; p=0.007); VAP (40.8±1.05 µm/sec vs. 38.4±0.4 µm/sec; p=0.033); and RAPID (28.1±2.16% vs. 24.5±0.87%; p=0.022). VCL (67.2±1.85 µm/sec vs. 63.3±0.74 µm/sec; p=0.050) and VSL (32.8±1.01 µm/sec vs. 30.7±0.37 µm/sec; p=0.050), parameters of sperm motility, showed some degree of difference that was not statistically significant. Differences in LIN, STR, BCF, and ALH were not statistically significant. After swim-up, semen analysis parameters were not significantly different between the two groups (Table 2).

According to the ROC curve, RAPID, motility, and concentration parameters in the unprocessed semen were predictive of pregnancy and the cut-off values were 30.1% for RAPID, 51.4% for motility, and 111×106/mL for concentration. Further analysis was performed according to the number of parameters that were consistent with these criteria. The pregnancy rate per cycle was 6.7% (7/104) if no criteria were met, 11.4% (13/114) for one criterion, 15.7% (11/70) for two criteria, and 24.2% (23/95) if all three criteria were met (Tables 3, 4, Figure 1).


Discussion

Intrauterine insemination with or without superovulation is usually the first-line treatment choice for patients with unexplained infertility who failed to conceive by conventional ovulation induction with timed coitus. Although the IUI procedure is less invasive and less expensive than IVF or ICSI, both infertile couples and doctors bear a psychological burden because of the relatively low success rate per cycle, leading to a repetition of the procedure and a risk of higher-order multiple pregnancy associated with superovulation [1,2,4,5].

Many investigators have attempted to determine the optimal predictor of success to help counsel patients before they make the decision to proceed with IVF/ICSI-ET. It is known that female age, duration of infertility, tubal patency, the number of induced follicles, the insemination technique, timing, and several semen parameters may affect the outcome of the IUI procedure [3,7]. Among semen parameters, sperm motility is believed to be one of the most important characteristics correlated with fertility [22,23].

Prospective studies have shown that the conventional semen profile of the WHO is unable to discriminate between fertile and infertile men, especially in idiopathic infertility [22,24-26]. Accordingly, additional functional tests, such as the zona-free hamster egg penetration test [27] and evaluation of acrosome reaction combined with assessments of cell viability [28], have been suggested to provide more information on the fertilizing potential of human spermatozoa in addition their number or appearance. However, many of the assays involved are technically complex.

CASA is an automated method that can measure several specific motility parameters that describe the movements of spermatozoa with the advantage of providing more objective, reproducible, and detailed data on sperm motility. Many studies have indicated that the direct quantitative assessment of sperm movement by CASA reflects the fertilizing ability of human spermatozoa in vitro under conditions where the conventional semen profile is of limited diagnostic value. A prospective follow-up study reported that the concentration of motile spermatozoa, defined as spermatozoa with a VCL>25 µm/sec, was the most significant and independent CASA parameter in predicting the chance of natural conception in the general male population [14]. Barratt et al. [29] found the total number of spermatozoa and VAP to be predictors of pregnancy. Other parameters such as ALH, VSL, VCL, and LIN have also been reported to be correlated with fertility [28,30].

The relative influence of semen parameters on the likelihood of a successful outcome in IUI remains controversial. Shibahara et al. identified three semen parameters including normal morphology ≥15.5% before sperm separation, rapid motility >25.5%, and VCL>102.6 µm/sec after sperm preparation as predictors of pregnancy by IUI in male infertility, and reported that no pregnancy was acheived if none of these three parameters were satisfied [15]. Freour et al. [20] found that the concentration and proportion of progressive motile spermatozoa, defined as cells with VAP>25 µm/sec and VSL>100 µm/sec, were higher in the pregnancy group than in the failure group, while respective sperm motility parameters measured by CASA failed to predict pregnancy in IUI cycles conducted with donor sperm. They also reported that ALH improvement during the sperm preparation is necessary for pregnancy in an IUI with frozen donor semen and speculated that it could help in improving pregnancy rates in IUI with donor semen programs, as ALH evolution before and after preparation can be easily calculated [21].

In a retrospective analysis of the relationship between the CASA estimates and fertilization rates in IVF-ET cycles, two parameters, including VCL and rapid, provide reliable estimations of the fertilizing ability of human sperm [19].

Most of the studies on CASA parameters and fertility outcome in IUI have been conducted in couples with male infertility, whereas few studies were conducted in couples with unexplained infertility. In this study, we assessed the sperm motility parameters by CASA in 383 IUI cycles with superovulation indicated in couples with unexplained infertility. Specific CASA parameters such as VAP, RAPID, VSL, VCL, as well as sperm concentration and motility were significantly different between the pregnant group and the non-pregnant group. We also found that the combination of parameters such as RAPID ≥30.1%, motility ≥51.4%, and concentration ≥111×106/mL before sperm preparation could be useful in predicting pregnancy in IUI. For instance, the pregnancy rate was highest if all three parameters were satisfied, while it was lowest if no parameters were satisfied.

Many studies reported that the improvement of sperm motility parameters during the preparation process or their value after preparation were correlated with their pregnancy outcome of IUI [15-21], whereas we did not find any difference between CASA parameters after sperm preparation according to cycle outcome. This may have been due to our use of fresh semen of normal values by WHO criteria, in contrast to previous studies that utilized semen with male factor infertility or frozen-thawed donor sperm.

In general, one of the most important movements associated with sperm fertilizing ability is known to be hyperactivated motility [31,32]. Hyperactivation is characterized by the development of high velocity, large amplitude, asymmetrical flagellar waves, and is thought to facilitate the detachment of spermatozoa from the oviductal epithelium and penetration of the matrix of the cumulus oophorus and the zona pellucida [32]. CASA has been used to detect hyperactivation, as it generates a characteristic non-progressive star-spin pattern of movement, and to identify the percentage of sperm in a sample that are hyperactivated. As for hyperactivation, we did not find any difference between the pregnant and the non-pregnant groups. Also, there was no difference in the pregnancy rate according to hyperactivation. It can be postulated that hyperactivation occurred when the spermatozoa reached the oocyte vicinity and CASA could only be of limited value to the assessment of hyperactivation in vitro before the IUI procedure. Unfortunately, factors triggering hyperactivation in vivo are poorly understood, and it has been suggested that the trigger may be hormones, ions, secretions of the oviductal epithelium, or a change in the pH of oviduct fluid or follicular fluid introduced into the oviduct by the oocyte-cumulus mass [33,34].

In conclusion, concentration and motility assessment of semen by CASA is a fairly useful method for predicting pregnancy outcome after IUI with superovulation, which could be helpful in planning strategies for the further treatment of unexplained infertile couples by proceeding with IVF/ICSI-ET.


Notes

No potential conflict of interest relevant to this article was reported.

References
1. ESHRE Capri Workshop GroupIntrauterine inseminationHum Reprod UpdateYear: 20091526527719240042
2. Stone BA,Vargyas JM,Ringler GE,Stein AL,Marrs RP. Determinants of the outcome of intrauterine insemination: analysis of outcomes of 9963 consecutive cyclesAm J Obstet GynecolYear: 19991801522153410368500
3. van der Westerlaken LA,Naaktgeboren N,Helmerhorst FM. Evaluation of pregnancy rates after intrauterine insemination according to indication, age, and sperm parametersJ Assist Reprod GenetYear: 1998153593649673879
4. Ransom MX,Blotner MB,Bohrer M,Corsan G,Kemmann E. Does increasing frequency of intrauterine insemination improve pregnancy rates significantly during superovulation cycles?Fertil SterilYear: 1994613033078299787
5. Dodson WC,Haney AF. Controlled ovarian hyperstimulation and intrauterine insemination for treatment of infertilityFertil SterilYear: 1991554574672001748
6. Verhulst SM,Cohlen BJ,Hughes E,Te Velde E,Heineman MJ. Intra-uterine insemination for unexplained subfertilityCochrane Database Syst RevYear: 20064CD00183817054143
7. Steures P,van der Steeg JW,Mol BW,Eijkemans MJ,van der Veen F,Habbema JD,et al. Prediction of an ongoing pregnancy after intrauterine inseminationFertil SterilYear: 200482455115236988
8. Shulman A,Hauser R,Lipitz S,Frenkel Y,Dor J,Bider D,et al. Sperm motility is a major determinant of pregnancy outcome following intrauterine inseminationJ Assist Reprod GenetYear: 1998153813859673883
9. Hauser R,Yogev L,Botchan A,Lessing JB,Paz G,Yavetz H. Intrauterine insemination in male factor subfertility: significance of sperm motility and morphology assessed by strict criteriaAndrologiaYear: 200133131711167514
10. van Voorhis BJ,Barnett M,Sparks AE,Syrop CH,Rosenthal G,Dawson J. Effect of the total motile sperm count on the efficacy and cost-effectiveness of intrauterine insemination and in vitro fertilizationFertil SterilYear: 20017566166811287015
11. World Health OrganizationWHO laboratory manual for the examination of human semen and sperm-cervical mucus interactionYear: 19994th edCambridgeCambridge University Press
12. Jørgensen N,Auger J,Giwercman A,Irvine DS,Jensen TK,Jouannet P,et al. Semen analysis performed by different laboratory teams: an intervariation studyInt J AndrolYear: 1997202012089401822
13. Brazil C,Swan SH,Drobnis EZ,Liu F,Wang C,Redmon JB,et al. Standardized methods for semen evaluation in a multicenter research studyJ AndrolYear: 20042563564415223853
14. Larsen L,Scheike T,Jensen TK,Bonde JP,Ernst E,Hjollund NH,et al. The Danish First Pregnancy Planner Study TeamComputer-assisted semen analysis parameters as predictors for fertility of men from the general populationHum ReprodYear: 2000151562156710875866
15. Shibahara H,Obara H,Ayustawati,Hirano Y,Suzuki T,Ohno A,et al. Prediction of pregnancy by intrauterine insemination using CASA estimates and strict criteria in patients with male factor infertilityInt J AndrolYear: 200427636815149462
16. Macleod IC,Irvine DS. The predictive value of computer-assisted semen analysis in the context of a donor insemination programmeHum ReprodYear: 1995105805867782436
17. Marshburn PB,McIntire D,Carr BR,Byrd W. Spermatozoal characteristics from fresh and frozen donor semen and their correlation with fertility outcome after intrauterine inseminationFertil SterilYear: 1992581791861624002
18. Sidhu RS,Sharma RK,Agarwal A. Effects of cryopreserved semen quality and timed intrauterine insemination on pregnancy rate and gender of offspring in a donor insemination programJ Assist Reprod GenetYear: 1997145315379401872
19. Hirano Y,Shibahara H,Obara H,Suzuki T,Takamizawa S,Yamaguchi C,et al. Relationships between sperm motility characteristics assessed by the computer-aided sperm analysis (CASA) and fertilization rates in vitroJ Assist Reprod GenetYear: 20011821321811432113
20. Freour T,Jean M,Mirallie S,Langlois ML,Dubourdieu S,Barriere P. Predictive value of CASA parameters in IUI with frozen donor spermInt J AndrolYear: 20093249850418399982
21. Fréour T,Jean M,Mirallié S,Dubourdieu S,Barrière P. Computer-Assisted Sperm Analysis (CASA) parameters and their evolution during preparation as predictors of pregnancy in intrauterine insemination with frozen-thawed donor semen cyclesEur J Obstet Gynecol Reprod BiolYear: 201014918618920074847
22. Bostofte E,Bagger P,Michael A,Stakemann G. Fertility prognosis for infertile men: results of follow-up study of semen analysis in infertile men from two different populations evaluated by the Cox regression modelFertil SterilYear: 199054110011062245836
23. Eimers JM,te Velde ER,Gerritse R,Vogelzang ET,Looman CW,Habbema JD. The prediction of the chance to conceive in subfertile couplesFertil SterilYear: 19946144528293843
24. Aitken RJ,Best FS,Warner P,Templeton A. A prospective study of the relationship between semen quality and fertility in cases of unexplained infertilityJ AndrolYear: 198452973036540770
25. Irvine DS,Aitken RJ. Predictive value of in-vitro sperm function tests in the context of an AID serviceHum ReprodYear: 198615395453818912
26. Ducot B,Spira A,Feneux D,Jouannet P. Male factors and the likelihood of pregnancy in infertile couples. II. Study of clinical characteristics--practical consequencesInt J AndrolYear: 1988113954043235208
27. Shibahara H,Mitsuo M,Inoue M,Hasegawa A,Shigeta M,Koyama K. Relationship between human in-vitro fertilization and intracytoplasmic sperm injection and the zona-free hamster egg penetration testHum ReprodYear: 199813192819329740451
28. Aitken RJ. Sperm function tests and fertilityInt J AndrolYear: 200629697516466526
29. Barratt CL,Tomlinson MJ,Cooke ID. Prognostic significance of computerized motility analysis for in vivo fertilityFertil SterilYear: 1993605205258375537
30. Paston MJ,Sarkar S,Oates RP,Badawy SZ. Computer-aided semen analysis variables as predictors of male fertility potentialArch AndrolYear: 19943393997818377
31. Sukcharoen N,Keith J,Irvine DS,Aitken RJ. Definition of the optimal criteria for identifying hyperactivated human spermatozoa at 25 Hz using in-vitro fertilization as a functional end-pointHum ReprodYear: 199510292829378747047
32. Suarez SS. Control of hyperactivation in spermHum Reprod UpdateYear: 20081464765718653675
33. Nichol R,Hunter RH,Gardner DK,Leese HJ,Cooke GM. Concentrations of energy substrates in oviductal fluid and blood plasma of pigs during the peri-ovulatory periodJ Reprod FertilYear: 1992966997071339849
34. Yao Y,Ho P,Yeung WS. Effects of human follicular fluid on the capacitation and motility of human spermatozoaFertil SterilYear: 20007368068610731525

Article Categories:
  • Original Article

Keywords: Insemination, Artificial, Infertility, Semen Analysis, Image Processing, Computer-assisted, Sperm Motility, Human.

Previous Document:  Endometrial histology and predictable clinical factors for endometrial disease in women with polycys...
Next Document:  Comparison of elective single cleavage-embryo transfer to elective single blastocyst-embryo transfer...